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Epigenetic plasticity of cultured female human embryonic stem cells and regulation of gene expression and chromatin by PR-SET7 mediated H4K20me1

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Epigenetic plasticity of cultured female human embryonic stem cells and regulation of gene expression and chromatin by PR-SET7 mediated H4K20me1

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Content EPIGENETIC PLASTICITY OF CULTURED FEMALE HUMAN EMBRYONIC STEM CELLS AND REGULATION OF GENE EXPRESSION AND CHROMATIN BY PR-SET7 MEDIATED H4K20ME1 by Chendhore Sai Veerappan ________________________________________________________________ A Dissertation Presented to the FACULTY OF THE USC GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA In Partial Fulfillment of the Requirements for the Degree DOCTOR OF PHILOSOPHY (GENETICS MOLECULAR and CELLULAR BIOLOGY) December 2012 Copyright 2012 Chendhore Sai Veerappan ii Acknowledgements This work is dedicated to my late father, Mr. Veeyen Rana Veerappan. His love, hard work, affection and commitment to his family and others will always reflect in all my scientific and personal endeavors. To my PI Dr. Judd Rice, who has been a constant source of support and guidance throughout my graduate career. He gave me the freedom to expand my knowledge in an unknown field and provided me with a very unique opportunity to pursue a specialty in both experimental and computational biology. I could not ask for a better boss. All of my work leaned on the constant support of my lab members, Dr. Tanya Spektor, Dr. Jennifer Sims, Dr. Shawn Rao, Dr. Creighton Tuzon and especially my colleague and friend Dr. Lauren Congdon. To my undergraduate and masters advisors, Dr. Etsuko Moriyama and Dr. Zoya Avramova for introducing me to science and encouraging me to always strive for bigger and better things. To my committee members, Dr. Peter Laird, Dr. Michael Stallcup, Dr. Woojin An and Dr. Richard Watanabe. You have given me excellent guidance and critical assessment for all my scientific work and future directions. I hope I can always communicate with you about science. To my immediate and extended family, and friends for always believing in me through the ups and lows of all these years. A dedication to my mom Padma for always putting iii education the first and foremost priority; my sister, Dhevi, brother in-law Krishna and my wonderful nephew Arjun for their unconditional love; my girlfriend Meagan Hubinger, Mr. and Mrs. Hubinger, and all the Hubinger family members for making my life much more easier and wonderful. Lastly, all my fury friends, Krishnakat, Jamalzer, Bella, Pookie, Pickles, Rosie, Sunny, Zoe and Quiddy, for being the ultimate stress relief one could wish for. i Table of Contents Acknowledgements ii List of Tables iv List of Figures v Abstract vii Introduction 1 Chromatin and epigenetic modifications 1 Active and repressive histone modifications 4 ‘Bivalent’ domains 6 Human embryonic stem cell lines and culture conditions 7 Embryonic stem cell-surface markers and master pluripotency associated genes 9 Embryonic stem cell heterogeneity 11 X-chromosome inactivation in female human embryonic stem cells 12 Roles of PR-SET7 and H4K20me1 in gene expression and chromatin regulation 17 Chapter 1: Epigenetic Plasticity of Female Human Embryonic Stem Cells 22 1.1 Introduction 22 1.2 Results 24 1.2.1 Processing and ChIP-sequencing of HES3 and H9 Samples 24 1.2.2. ChIP-Seq analysis 26 1.2.3 Quantile normalization 29 1.2.4 H3K27me3: X-chromosome differences between LP, HP and H9 32 1.2.5 H3K27me3: Genome-wide differences between P2 and P5 35 1.2.6 Identification of conserved quantitative H3K27me3 enrichment differences between P2 and P5 39 1.2.7 Identification of conserved quantitative H3K27me3 enrichment differences based on Xi status of H9 41 1.2.8 Unique H3K4me3 epigenetic signature in low passage HES3 cells 50 1.2.9 Conserved quantitative H3K4me3 changes occur at key developmentally regulated genes 55 1.2.10 Independent quantitative H3K4me3 changes in LP and HP 58 1.2.11 A subset of epigenetic changes at key regulated genes correspond with gene expression 61 1.3 Discussion and Future Directions 68 ii Chapter 2: Role of PR-SET7 mediated H4K20me1 in Gene Expression Regulation 76 2.1 Introduction 76 2.2 Results 77 2.2.1 H4K20me1 and H4K20me3 enriched regions on chromosomes 21 and 22 77 2.2.2 Profile of H4K20me1 and H4K20me3 at target genes 85 2.2.3 H4K20me1 is needed for transcriptional repression of specific target genes 88 2.2.4 Global role of PR-SET7 and H4K20me1 in gene expression repression 93 2.2.5 Small molecule inhibitors of PR-SET7 108 2.3 Discussion and Future Directions 114 Chapter 3: Materials and Methods 121 3.1 Materials and Methods: Chapter 1 121 3.1.1 Cell Culture, passaging and cell sorting 121 3.1.2 Chromatin immunoprecipitation and sequencing 122 3.1.3 Sequence alignment and processing 123 3.1.4 Peak calling and quantitative analysis 124 3.1.5 Gene expression and heatmap 125 3.2 Materials and Methods: Chapter 2 126 3.2.1 Chromatin immunoprecipitation 126   3.2.2 ChIP-chip and ChIP-cloning 127   3.2.3 Peak detection and gene identification 129   3.2.4 Peak distribution analysis 129 3.2.5 Gene expression analysis 130 3.2.6 PR-SET7 small molecule inhibitor, cell growth and cell-cycle characterization 131 3.3 Perl Code 131 3.3.1 Background Subtraction 131 3.3.2 Comparing between multiple gene sets 132 3.3.3 Counting number of tags in a given window from a database 133 3.3.4 Creating equivalent database files 134 3.3.5 Graph Meta-plot from ChIP-Chip Peak data 136 3.3.6 Plot meta-analysis of ChIP-seq count/ChIP-chip intensity data 140 3.3.7 Peak file annotation to gene 142 3.3.8 Splitting bed file to chromosome files 144 Bibliography 145 Appendix A 157 Appendix B 185 Appendix C 214 iii Appendix D 226 Appendix E 248 iv List of Tables Table 1: Summary of ChIP-seq uniquely aligned reads in each library and associated peak number 27 Table 2: Conserved differentially enriched H3K27me3 predicted enhancer regions 49 Table 3: Lowly enriched H3K4me3 domains in LP P2 55 Table 4: H4K20me1/me3 peak and gene target summary 78 Table 5: H4K20me1 enriched genes 79 Table 6: H4K20me3 enriched genes 81 Table 7: Genes with expression change >2-fold in HELA cells lacking PR-Set7 and global loss of H4K20me1 94 Table 8: List of primers used for ChIP-PCR/cloning and gene expression 127 v List of Figures Figure 1: FACS profile of HP, LP and H9 25 Figure 2: Representation of read-count and quantile normalized data 31 Figure 3: H3K27me3 pan-X chromosome enrichment in LP, HP and H9 33 Figure 4: Quantile normalized H3K27me3 distribution between P2 and P5 of HP, LP and H9 38 Figure 5: Relationship of quantitatively over-enriched loci between LP, HP and H9 44 Figure 6: GREAT GO analysis of conserved differentially enriched H3K27me3 loci 46 Figure 7: H3K4me3 quantile normalized peak distribution between P2 and P5 in HP and LP 53 Figure 8: Conserved differentially enriched H3K4me3 loci between HP and LP 58 Figure 9: GREAT GO analysis of differentially H3K4me3 enriched loci in P2 and P5 from HP and LP 61 Figure 10: Differential expression of gene targets between P2 and P5 64 Figure 11: GREB1L enrichment data and genome-wide epigenetic changes correlating with gene expression differences 67 Figure 12: H4K20me1 is preferentially enriched within genes while H4K20me3 is typically located within repetitive elements 85 Figure 13: Identification of H4K20me1- and H4K20me3-associated genes 87 Figure 14: Meta-analysis of H4K20me1/me3 enrichment at gene loci 88 Figure 15: Depletion of the PR-Set7 H4K20 monomethyltransferase results in the selective derepression of H4K20me1-associated gene 90 Figure 16: H4K20me1 is essential for repression of endogenous H4K20me1-associated genes 92 Figure 17: Cell type specific distribution of H4K20me1 98 vi Figure 18: Histone modification changes during mESC differentiation mESCs treated with 100nM retinoic acid 99 Figure 19: K-means clustering heatmap of H4K20me1 positive genes 104 Figure 20: PR-SET7 and H4K20me1 distribution in Drosophila 107 Figure 21: NIH293T cells treated with SP, FH and FM drugs 110 Figure 22: Propidium Iodide stained cell-cycle analyses of FM drug treated NIH293T cells 113 vii Abstract Epigenetics is the study of changes in gene expression that occur in cells without alterations to DNA sequence. Epigenetic modifications are critical components of eukaryotic gene regulation and chromatin organization. Different epigenetic mechanisms, including the post-translational modifications of DNA-associated histone proteins play a role in the activation or repression of genes. One of my research goals was to define the epigenetic signature of cultured human embryonic stem cells (hESCs) and to determine how their epigenomes change during lineage commitment. Pluripotent hESCs are capable of self-renewal and have the capacity to differentiate into any lineage of the embryo. However, hESCs grown in culture are heterogeneous in nature, consisting of a mixture of pluripotent to differentiated cells, making investigation of pluripotent hESCs difficult. Therefore precise definition of pluripotent cells present in culture is critical in order to use these cells for future stem cell based therapies. Using a FACS based approach, I demonstrated that I was able to selectively isolate sub-populations from the bulk culture that expressed high levels of pluripotency factors (P2) and those that lacked these factors (P5). For both populations, I performed high resolution ChIP-sequencing for 2 different histone modifications, indicative of transcriptionally active regions (H3K4me3) and repressed regions (H3K27me3) in order to define, compare and contrast their epigenomes. In the widely used female H9 hESC line, I found that the X-chromosome of the P5 population was enriched for H3K27me3 but was not in the P2 population. These findings strongly viii suggest that P2 represents the more naïve pluripotent stem cells, whereas, P5 has committed to differentiate, consistent with X-chromosome inactivation (Xi). In a separate female hESC line, HES3, I discovered that low passage cells (LP) are devoid of H3K27me3 on the X-chromosome but high passage cells (HP) are enriched for H3K27me3 on the X-chromosome. These findings indicated that extended passage length of hESCs in culture can have a dramatic effect on their epigenetic signature. My detailed analysis of these data sets revealed many novel findings. In LP P2 cells, I defined for the first time, the presence of a non-canonical H3K4me3 profile which is characterized by lowly enriched H3K4me3 domains many kilobases long, and spanning protein families such as zinc finger, keratin, olfactory and extra cellular matrix protein families. The function of these long domains in hESCs are unknown and previously undefined. I have also detected many stochastic quantitative differences in H3K4me3 and/or H3K27me3 between P2 and P5 that are not conserved between cell types, suggesting that embryonic stem cells are epigenetically plastic. However, more importantly, I have identified a core subset of genes, promoters and regulatory regions that contain quantitative epigenetic differences between P2 and P5 and correlate with gene expression changes. These conserved regions may play a critical role in early differentiation or maintenance of pluripotency. Overall, I have defined a unique epigenetic signature of purified pluripotent stem cells and identified conserved epigenetic changes that likely play an important role in the maintenance of pluripotent state or play a role in the commitment to differentiation (Chapter 1). ix My second research goal was to investigate the role of the PR-SET7 H4K20 mono- methyltransferase (H4K20me1) in the transcriptional regulation of specific genes. Although PR-SET7-mediated H4K20me1 was previously shown to be involved in several DNA-templated processes including chromatin compaction, DNA damage response, DNA replication and cell cycle progression, the role of H420me1 in transcriptional regulation remains controversial. Initial studies showed that H4K20me1 functioned as a repressor but newer studies suggested a role in activation. Using conventional molecular biology techniques, I found that PR-SET7 and H4K20me1 predominantly functions as a transcriptional repressor of specific sets of genes. Consistent with this, my bioinformatics analysis indicated that H4K20me1 associated genes are largely devoid of acetylated histones; marks of transcriptionally active genes. In addition, I discovered that H4K20me1-associated genes are typically cell-type specific, but also, ~500 highly transcribed metabolic genes are conserved for H4K20me1 across cell types. Also, I showed that in genes enriched with PR-SET7 and H4K20me1, the two enrichments were physically distinct on gene bodies and they are highly expressed compared to genes modified for either H4K20me1 or PR-SET7. My results demonstrated that PR-SET7-mediated H4K20me1 functions to repress certain genes in a cell-type specific manner, regardless of basal levels of expression. However, my study also suggested that H4K20me1 together with localization of PR-SET7 might have a distinct function associated with highly expressed genes, compared with genes modified with only H4K20me1 or PR-SET7, which are expressed at lower levels (Chapter 2) 1 Introduction Chromatin and epigenetic modifications One of the most fascinating phenomena in science is the systematic and highly precise development of organisms. The amazing complexity and diversity of living organisms is due to the various distinct types of cells and tissues in the body, responsible for all the various functions and roles needed for life. One of the great challenges in Biology is to understand this complex diversity. Most challenging in trying to understand the development of organisms is the inherent capacity of a single cell fertilized zygote from the parents to then develop, over the course of time, into all the necessary cells, tissues and organs that are required for the makeup of an organism. A totipotent stem cell is defined as a cell capable of differentiating into all tissues of the organism and extra- embryonic tissue such as the placenta, umbilical chord etc. A single cell eventually divides and develops into an adult organism; from a totipotent single stem cell to the developed adult organism, every cell consists of the same DNA genetic code. The genetic code contains information for producing genes. Genes produce all the building blocks needed for the identity and survival of the cell. The fundamental question is, what regulates the differentiation and development of a single cell into a developing fetus? The DNA in a single cell contains all the information needed to produce the necessary building blocks of life such as genes that produce RNA and proteins. However, in the cell, DNA is organized in a fashion in which the access to the information is controlled, so that only information needed to produce a cell with a specific identity and function is 2 accessible and other information is hidden and not processed. When fully stretched out, DNA in a single cell is approximately 1.8 meters long but compacted in the nucleus of a cell approximately 6µm (6 millionth of a meter). The compaction and regulation is carried out by a set of proteins and chemical modifications that allow for the vast information to be compacted and processed in a highly precise manner. In eukaryotes, DNA is compacted in the cell in the form of chromatin. Chromatin consists of repeating units of protein and DNA called nucleosomes. A nucleosome consists of a histone octamer protein complex (2 copies of histone H2A, H2B, H3 and H4) with approximately 147 bases of DNA wrapped around it (Margueron et al., 2010). The organization of the nucleosomes into chromatin and its regulation in allowing access of DNA to transcription and translation, which produce RNA and protein, is the central process that characterizes cell identity and function. The information needed to regulate chromatin is coded on the DNA itself and the histone protein complexes that are associated with the DNA. The study of these modifications that occur on DNA molecules and histones is called Epigenetics. More formally, Epigenetics is the study of changes in the genome that turn genes off or on, which cannot be explained by changes in the DNA sequence. Epigenetic modifications are modifications on DNA molecules by methylation or post- translational modifications of histone found in chromatin. Post-translational modifications include acetylation, methylation, phosphorylation, among other covalent 3 modifications of histones. Each modification, or in combination with others, are read by effector proteins which then function in gene expression regulation, chromatin structure modification, and other functions. Histone variants also play a role in chromatin organization (Kouzarides, 2007). DNA methylation occurs on islands of CpG di- nucleotides and has been shown to be associated with repressed chromatin and gene expression (Bird, 2002). The core of histones forms a tight globular structure around which DNA is wound. However the N-terminal tails of histones do not tightly interact to the core nucleosomal structure and are exposed to the nuclear environment (Luger, 2003). The tails of histones carry many of the acetylation, methylation and phosphorylation modifications . As many as 60 modifications have been characterized in histones; however, lysines and arginine amino-acid residues of histone tails can be modified into different forms. Lysines can be mono- di- are tri- methylated and argnines can be mono- or di- methylated. Therefore the number of combinations and levels of histone modifications at a given nucleosome is very large and highly complex (Kouzarides, 2007). Some histone modifications are found at gene promoters, 5’ end of genes and gene bodies and have been correlated with active gene transcription or repressed gene expression (Barski et al., 2007). Histone methylation is associated with both repressed and active loci depending on the histone tail substrate modified. In this thesis, my work focuses on the role of histone modifications involved in the regulation of gene expression, by forming active regions of 4 chromatin and repressed regions of chromatin and thus allowing or precluding access of DNA to transcription factors. I chose two critical and well-studied active and repressive marks, H3K4me3 and H3K27me3 respectively (described below), to understand the regulation of genes in embryonic stem cells and the changes that occur during early differentiation of embryonic development (next section). Active and repressive histone modifications Histone three-lysine four trimethylation (H3K4me3) is found in the 5’ end of actively transcribed genes in eukaryotes and is a marker for open chromatin. Trithorax group (TrxG) of methyltransferases in mammals catalyzes H3K4me3 in mammals. Initiation of transcription is linked to this post-translational modification (Mills, 2010). H3K4me3 methyltransferases have been shown to interact with RNA polymerase II (Pol II) and is required for normal transcription (Milne et al., 2005). Another modification, histone H3, lysine 36 trimethylation (H3K36me3) is found towards the 3’ end of actively transcribing genes. Studies have suggested that the H3K36me3 methyltransferase is targeted to the gene body by the elongating Pol II complex (Bannister et al., 2005). Other histone methylations such as H3K79me2 (Steger et al., 2008) and acetylation marks such as H3K27ac (Kurdistani et al., 2004) are also associated active gene transcription . Levine et al. also showed that H3K4me3 and Pol II occupy the promoter of most protein- coding genes in hESCs regardless of their activity status. Non-actively transcribing genes lack the elongating Pol II and H3K36me3 but are enriched in the initiating form of Pol II 5 along with H3K4me3. Active genes are positive for H3K4me3, the elongating Pol II version and H3K36me3 (Guenther et al., 2007). Current literature, suggests that open chromatin and transcription initiation by H3K4me3 and initiating form of Pol II, but not necessarily elongation, is a general phenomenon in most protein-coding promoters and renders the transcription in these genes in a ‘poised’ state, ready for active transcription. Negative regulators of gene expression include histone modifications that result in chromatin compaction and subsequent gene expression repression. The Polycomb group of genes is one such negative regulator of gene expression. They were initially characterized in Drosophila and were found to repress homeotic genes in the fly embryo by complexes known as Polycomb Repressive Complexes (PRCs) (Nusslein-Volhard, Kluding, & Jurgens, 1985). PRCs are evolutionarily conserved and found in higher eukaryotes. There are two major components of the PRC: PRC1 and PRC2. PRC2 complex is responsible for the trimethylation of histone H3 lysine 27 (H3K27me3) (Cao et al., 2002).This modification serves as a substrate for the binding of the PRC1 complex. The binding of PRC1 promotes chromatin condensation and antagonizes nucleosome remodeling to allow access of DNA to transcription factors. Together, these two complexes render the genes silent and chromatin repressed. Further studies have shown the PRC2 component EZH2, which is the H3K27me3 HMT, is required for the maintenance of the embryonic stem cells in the undifferentiated state (Simon et al., 2009). 6 ‘Bivalent’ domains Until recently, the active marks and the repressive marks were thought to play a mutually exclusive role in distinct regions of the genome. In embryonic stem cells for example, conventional thinking was that the active histone mark H3K4me3 is found promoting the active transcription of genes involved in the maintenance of the undifferentiated state; whereas the Polycomb group catalyzed H3K27me3 serve to repress genes involved in lineage specification and differentiation. In 2006, two studies revealed a surprising epigenetic feature in ESCs. Initially shown in mouse ESCs (Azuara et al., 2006; Bernstein et al., 2006) and then in hESCs (Pan et al., 2007; Zhao et al., 2007), is the existence of both active and repressive histone modification in genes involved in development and expressed in low levels. The studies show that during differentiation, these domains are resolved by loss of one of the modifications. For instance, investigators have shown that during differentiation, bivalent early lineage genes such as GATA2, GATA3, GATA6, MSX1 and HAND1 genes lose H3K27me3 and retain H3K4me3 (Pan et al., 2007). In general, there is a loss of H3K27me3 at co-modified genes and formation of new repressive H3K27me3 marks in other genes. Another important observation made by these studies is that a proportion of these ‘bivalent’ domains are retained in differentiated cells while others are resolved to H3K4me3 or H3K27me3. Another study conducted high-throughput ChIP sequencing in mouse ES cells, neural precursor cells (NPCs) and mouse embryonic fibroblasts (MEFs) for H3K4me3 and H3K27me3, as well as other modifications (Bernstein et al., 2006). 7 The study revealed that in high CpG island promoters (marks housekeeping genes and genes involved in development), nearly all H3K27me3 in ES cells exist with H3K4me3. During differentiation to NPCs and MEFs, these ‘bivalent’ genes are resolved to either H3K4me3, H3K27me3 and lose both marks or remain bivalent. Human embryonic stem cell lines and culture conditions Human embryonic stem cells (hESCs) are derived from the inner cell mass of the blastocyst and are invaluable in-vitro systems to understand the development of the embryo (Thomson, 1998). hESCs are capable of indefinite proliferation and capable of differentiating into all the various tissues of the developing embryo (Martin, 1981). These cells are also called pluripotent stem cells. They are derived from the totipotent zygotic cells and have lost the ability to form extraembryonic tissues. The first human embryonic stem cell line was derived in the late 90’s (Thomson, 1998). Since then, over 100 hESC lines have been derived in various labs around the world and are being used as model systems for studying embryonic development (Adewumi et al., 2007). The primary use of stem cell lines is to serve as a model system to understand the developmental program and biology of pluripotent cells and their ability to differentiate into the all the cell types of the developing fetus. Understanding the biology of these cells is critical if they are to be used for clinical purposes. The potential for stem cell therapy is enormous. With full understanding of the biological properties of stem cells and how they 8 specialize and form various tissues, cells can then be accurately engineered on the petri dish to produce cells to replace diseased or damaged cells in a patient. Stem cells cultures have many different origins and have been cultured in various environments to grow and propagate in the pluripotent state. Because of various culturing conditions, ESCs tend to be biologically and phenotypically variable. Many different environmental factors were used to maintain cells in their pluripotent state. Initially stem cell lines were grown on mouse embryonic feeder cells (MEFs) and serum containing media, such fetal bovine serum (Thomson, 1998). Soon thereafter, cells were grown in serum-free and feeder-free conditions. Initially, feeder free systems were developed by hESC culture on matrigel, using hESC media grown separately in the presence of MEFs (conditioned media; CM) (C. Xu et al., 2001). However, due to the inherent lack of definition of MEF factors, mixing of animal systems and inherent variability in CM media, several hESC media, free of serum or MEF feeder factors, were developed. These serum free media were derived using various combinations of growth factors targeting various pathways such as the TGF-beta and tyrosine kinase inhibitor pathways. Cell are grown on various surfaces such as Matrigel, Laminin and others (Adewumi et al., 2007; Amit et al., 2004; Y. Li et al., 2005; Lu et al., 2006; Ludwig et al., 2006; L. Wang et al., 2007). A comprehensive review of serum-free and feeder-free media compositions and surface coating factors have been published recently (Akopian et al., 2010; Oh, 2012). In 9 summary, two commercially available serum and feeder-free media compositions, mTeSR and StemPro, have been shown to be good alternatives for CM media for long term hESC propogation (Ludwig et al., 2006; L. Wang et al., 2007). A minimum set of factors such as lipids and insulin, as well as, growth factors such as FGFB and TGF-b appear to be critical for long-term hESC propagation and maintain pluripotency. However, there are other defined sets of factors that can also maintain hESCs in the long term and these methods have been reviewed (Adewumi et al., 2007; Akopian et al., 2010). Embryonic stem cell-surface markers and master pluripotency associated genes Several cell surface markers have been characterized in hESCs that uniquely identified undifferentiated stem cells. These markers are critical to correctly identify cells and also separate undifferentiated cells from differentiated cells. These include glycolipid surface markers such as SSEA3 and SSEA4, kertain surface markers such as TRA-181, alkaline phosphatases antigens and others (Andrews et al., 1984; Adewumi et al., 2007; Stewart et al., 2006). Along with the above immunologically derived undefined stem cell markers, recently, in order to find well defined surface markers, immuno-transcriptional and translation status assays were employed to characterize several new stem cell markers such as EPCAM and CDH36 were described (Kolle et al., 2009). The signal and intensity of several key cell surface markers have also been shown to be variable in culture. Various studies have shown that intensity of signal from markers such 10 as SSEA3 and GCTM-2 are directly proportional to the ‘stemness’ of a cell or a given population of cells with similar intensity. The stemness of a cell can be determined by the expression of master pluripotency markers and the ability to form colonies and differentiate into the three germ layers (Hough et al., 2009; Kolle et al., 2009; Stewart et al., 2006). Overall, these studies suggest that although hESC surface markers define pluripotent embryonic cells; clonal population of cells are heterogeneous in nature in the level of expression of pluripotency associated markers. Taking this into consideration, we have to consider the use of these markers to separate cells based on their stemness for future use and studies. Transcriptional factors form one of the core components of the pluripotency programming of hESCs. Three core genes; OCT4, NANOG and SOX2, of this machinery were elucidated to be master regulators of pluripotency (Chambers et al., 2003; Nichols et al., 1998). Transcriptional factors OCT4, NANOG and SOX2 work in concert and are often found associated at the same loci and regulation of genes involved in maintaining pluripotency and development (Boyer et al., 2005a; Kashyap et al., 2009; Pan & Thomson, 2007). Ectopic expression of these factors on differentiated adult somatic cells can reprogram cells into pluripotent stem cells (induced pluripotent stem cells, iPSCs) (Takahashi et al., 2007; Takahashi et al., 2006). The master regulators of pluripotency, such as NANOG, OCT4 and SOX2 play a role in the regulation of epigenetic modifiers and pluripotency genes including their own promoters and differentiation associated genes (Boyer et al., 2005b). Therefore, the expression and function of NANOG, OCT4 11 and SOX2 are critically important in the maintenance of pluripotency. Changes in the expression of these genes result in changes in their binding pattern to target promoters and results in changes in the transcription of genes responsible for chromatin regulation and development. Embryonic stem cell heterogeneity However, studies have shown that the expression of pluripotency-associated markers is not homogenous in a stem cell culture. NANOG expression in colonies has been shown heterogeneous in culture. Terada et al., have shown that cell expression high levels of NANOG display higher levels of pluripotency associated markers compared to cells with low NANOG cells, which have higher levels of endodermal marker GATA6 (Singh et al., 2007). In another study, OCT4 along with other pluripotency associated markers were shown to be differentially regulated during differentiation depending on the lineage specification of given cell to the 3 different germ layers (Ramirez et al., 2011). Importantly, the authors show that during early differentiation, OCT4 expression is the earliest gene to be down regulated. Single cell transcription analyses of iPSCs have established gene expression heterogeneity of these cells in culture (Narsinh et al., 2011). Two recent studies have shown that hESCs grown in culture display heterogeneous gene expression patterns. The authors isolated cells at various degrees of expression two pluripotency-associated cell surface markers, GCTM-2 and TG30 (CD9). They showed that cells isolated with the highest levels of GCTM-2 and TG30 also expressed the highest 12 levels of OCT4. Cells with the lowest levels of the cell surface markers expressed the lowest levels of OCT4 and higher expression developmentally associated genes in a gradient fashion (Hough et al., 2009; Laslett et al., 2007). The studies also showed that cells with the highest expression of OCT4, when isolated and re-plated in culture, more readily formed ES colonies compared to cells with the lowest expression of OCT4, which are generally abortive in colony formation. Therefore, the authors suggest that cells with the highest levels of GCTM-2 and TG30 represent hESCs express the highest levels of pluripotency-associated genes and the lowest levels of lineage specification genes; as a result these cells retain a higher capacity to proliferate, form colonies and display other characteristics of pluripotent stem cells. However with lower levels of GCTM-2 and TG30, cells express lower levels of pluripotency-associated genes, higher levels of lineage specification factors and diminished potential for proliferation and colony formation, suggesting that these cells are undergoing early differentiation. Therefore this study provides a methodology to isolate a pure population of hESCs for highly resolved characterization of pluripotent stem cells. X-chrosomosome inactivation in female human embryonic stem cells Compared to male cells that only consist of one X-chromosome, female cells carry two X-chromosomes. Therefore, in order to maintain the same gene dosage of X-chromosome gene expression in mammals, female cells randomly inactivate one of the inherited X- chromosomes. During early differentiation of female hESCs, one of the earliest epigenetic changes that occur is X-chromosome inactivation (XCI). XCI has been widely 13 studies in both female mouse and human models (Brown et al., 1991; Krietsch et al., 1986; Lyon, 1961). In ESCs, female lines carry two active X-chromosomes (Xa). During early differentiation, the X-chromosome non-coding RNA gene, XIST, is expressed and leads to binding and spreading of the XIST RNA one of the X-chromosomes in a random fashion (Epstein et al., 1978; Monk et al., 1979; Penny et al., 1996). The binding of spreading of XIST results in the inactivation of that particular X-chromosome (Xi) by a conserved series of histone and epigenetic changes (Brown et al., 1991; Heard et al., 2001; J. Silva et al., 2003). One of the earliest epigenetic changes that results in inactivation is the enrichment of the repressive mark H3K27me3 (J. Silva et al., 2003). It has also been shown that during reprogramming of somatic cells to ESCs, the silenced X- chromosome is reactivated late during reprogramming (Tchieu et al., 2010). Therefore a hallmark of female ESCs, in vivo, is the presence of two active X-chromosomes (Xa). In the mouse models it has also been shown that Oct4, NANOG and SOX2 bind to the promoter region of XIST and work together along with other factors in the repression of XIST. The loss of these factors results in the up-regulation of XIST (Nesterova et al., 2011). In human ESCs (hESCs), it remains unclear when XIST is expressed and coats the surface of the X-chromosome. In some cases the cloud has been documented in the early zygote at the 8-cell and morula stage (Daniels et al., 1997; van den Berg et al., 2009). In other studies, the inner cell mass of the blastocyst, from where ESCs are derived, consists of two Xa (Okamoto et al., 2011). Importantly, a recent paper highlighted the 14 heterogeneous nature of the XCI in cultured hESCs (S. S. Silva et al., 2008). In this study the authors describe effects of culturing and repeated passaging on hESCs in terms of XCI. They presented three observable states: In the Class I state, the hESCs have two Xa and upon differentiation XIST is expressed and they undergo random XCI. In the Class II state, the cells have undergone inactivation with one Xa and Xi along with XIST expression and the RNA cloud. The authors noted, that hESCs tend to readily transition from Class I to Class II. The Class III state consists of a Xa and Xi but without XIST expression and H3K27 methylation on the histones. There have been some studies which showed that re-programmed hESCs (iPSCs) can be maintained with two Xa by varying the culture conditions (Pomp et al., 2011). However, hESCs in culture are not stable in the maintenance of Xa; the instability maybe due to a lack of knowledge needed to support these cultures with two Xa or the inherent heterogeneity of hESCs and passage length and cell of origin differences (Dvash et al., 2010; Hoffman et al., 2005). Therefore by understanding early transcriptional and epigenetic changes that occur during early differentiation coupled with early X- chromosome inactivation, we may be able to determine factors and genes responsible in keeping cells in a state of pluripotency and maintaining two active X-chromosomes. Human embryonic stem cell cultures are invaluable systems to study the complex regulatory networks and gene expression programming that are critical in the development of organisms. However, hESCs colonies grown in culture are 15 transcriptionally heterogeneous. Studies have shown that hESCs spontaneously differentiate in culture and repress the expression of critical pluripotency associated genes. Early differentiating cells express lower levels of pluripotency-associated markers. However, the role of epigenetics in the early differentiation program of cells remains unclear. I hypothesized that by purifying a pure population of stem cells based on the highest levels of pluripotency-associated factors, I will be able to define an unique epigenetic profile of pluripotent cells and detect early epigenetic changes that accompany cells during early differentiation. One of the hallmarks of early differentiation is the expression of differentiation-associated factors and the inactivation of the X- chromosome. I hypothesized that cells expressing the highest level of pluripotency- associated stem cells markers will carry an epigenetic signature that is consistent with pluripotent stem cells with two active X-chromosomes, active epigenetic modification (H3K4me3) at loci associated with pluripotency and repressive epigenetic marks (H3K27me3) at differentiation associated genes. In my study, using previously described cell surface markers, GCTM2 and TG30, I isolated cells expressing the highest and lowest levels of these markers. I expected that cells with the highest levels of GCTM2/TG30 will carry an epigenome characterized by no enrichment of H3K27me3 on the X-chromosome and GCTM2/TG30 low cells will carry enrichment of H3K27me3 on the X-chromosome, consistent with early differentiating cells and X-chromosome inactivation. Based on this method, I also expected to find quantitative epigenetic differences in H3K4me3 and/or H3K27me3 at loci that play an important role in early differentiation or maintaining pluripotency. Together, I defined a unique epigenetic 16 profile of a pure population of hESCs expressing the highest level of pluripotency- associated markers and early epigenetic changes that accompany early differentiation. My results revealed that low passage and high passage cells are epigenetically very different. Low passage cells lack appreciable H3K27me3 enrichment across the X- chromosome. However repeated passaging resulted in the accumulation of H3K27me3 on the X-chromosome in both the GCTM-2/TG30 high and low populations, indicative of early X-chromosome inactivation as cells are cultured through time. However, in an independent high passage cell line, I defined an intermediate epigenetic phenotype where GCTM2/TG30 high cells are devoid of X-chromosome H3K27me3 enrichment and GCTM2/TG30 low cells carry X-chromosome H3K27me3 enrichment, suggesting that GCTM2/TG30 low cells were undergoing X-chromosome inactivation and early differentiation. In low passage GCTM2/TG30 high cells, I defined for the first time, the presence of a non-canonical H3K4me3 profile which is characterized by lowly enriched long domains, many kilobases long, and spanning protein families such as zinc finger, keratin, olfactory and extra cellular matrix protein families. The function of these long domains in hESCs are unknown and previously undefined. I have also detected many stochastic quantitative differences in H3K4me3 and/or H3K27me3 between GCTM2/TG30 high and low cells in the three cells types analyzed, suggesting that embryonic stem cells are epigenetically plastic. However, I have identified a core subset of genes, promoters and regulatory regions that contain quantitative epigenetic differences, conserved between all cell types that correlate with gene expression changes 17 between GCTM2/TG30 high and low cells. These conserved regions may play a critical role in early differentiation or maintenance of pluripotency. Overall, I have defined a unique epigenetic signature of purified pluripotent stem cells and identified conserved epigenetic changes that occur during early differentiation. Understanding the true epigenetic profile of pure populations of hESCs is critical for accurate use and manipulation of these cells in future studies and in the clinic. Roles of PR-SET7 and H4K20me1 in gene expression and chromatin regulation Extensive studies have been conducted on histone modifications that have been involved in gene expression regulation, as discussed in the previous sections. However, histone modifications have also been implicated in other critical cellular processes such as cell- cycle regulation, DNA damage response, DNA replication and gene expression regulation (D. B. Beck et al., 2012). One of the earliest identified modified histone residue was H4 at lysine 20 (H4K20), which was discovered 60 years ago (DeLange et al., 1969) but still continues to be enigmatic. The methylation(s) of this residue has been associated with various cellular processes, including gene transcription as described below. The ε-amino group of lysine residues have the capacity to be mono-(me1), di- (me2), or trimethylated(me3). In humans, different proteins are responsible for specific degree of methylation at H4K20. Suv4-20h1 and Suv4-20h2 catalyze H4K20me2 and H4K20me3, which are linked to DNA repair and heterochromatin respectively (Sakaguchi et al., 2008; 18 Schotta et al., 2004). It has been shown by structural and biochemical analysis that 53BP1 DNA damage checkpoint protein binds to H4K20me2 using 2 TUDOR domains. Loss of H4K20me2 results in the loss of binding of 53BP1 and loss of checkpoint signaling during DNA damage/repair. H4K20me3, along with another modification H3K9me3, has been shown to interact with constitutive heterochromatin and is involved in the regulation and stablility of telomeres and heterochromatin. Knockout mice, lacking Suv4-20 proteins show decreased levels of H4K20me2/me3 and are prone to DNA damage, chromosome alternation and are embryonic lethal (Wongtawan et al., 2011). PR-SET7 is the only known protein responsible to specifically mono-methylate H4K20 (Nishioka et al., 2002); PR-SET7 is an essential enzyme because loss of PR-SET7 and H4K20me1 results in multiple deleterious phenotypes in cells such as chromatin condensation aberrations, spontaneous DNA damage, genome instability and has been shown to be embryonic lethal earlier than the 8-cell stage (Houston et al., 2008; Nishioka et al., 2002; Sakaguchi et al., 2007). Similarly, in Drosophila, loss of PR-SET7 results in embryonic lethality at the third instar stage (Karachentsev et al., 2005). Importantly, it was shown that the loss of PR-SET7 in mice caused embryonic lethality (Oda et al., 2009). Therefore PR-SET7 mediated H4K20me1 is critically important in survival of stem cells and plays a critical role in development. A recent paper also demonstrated the role of PR-SET7 in epidermal stem cell maintenance (B. Beck et al., 2012). 19 In addition, recent work demonstrates that PR-SET7 and H4K20me1 is an essential regulator of the cell cycle. PR-SET7 undergoes dynamic regulation according to the phase of the cycle, which in result affects levels of H4K20me1. H4K20me1 and PR- SET7 have been shown to be critical modulators of S-phase progression and the completion of mitosis (Centore et al., 2010; Houston et al., 2008; Jørgensen et al., 2007; Rice et al., 2002; Tardat et al., 2007; Wu et al., 2010). Recently, a study demonstrated that PR-SET7 functions in non-histone methylation of proteins. PR-SET7 has been shown to be involved in carcinogenesis by methylating the non-histone protein PCNA and resulting in its degradation (Takawa et al., 2012). One major focus of my thesis work was to determine the role of PR-SET7 and H4K20me1 in transcriptional regulation. Initial studies have shown that H4K20me1 is associated transcriptionally repressed chromatin and also at the inactive X-chromosome (Heard, 2005). Transcriptional repression could be due to direct chromatin compaction or by binding of effector transcriptional repressors such as L3MBTL1 (Trojer et al., 2007). L3MBTL1 contains three methyl-binding domains. These proteins have been shown to preferentially bind H4K20me1/2 and binding results in chromatin compaction, the formation of facultative heterochromatin and target gene expression repression. Therefore it was thought that PR-SET7 and H4K20me1 might play a role in transcriptional repression; H4K20me1 also prevents the induction of active histone acetylation marks such as H4K16 acetylation; in Drosophila, the targeted insertion of an active gene into an H4K20me1 region, resulted in the spreading of the mark into the inserted loci and 20 resulted in gene repression (Karachentsev et al., 2005; Nishioka et al., 2002; Plath et al., 2003). Another study showed that H4K20me1 is found at repressed genes regulated by E2F transcription factor (Abbas et al., 2010). With the advent of high-throughput sequencing, the genome-wide location of H4K20me1 in the genome has been mapped in many cell types. These studies have revealed that H4K20me1 is localized on the gene body with the highest levels found at the 5’ end of genes. These studies also revealed that H4K20me1 methylated genes are highly expressed and found associated with active acetylated histone H3 and H4 modifications, suggesting a role in active genes (Barski et al., 2007; Talasz et al., 2005). However, a recent study demonstrated a dual role for PR-SET7, in complex with other proteins, in both gene activation and gene repression (Yang et al., 2012). This study found that PR-SET7 formed a complex with master epithelia to mesenchymal transition (EMT) master regulator TWIST, and found at genes such as N-cadherin and E-cadherin which are then H4K20me1 methylated. However, the PR-SET7 and TWIST complex along with H4K20me1 functioned to repress E-cadherin and activate N-cadherin. Therefore PR-SET7 may have a dual role in gene expression or repression depending on its binding factors. Another study showed that H4K20me1 and PR-SET7 plays a positive role in the upregulation of Wnt3a responsive genes upon stimulation (Li et al., 2011). 21 Therefore the role of PR-SET7 and H4K20me1 in gene transcription regulation remains unclear. One of the goals of my dissertation was to elucidate the role of PR-SET7 mediated H4K20me1 in gene transcription regulation. Given the role of PR-SET7 in chromatin compaction, I hypothesized that by knocking down PR-SET7 and subsequently H4K20me1 at target genes, I expected up-regulation of gene expression consistent with previous studies that have shown that H4K20me1 is a repressive mark. My findings demonstrated that the loss of PR-SET7 and H4K20me1 at a subset of H4K20me1 target genes resulted in an approximately 2-fold up-regulation of transcription. I also showed that the there is an inverse correlation between H4K20me1 enrichment levels and active transcription associated H4K16 and H3K9 acetylation enrichment levels at target genes. H4K20me1 was also found in repressed developmentally regulated genes in embryonic stem cells. On the contrary, I found that H4K20me1 targeted genes are largely cell-type specific but a subset of highly transcribed metabolism-associated genes are conserved in all cell-types analyzed. Also, I showed that genes enriched with PR-SET7 and H4K20me1 were physically distinct on gene bodies and highly expressed in the genome, compared to genes modified for either H4K20me1 or PR-SET7. These results indicated that PR-SET7 and H4K20me1 together were associated with active genes but a modulator role to repress expression cannot be ruled out. Together my study indicated that at a subset of genes H4K20me1 acts as a transcriptional repressor. However, my study also suggested that H4K20me1 along with PR-SET7 might plays a distinct function associated with highly expressed genes. 22 Chapter 1: Epigenetic Plasticity of Female Human Embryonic Stem Cells 1.1 Introduction hESCs grown in culture are heterogeneous for the expression of pluripotency and differentiation associated genes. Previous studies have shown that purified populations of hESCs from culture based on the highest levels of GCTM-2 and TG30 cell surface markers contain the highest levels of pluripotency-associated genes and the lowest levels of differentiation associated factors. On the contrary, cells with the lowest levels of GCTM-2 and TG30 display the lowest levels of pluripotency-associated genes and highest levels of differentiation associated factors and are in the early stages of differentiation. I hypothesized that populations of female hESCs purified based on the levels highest levels of GCTM-2 and TG30 will contain a unique epigenetic signature consistent with cells in the pluripotent state. In contrast, cells from the same culture with low expression of GCTM2/TG30 will have a very different epigenetic signature indicative of cells exiting the pluripotent state. Based on previous studies, I hypothesized that GCTM- 2/TG30 high cells will be characterized by low enrichment of H3K27me3 on the X- chromosome, strongly suggesting that these cells have not initiated early differentiation and X-chromosome inactivation. In contrast, GCTM-2/TG30 low cells will display higher levels of H3K27me3 on the X-chromosome indicating that these cells have undergone early X-chromosome inactivation during early differentiation. I also expected 23 higher active H3K4me3 enrichment at loci associated with high expression of pluripotency-associated markers and open chromatin in the GCTM-2/TG30 high cells, compared to GCTM-2/TG30 low cells. Similarly I expected to find high H3K27me3 enrichment at developmental genes in undifferentiated cells and the resolution of these marks during early differentiation, along with higher enrichment of H3K27me3 at pluripotency-associated genes. One of my goals was to understand epigenetic signature that defines pluripotent hESCs and the epigenomic changes that occur as cells exit the pluripotent state. I also wanted to determine if there were specific differences in epigenetic regulation that occur in different hESC lines and also the consequences due to repeated passaging and different growth conditions of cultured hESCs. To this end, I used low passage female HES3 cells (defined as LP) and high passage HES3 (defined as HP), as well as a completely different female hESC line, H9. The two different cell lines were grown in different culture conditions and therefore provide a useful model to define epigenetic differences observed between hESCs of different origin, culture conditions and passage lengths. Furthermore, LP, HP and H9 cells were sorted into 2 separate populations for analysis based on the highest (P2) or lowest (P5) levels of the pluripotency-associated markers, GCTM-2 and TG30. My results revealed large stochastic differences in the epigenomes of the LP, HP and H9 cells, as well as, unique epigenetic signatures between the P2 and P5 populations that are conserved between cell types. Results indicated that cultured 24 embryonic stem cells were prone to epigenetic changes between P2 and P5 suggesting that the epigenomes of hESCs are very plastic. The plasticity observed between P2 and P5 appear to largely vary based on the cell type, passage length and culture conditions and appeared to be stochastic. However, a core set of potential pluripotency and differentiation-associated loci were differentially enriched between P2 and P5 and conserved across all cell types. These loci may play an important role in the maintenance of the pluripotent phenotype or may play role in the early differentiation of hESCs. These genes are good candidates for further investigation to further understand the developmental programming of embryonic stem cells and accurately characterize the nature of homogeneous populations of hESCs. 1.2 Results 1.2.1 Processing and ChIP-sequencing of HES3 and H9 samples HES3 and H9 cells were grown in culture conditions using previously established conditions (Hough et al., 2009; Laslett et al., 2007). Briefly, HES3 cells were grown on media containing serum and with physical contact with mouse embryonic feeder cells (MEFs). In contrast, H9 cells were grown in media with knockout serum replacement (KSR) and MEF conditioned media (CM). CM media is media transferred from culture containing MEFs. Therefore H9 cells are grown without serum and without physical contact with MEFs. In order to be consistent with previously conducted studies, I used cells cultured between 45 and 60 passages. These cells were labeled as high passage HES3 cells, HP. Low passage HES3 cells (LP) were also isolated in order analyze 25 passage dependent epigenetic changes (10-25 passages). As an independent sample, the H9 cells are high passage cells comparable to the HP. Figure 1: FACS profile of HP, LP and H9. Representative FACS profile of TRA-181 positive HES3 and H9 cells sorted for GCTM2 and TG30. The gates used to isolate the top 20% and bottom 20% cells as P2 and P5, respectively, are indicated. As previously established, I isolated cells based on the highest and lowest levels of GCTM-2 and TG30 (Laslett et al., 2007) and positive for pan human embryonic stem cell marker TRA1-81 to remove potentially contaminating MEF. The top ~20% of cells were designated as P2 population and the bottom ~20% of cells were called the P5 population (Figure 1). Every week, 6 35mm plates of HES3 and H9 cultures were used to isolate cells. Trypsin was initially used to remove MEFs (for HES3) and re-trypsinized to isolate both HES3 and H9 cells. Cells were fixed using formaldehyde and triple stained with GCTM-2, TG30 and TRA-181. Six 35mm plates typically yielded between 100,000- 250,000 cells per week. Cells were passaged weekly. The process was repeated every week until approximately 3 million cells were isolated. Approximately two million cells 26 were isolated for each population per H3K27me3 ChIP experiment and one million cells from each population were used for every H3K4me3 ChIP experiment. ChIPs were performed and libraries were then constructed on the ChIPed material and sequenced using next generation Solexa sequencing. 1.2.2. ChIP-Seq analysis The sequences were then aligned to the human genome. Uniquely aligned sequences were used for downstream bioinformatics analysis; uniquely aligned sequences were defined by, at any given location in the genome the end of only one read is aligned and the other sequences starting at the same location were discarded. In order to maintain consistency, we sequenced and obtained aligned reads such that read count between P2 and P5 in a given cell type and experiment was comparable. All experiments were conducted in similar experimental conditions and methodologies to minimize variation between experiments. I conducted an initial analysis of the data using the Homer package (Heinz et al., 2010a) to call statistically significant peaks of enrichment. The uniquely aligned reads and Homer peak output summary data is provided in Table 1. The main focus of my analysis was to analyze the epigenetic changes between the P2 and P5 populations in both the HP, LP and H9 cell lines, for each modification. In Table 1, I observed that compared to the HP, LP samples show drastic epigenetic differences between the P2 and P5 for both modifications. The LP P2 population has significantly higher number of peaks compared to the LP P5 for both H3K4me3 (27,110 vs. 16,539 27 peaks) and H3K27me3 (14,192 vs. 9572 peaks). In light of this observation, I started my analysis with the LP H3K27me3 to further understand these differences. Table 1: Summary of ChIP-seq uniquely aligned reads in each library and associated peak number. Table contains the number of uniquely aligned reads for each population and the number peaks detected using the non-quantile normalized read count data using SICER. I constructed visualization (wig) files by counting the number of unique reads in a 400 bp window and normalized the numbers to read counts per window, per 10 millions total reads. The read count normalized data was used to view the enrichment of H3K27me3 in the P2 and P5. The normalized read counts were used to construct the wig files. In order to assess the quality of my experiments, I looked at a well-characterized H3K27me3 enriched gene, Neurog1. I chose this gene because of the wide range of enrichment levels observed in the neighboring loci (Figure 2A). At Neurog1 and at other neighboring loci, it was evident that LP P2 and LP P5 displayed a similar H3K27me3 enrichment profile. 28 However, regardless of enrichment levels in the loci analyzed, LP P5 levels were quantitatively lower at every peak, compared to LP P2. I analyzed the genome-wide distribution of normalized H3K27me3 read-counts between P2 and P5. The genome was divided into 2.5 KB windows. The normalized read counts were calculated for each matched window in P2 and P5. The read counts are plotted in Figure 2C. The distributions were comparable but I found a shift in the distribution from the 45-degree line towards the P2 (x-axis). The distribution suggests that distribution of H3K27me3 in P2 and P5 are very similar in the LP population with the exception of skewed quantitative enrichment in nearly every enriched loci towards the P2 population. Given the close similarity between P2 and P5 except for the skewed quantitative differences at enriched regions towards P2, I reasoned that those differences detected between P2 and P5 were due to technical variation between experiments. I also expected a small number of subtle epigenetic changes at enriched loci given that the two populations are from the same cell colony and large quantitative differences in the chromatin modifications were not expected. Therefore I had to find an appropriate method to normalize the data in order to accurately define quantitative differences in histone methylation enrichment between P2 and P5. 29 1.2.3 Quantile normalization Quantile normalization is a method that is frequently used in microarray and other gene expression assays to normalize multiple experimental samples (Bolstad et al., 2003). The fundamental assumption behind quantile normalization is that the biological experiments share very few biologically differences between each other and majority of the variation between the samples are due to technical variations between the experiments. In the case of my study, my goal was to detect quantitative differences between P2 and P5 cells, which were isolated from the same colony of stem cells and therefore, I expected to find very few biologically significant quantitative differences between them. Therefore, I determined that quantile normalization is an appropriate method to normalize the distributions of P2 and P5 in any given enrichment and cell type. For the first time, the quantile normalization method was used to normalize ChIP-seq library datasets. I used this novel approach to normalize closely related P2 and P5 data for a given modification and cell type. In order to detect quantitative changes between P2 and P5, I normalized the library using the quantile normalization method. Briefly, I divided the genome into equivalent 400 base pair windows (in P2 and P5) and counted the non read-count normalized sequence reads in them. The windows were then ordered from lowest to highest read count for each library. The value for the lowest read count window in both P2 and P5 were set to the average value between the lowest window count between the P2 and P5 libraries. Similarly the second lowest read count number for P2 and P5 was set the average between the second lowest read count numbers between 30 P2 and P5, etc. The rationale behind this approach is to force the samples to follow the same distribution. After quantile normalizing the data, the two libraries had the same distributions (Figure 2). I then used the normalized libraries to conduct peak distribution analysis and also constructed visualization wiggle files. Figure 2A-2B and Figure 2C-2D show the H3K27me3 read count distributions in P2 and P5 before and after quantile normalization. The distributions between the P2 and P5 were comparable after quantile normalization I then used this data to call peaks and determined subtle and significant quantitative differences between the normalized libraries for all matched P2 and P5 libraries in my study. 31 Figure 2: Representation of read-count and quantile normalized data. (A) Representative enrichment of the Neurog1 (highlighted by light red rectangular box). Read count normalized H3K27me3 enrichment between P2 and P5 of the LP population is indicated. (B) Quantile normalized H3K27me3 between P2 and P5 of the LP population. (C) Genome-wide distribution of reads in 2500 bp tiled windows between P2 and P5. (D) Read count distribution in 2500 bp tiles windows between P2 and P5 using the quantile normalized data. 32 1.2.4 H3K27me3: X-chromosome differences between LP, HP and H9 To assay H3K27me3 changes that occur between the P2 and P5, I took advantage of the inherent heterogeneity in the XCI status in hESCs. H3K27me3 enrichment on the X- chromosome is an early marker of early differentiation associated X-chromosome inactivation (J. Silva et al., 2003). Earlier studies have indicated the presence of long domains of H3K27me3 enrichment across the X-chromosome (Chadwick et al., 2004). In culture, XCI status is heterogeneous. X-chromosome inactivation in hESCs is generally occurs in culture without ectopic expression of pluripotency factors and specific environmental conditions (S. S. Silva et al., 2008). Cells grown in culture are prone to spontaneously undergo random X-chromosome inactivation. Therefore it is likely that repeated passaging of cells in culture will lead to spontaneous XCI. Therefore in my study, I used the H3K27me3 data to examine the enrichment of the mark on the X- chromosome in LP and HP cells, as well as, late passage H9 cells. I hypothesized that cell populations purified for the highest levels of GCTM-2 and TG30 (P2) are representative of cells, which have not undergone early differentiation and therefore, I expected the X- chromosome in the P2 population to be devoid of H3K27me3 and P5 populations will contain enrichment of H3K27me3 across the X-chromosome. I tested this hypothesis in the LP, HP, and H9 populations. 33 Figure 3: H3K27me3 pan-X chromosome enrichment in LP, HP and H9. (A) Pan H3K27me3 across the X-chromosome in the P2 and P5 populations of the HP, LP and H9 cells. The scales are indicated in square brackets. (B) Hypothetical model of the relationship between P2 and P5 of HP, LP and H9 cells based on X-chromosome H3K27me3 and passage length differences. Contrary to my hypothesis, my analysis revealed that P2 and P5 in the LP and HP cells were not significantly different in terms of H3K27me3 on the X-chromosome. However, comparison of LP and HP cells show dramatic differences in the enrichment of H3K27me3 on the X-chromosome, indicating passage length depended epigenetic changes. In P2, HP showed a dramatic increase in enrichment of H3K27me3 on the X- 34 chromosome, compared to LP: 45.22% vs. 2.52% (>20-fold increase) respectively (Figure 3A). A similar trend was also observed between the LP P5 and HP P5 population. My data indicated that continual passaging of HES3 cells results in the enrichment of H3K27me3 of the X-chromosome, indicative of Xi. This suggested that LP cells may represent a more naïve pluripotent stem cell, whereas HP had begun to move away from the pluripotent state, as evidenced by X-chromosome H3K27me3 enrichment and Xi. Purification of hESCs failed to obtain a population of cells with differential X- chromosome H3K27me3 enrichment. In order to determine if a similar profile of H3K27me3 occurs in an independent cell line, I analyzed the early X-chromosome inactivation status of an independent high passage hESC cell line, H9. In contrast to HES3 cells, I observed significant differences between the H9 P2 and H9 P5 samples, as I originally hypothesized. There was a ~40% difference in the percentage of total peaks represented in the X-chromosome in the P2 (2.62%) vs. P5 (4.2%). Although a slight increase overall, these differences occurs at lowly enriched peaks that span the entire X-chromosome in H9 P5 (Figure 3A). These patterns are strikingly similar to the differences observed between the LP and HP datasets, albeit at lower enrichment levels. Therefore, my findings indicated that the H9 P2 cells lack high enrichment of H3K27me3 on X-chromosome compared to HP P5 cells. Purified cells with high GCTM2/TG30 cells appear to have not undergone early X-chromosome inactivation compared to cells with low GCTM2/TG30 expression. Therefore these findings suggested that the sorting of hESCs based on the expression of pluripotency- 35 associated markers is a useful approach to isolate especially naïve pluripotent stem cells from those moving away from the pluripotent state. For the first time, I have demonstrated that our protocol is essential to obtain a population of cells with an epigenetic signature consistent with pluripotent cells with no evidence early X- chromosome inactivation. My observation of H9 cells indicated that these cells may represent a “intermediate” hESC cell type with a subpopulation of cells, lacking Xi (P2), similar to LP, and another subpopulation undergoing X-chromosome inactivation (P5), similar to HP (Figure 3B). The above analysis revealed that there are large-scale changes in H3K27me3 on the X- chromosome in LP, HP and H9. My initial analysis only focused on the bulk H3K27me3 levels on the X-chromosome, however, I also predicted local quantitative differences in H3K27me3 at functional loci; loci that may play a role in the pluripotency status of hESCs. To further investigate local changes in H3K27me3 at functionally significant locations in HES3 and H9 cell lines, I conducted a thorough analysis of genes and putative regulatory elements that contain statistically significant quantitative differences in H3K27me3 between P2 and P5, for each cell type. 1.2.5 H3K27me3: Genome-wide differences between P2 and P5 In order to determine quantitative differences in the levels of H3K27me3, I first analyzed the H3K27me3 peak distribution, using SICER (Zang et al., 2009), between pair-wise quantile normalized P2 and P5 libraries in the LP, HP and H9 hESCs. In contrast to 36 transcription factor enrichment, H3K27me3 is diffused in nature with gaps of no enrichment within loci or peaks of significant enrichment. SICER was used to identify peaks because it allows for gaps of no-enrichment for detecting peaks. For the my analysis, I searched for peaks in 400 bp windows and allowed for a non-peak gap of 800 base pair and a stringent e-value cutoff of .0005. As shown in Figure 4A the peak distribution between P2 and P5 in LP, HP and H9 hESCs displayed drastic differences in peak distribution. In the LP cells, P2 has 2x over representation of unique peaks (1542) compared to the P5 (730). In contrast, I observed the opposite trend in HP cells with a 2x over representation of peaks from the P5 population (3024) compared to P2 (1390). The numbers of unique reads represented in the P2 and P5 in the H9 cell line are equally distributed (1318 vs. 1235). As observed in the X-chromosome analysis, LP and HP cell types have undergone dramatic epigenetic changes in the course of continual passaging at local H3K27me3 peaks. However, the H9 cells appeared to display relatively fewer H3K27me3 peak distribution differences between P2 and P5. I then proceeded to determine the statistically significant quantitative differences, using the ChIPDiff software (H. Xu et al., 2008), in H3K27me3 between the P2 and P5 populations in each of the different cell types. ChIPDiff calculates statistically significant enrichment differences between two given sample libraries using the hidden markov model (HMM) method. As an initial analysis, I determined peaks that were over-enriched 37 by 50% or greater in either the P2 or P5 population (p<.005). These peaks of enrichment were then mapped to predicted poised promoters, weak promoters, active promoters, and enhancers defined in other hESCs from a previous study (Ernst et al., 2011). Congruent with my initial peak distribution analysis (Figure 4A), in the LP, I found a 3.5 fold over- representation of over-enriched peaks in the P2 compared to the P5 (1417 peaks vs. 396 peaks). The largest functional categories in the LP P2 over-enriched category were poised promoters, weak promoters, weak enhancers and strong enhancers, in order (Figure 4B). This observation is consistent with the hypothesis of poised lowly expressed promoters and enhancers in embryonic stem cells and resolution of the poised status by the loss of H3K27me3 during differentiation (Bernstein et al., 2006). 38 Figure 4: Quantile normalized H3K27me3 distribution between P2 and P5 of HP, LP and H9. (A) H3K27me3 SICER peak comparison analysis between P2 and P5 from LP, HP and H9. The peaks were identified using the pair-wise P2 and P5 quantile normalized libraries. (B) Loci distribution of quantitative differences in H3K27me3 (>= 50%) between P2 and P5 in the LP, HP and H9 cell types. The overlap between the quantitatively different loci and predicted promoter and enhancer categories from the H1 data are color-coded. The overlap between all P2 over- enriched datasets and all P5 over-enriched datasets across LP, HP and H9 are indicated Similarly, consistent with my initial peak distribution analysis (Figure 4A), I found a ~12.5 fold over-representation of H3K27me3 over-enriched peaks in P5 compared to P2 (1602 vs. 126 peaks) in HP cells. Largest functional categories were poised and active promoters, followed by enhancers (Figure 4B). In the H9, I found a similar distribution of P2 over-enriched and P5 over-enriched peaks (578 and 796 peaks respectively). With the largest functional categories, in both P2 and P5 over-enriched peaks, represented from 39 poised promoters and enhancers and followed by active promoters. Appendix A-C contains the peaks from each over-enriched category in the HP, LP and H9, along with loci mapping to predicted enhancer regions. My initial results indicate that the HP and LP cells are significantly different in the distribution of the over-enrichment loci between P2 and P5, regardless of Xi status. 1.2.6 Identification of conserved quantitative H3K27me3 enrichment differences between P2 and P5 One of the main goals of this study was to determine quantitative differences at specific loci in the P2 and P5 populations that were consistent between LP, HP and H9. I hypothesized that these differences may define a unique epigenetic identity of P2 vs. P5 cells and may also provide valuable insights into specific epigenetic changes at specific loci important for the maintenance or exit from pluripotency. To this end, I determined P2 over-enriched and P5 over-enriched loci that were common between the three different cell types. I found peaks with direct overlap in the over-enriched categories in the P2 over-enriched datasets from all the three cell types. I repeated the same method for the P5 over-enriched categories. There was only a single locus in which H3K27me3 was over-enriched in the P2 population in all the three cell types. This locus is mapped to the promoter region of GREB1L. GREB1L is similar to a known estrogen depended oncogene found in breast cancer, GREB1 (Rae et al., 2005). However the role of GREB1L in hESCs or other model 40 systems remains unknown. Given its similarity with a growth factor, I predict that GREB1L may play a role in the proliferation or growth of embryonic stem cells. There were 13 loci defined in which H3K27me3 was over-enriched in P5 population of all three cell types. The loci were mapped to the promoter region of TDGF1, ETS1, ETV4 and MIR663B. Three loci were mapped to the gene bodies of IFLTD1, PKM2 and HS3ST4 and 4 loci mapped predicted H1 enhancer or repressed intergenic regions. TDGF1 is an important gene that is critical in maintaining cells in the pluripotent state; consistent with a transcriptional study that demonstrated lower expression of TDGF1 in the P5 (Hough et al., 2009), my analyses revealed over-enrichment of repressive mark H3K27me3 at TDGF1 in the P5 cells. ETS1 and ETV4 belong to the ETS family of transcription factors and have been shown to play a role in hESCs (Rho et al., 2006). ETS1 has been shown to be highly expressed in hESCs and is expressed at lower levels during differentiation. Therefore this family of transcription factors may play an important role in the regulation of their target genes involved in early differentiation. The role of MIR663B remains unclear in hESCs. IFLTD1 has been implicated in a disease risk locus, which is involved in lung tumourigenesis (Manenti et al., 2008), and it is possible that this gene plays a role in the growth and proliferation of hESCs. Interestingly, transcription factor, PKM2 has been shown to be an interacting partner with OCT4 and positively regulates genes (Lee et al., 2008). Therefore PKM2 H3K27me3 over-enrichment in the P5 may affect gene transcription downstream of the OCT4/PKM2 regulatory pathway. HS3ST4 is a heparin sulfate sulfotransferase. Heparin sulfate chains 41 are found on cell surface proteoglycans and have been shown to play a regulatory role in self-renewal and pluripotency in hESCs (Sasaki et al., 2008). Therefore differentially modified herparin sulfate molecules may play a role during early differentiation by possibly regulating cell-cell interaction and the extra-cellular matrix. Overall, analyzing common H3K27me3 differential modification between P2 and P5 cells regardless of passage length, growth conditions or Xi status, I was able to define loci that displayed conserved changes that may likely play a direct role in the transcriptional regulation of their targets and play a role in the maintenance/exit of pluripotency. Consistent with this, the function of several of these targets genes suggests that they plan an important role in hESCs and maintaining pluripotency. Therefore, these genes may be potential markers of early differentiation of hESCs in culture. 1.2.7 Identification of conserved quantitative H3K27me3 enrichment differences based on Xi status of H9 The findings above strongly suggest that H3K27me3 enrichment, both globally (Xi) and locally, is significantly different between LP and HP. To test this I compared LP P2 and HP P5 H3K27me3 over-enriched loci (1241 vs. 1068) and 337 loci were common between the two datasets. Approximately 31.5% of all HP P5 over-enriched loci were over-enriched in the LP P2. This data suggests that during repeated passaging approximately a third of all over-enriched peaks have undergone significant quantitative changes and have ‘flipped’ from being P2 over-enriched in the LP to being HP-over- 42 enriched in the P5. Therefore, LP and HP are epigenetically very different. Although they are derived from the same origin, because of repeated passaging and/or long-term culture conditions, LP and HP have acquired significantly different epigenomes. My initial analysis of H9 cells strongly suggested that they are an “intermediate” cell type with H9 P2 similar to LP and H9 P5 similar to HP (based on status of H3K27me3 enrichment in X-chromosome). Therefore, I hypothesized that this relationship will also hold true at local regions of enrichment also. I tested this hypothesis by identifying loci that were common between LP and H9 in the context of P2 over-enrichment. Similarly, I identified the common loci between HP P5 and H9 P5 over-enrichment. I predicted H9 P2 over-enriched loci to specifically overlap with LP P2 over-enriched loci and not HP P5 over-enriched loci. Similarly, I expected H9 P5 over-enriched loci to specifically overlap with HP P5 over-enriched loci and not HP P2 over-enriched loci. In order to conduct a more stringent analysis, I only used loci that were 2-fold or greater over- enriched at p-value < 0.05. Once identified, I then compared the overlap and distribution of P2 and P5 over-enriched loci in the H9 to the LP P2 and HP P5 over-enriched loci, respectively (Figure 5). Consistent with my hypothesis, my analysis revealed a specific and significant overlap between H9 P2 and LP P2 over-enriched loci (25 loci; p-value <4.388e-6). Similarly, a specific and significant overlap between H9 P5 and HP P5 over-enriched loci were identified (67 loci; p-value <5.163e-19). Importantly, I found that H9P2 is not similar to 43 HPP5 and H9P5 is not similar to LPP2. I found just one exception where H9 P5 over- enriched loci overlapped with a LP P2 over-enriched loci. Figure 5A shows the genes, in square brackets, which were mapped to the overlapped loci. Therefore, the analysis is consistent with my initial hypothesis that H9 represents an intermediate epigenetic phenotype, where P2 (=LP) with no Xi and P5 (=HP) with Xi. Therefore further understanding the genes with conserved quantitative differences between HES3 and H9 will allow investigators to further understand the early epigenetic changes that occur during early onset of Xi and differentiation. 44 Figure 5: Relationship of quantitatively over-enriched loci between LP, HP and H9. (A) Four-way comparison between LP P2 over-enriched, HP P2 over-enriched, H9 P2 and P5 over- enriched H3K27me3 loci (2-fold enrichment or greater). Mapped genes in the significantly overlapping categories are indicated in square brackets along with p-value for the overlap significance. (B) Representative tracks of quantitative differences in H3K27me3 from the list of overlapping genes shown in (A). I conducted gene ontology (GO) analysis using the GREAT tool (Valouev et al., 2008) in order to functionally classify the differentially methylated loci common between the P2 population of LP and H9 and the P5 population of HP and H9. This program maps loci, which may include promoters and enhancer elements to the nearest gene and classifies the mapped genes into functional categories that are significantly represented in the 45 dataset. In my analysis, I mapped the over-enriched loci within 10 kb of transcription start site and listed the top 10 biological process categories in order of statistical significance in Figure 6. For the conserved P2 over-enriched loci in the H9 and LP, the top 5 biological categories were related to development and the next 5 categories related to neurogenesis. These loci were expected to be relatively upregulated in the P5 since these genes were over-enriched with repressive H3K27me3 in P2 compared in P5. The mapping of these loci to neuronal genes was not unexpected because during spontaneous differentiation of hESCs cells tend to differentiate into the neuronal-like lineage (Hough et al., 2009). In contrast, for the loci common between HP P5 and H9 P5 H3K27me3 over-enriched datasets, only 1 out of 10 biological process categories represented was neurogenesis (Figure 6); the others represented general developmentally related biological processes. Therefore my data suggests that P5 cells have already changed their epigenomes from the naïve pluripotent state and have moved to an epigenome consistent with cells that have exited the naïve pluripotent state. 46 Figure 6: GREAT GO analysis of conserved differentially enriched H3K27me3 loci. GREAT analysis of significant biological process categories in loci common between H9 P2 and LP P2 over-enriched datasets and also H9 P5 and HP P5 over-enriched loci. Identification of genes conserved for H3K27me3 P5 over-enrichment in HP and H9 included TG30, TDGF1 and NODAL. These genes have been shown to be important in maintaining pluripotency and expressed at lower levels in the P5 (Hough et al., 2009). However, I have defined other genes conserved for P5 H3K27me3 over-enrichment (BCL11B, BFSP1, COL161, ETV4, FGF19, FOXD3, HEY2, NFIB, PCDH8, TAL2 and 47 WNT7A), which may also play an important role in maintaining pluripotency and/or early differentiation and open for further investigation. Similarly, among the 25 loci P2 over- enriched conserved between LP and H9, I mapped 10 genes (DRD4, EFNB1, EPHA4, FGFR3, GAS1, METRN, NTF3, OTX2, SFRP1 and SOX21). A number of these genes play a role in neuronal development (GO analysis; Figure 6). Eight loci (33%) out of the 25 conserved P2 H3K27me3 over-enriched loci were mapped to predicted enhancer regions. Similarly, 39 out of the conserved 67 (over 50%) P5 over-enriched loci were conserved at predicted enhancer regions. However, overall H3K27me3 changes in enhancer enrichment between P2 and P5 in the LP and H9 appear to largely be stochastic. Out of all the predicted enhancer elements mapped to P2 H3K27me3 over-enriched loci in LP (162 loci) and H9 (106 loci), only 7.5% (8 loci) are conserved between LP and H9. Similarly, only 39 (28.7% of H3K27me3 H9 P5 over-enriched loci) of the predicted enhancer elements P5 over- enriched in HP (296 loci) and H9 (136 loci) are conserved (Table 2). My results suggested that there is regulatory element H3K27me3 heterogeneity between P2 and P5 within individual cell lines with only a small subset of loci conserved between cell types. The conserved enhancer elements may play a critical role in the regulation of genes involved in maintenance of pluripotency. Overall, I have identified loci over-enriched for H3K27me3 in P2 or P5 conserved between LPP2/H9P2 and HPP5/H9P5 respectively. I predicted that these quantitative 48 epigenomic differences will result in differential expression of their target genes and result in changes in the developmental programming between the P2 and P5 populations. I also predicted that the conserved set of loci may play a critical role in maintaining pluripotency or involved in early differentiation across very different cell types (Figure 5A and Table 2). 49 Predicted enhancer P2 over-enriched loci conserved between LP and H9 Chr Start End chr8 41281720 41299720 chr1 5973775 5977775 chr11 117250816 117253816 chr12 6797900 6804900 chr19 55741600 55752600 chr16 78189650 78192650 chr13 94162175 94167175 chr20 42677360 42680360 Predicted enhancer P5 over-enriched loci conserved between HP and H9 Chr Start End chr8 25598975 25605975 chr7 30990675 30992675 chr17 7103925 7106925 chr4 179908300 179915300 chr4 88657300 88665300 chr8 5504916 5520916 chr13 52323925 52326925 chr19 11663237 11668237 chr21 46218325 46221325 chr1 44742480 44751480 chr7 68621550 68622550 chr3 62369837 62380837 chr3 191003983 191009983 chr8 95720400 95724400 chr1 22406783 22409783 chr3 140062083 140066083 chr9 7767600 7773600 chr13 111108450 111111450 chr18 5263925 5270925 chr20 11175525 11180525 chr9 95148075 95151075 chr3 57167025 57170025 chr11 82232375 82237375 chr3 46592925 46597925 chr4 185961216 185970216 chr9 14305275 14314275 50 Chr Start End chr20 17457416 17464416 chr12 6179225 6181225 chr5 151273150 151282150 chr11 133649100 133655100 chr2 3245750 3254750 chr2 118947487 118954487 chr20 44681250 44687250 chr2 118844283 118852283 chr10 103029500 103032500 chr7 31046150 31051150 chrX 152746587 152751587 chr2 223318216 223330216 chr9 107451200 107458200 Table 2: Conserved differentially enriched H3K27me3 predicted enhancer regions. Conserved over-enriched predicted enhancer regions conserved between LP P2 and H9 P2 and HP P5 and H9 P5 for H327me3. 1.2.8 Unique H3K4me3 epigenetic signature in low passage HES3 cells In the previous section, my analyses demonstrated that both bulk (Xi) and local H3K27me3 distribution patterns in the P2 and P5 populations are significantly different between the LP and HP population, suggesting large chromatin structure differences in the X-chromosome and at functional autosomal loci. I predicted that the other histone modifications might also be similarly altered. Using the same isolated P2 and P5 populations used to ChIP for H3K27me3 in the HES3 cells, I ChIP-sequenced H3K4me3 in the LP and HP cells. As previously described, H3K4me3 is associated with active and open chromatin. I hypothesized that GCTM-2/TG30 high cells (P2) will be characterized by high enrichment of H3K4me3 consistent with open chromatin compared to GCTM- 2/TG30 low cells. I expected higher active H3K4me3 enrichment at loci associated with high expression of pluripotency-associated markers and open chromatin in the GCTM- 51 2/TG30 high cells (P2). Similarly, I expected enrichment of H3K4me3 at developmentally regulated genes in the GCTM2/TG30 low cells (P5). My goal was to find quantitative epigenetic differences in the active chromatin mark at functional loci between P2 and P5 with which I can gain further understand the active epigenetic modification changes that occur during early differentiation. As described above, I used uniquely aligned and pair-wise (P2 and P5) quantile normalized the data in the LP and HP population. Peaks were called for these datasets using SICER as previously described. Since H3K4me3 is mostly promoter bound and not diffused like H3K27me3, a gap of 400 bp instead of 800 bp with e-value of .0005 was used. In the HP, approximately 13,000 peaks were conserved between P2 and P5 in both cell types with >90% overlap between the datasets. However in the LP, I observed a large number of peaks that did not overlap with the P5 population. There were 6348 peaks unique to LP P2 compared to the 1902 unique loci in the LP P5 population (Figure 7A). To further investigate the distribution of these peaks, I determined the distribution of the peak score of LP P2 unique and LP P5 unique loci. As indicated by the increased area under the curve for LP in Figure 7B, I found that LP P2 unique loci are mostly representative from the lowly enriched (low peak score) regions of the genome compared to the LP P5 unique loci. Visual inspection of the lowly enriched P2 over-enriched H3K4me3 loci revealed that these peaks usually occur in a contiguous fashion across long stretches of the genome. 52 Canonical H3K4me3 peaks are found at transcription start sites of genes. Given the distance between genes in a genome, on average I expected adjacent peaks to occur at a fairly large distance from each other. Figure 7C shows the distance between adjacent peaks in an ordered list of peaks from the telomeres of the q-arm to the telomeres of the p-arm on Chromosome 1. Compared to the LP P5 (Figure 7C), H3K4me3 peaks, LP P2 H3K4me3 have sets of contiguous peaks that are close to one another (defined by log [adjacent peaks distance] < 8); clusters of these peaks are highlighted using green boxes. I next determined if the long stretches of lowly enriched H3K4me3 was functionally significant in embryonic stem cells. In order to answer this question, I developed an algorithm to determine the exact genomic locations of these domains. After identifying the location of these long domains, I mapped them to known gene locations. To map domains of lowly enriched H3K4me3 peaks unique to the LP P2 dataset, I excluded highly enriched peaks (log peak score >3.5) from the analysis and concatenated 2 or more peaks that are less that 3000 bp from each other into a single domain classification. I then used the mapping information from the concatenated peaks to determine the distribution of these long lowly enriched H3K4me3 loci in the genome. A complete list of these mapped domains is listed in Table 3. The long lowly enriched H3K4me3 domains typically span hundreds of kb of DNA. Figure 7D shows one such example of a long domain encompassing the entire length of a group of genes that represent the epidermal differentiation complex (late cornified envelope proteins). My analysis revealed that 53 Figure 7: H3K4me3 quantile normalized peak distribution between P2 and P5 in HP and LP. (A) H3K4me3 peak distribution of quantile normalized P2 and P5 libraries from LP and HP cell type. (B) Peak score distribution between the P2 and P5 libraries of the LP populations. (C) Distance between adjacent H3K4me3 peaks in the LP P2 and LP P5 populations in chromosome 1. The adjacent log peak distances are plotted in order starting from the telomeric end of the q- arm through the centromeric region (indicated by orange box) to the telomeric end of the p-arm. these long domains extended through regions belonging to a specific gene family, suggesting that they maybe functionally conserved. These included gene families from olfactory receptor, keratin, zinc-finger, LCE (epidermal growth factor) genes, extracellular matrix (ECM) genes etc. It is important to note that these domains are unique to only the LP P2 H3K4me3 dataset. 54 The functional significance of these H3K4me3 remains unclear. It is important to note that they were unique to the LP P2 and not observed in the HP P2, which has already undergone bulk changes in H3K27me3. However, I speculated that these domains allow the chromatin to maintain an open confirmation, along with other unknown epigenetic and genetic factors, to regulate gene families during early differentiation or the general loss of open chromatin observed during the exit from pluripotency. For example, zinc- finger families of proteins are transcription factors that may play an important role in maintenance of pluripotency. Similarly, extra-cellular and cell-surface proteins may also play an important role in cell-cell communication required to maintain pluripotency. The clusters of genes may have a common regulatory pathway and during early differentiation, chromatin confirmation changes may occur due to the lack of H4K4me3 in the P5 at these loci. 55 Chromosome Start End Annotation chr1 150600000 151600000 LCE Domain chr17 2900000 3250000 OLFAC-17; Olfactory Receptor chr20 29250000 29450000 DEFENSIN-20; Defensin Proteins chr19 8700000 8950000 OLFAC-19-A; Olfactory Receptor chr19 9050000 9500000 OLFAC-19-B; Olfactory Receptor chr8 143760000 14000000 CELLCELL-8; Cell-cell interactions chr7 57200000 57600000 ZNF-7; Zinc Finger Proteins chr8 142500000 145700000 SPANK chr11 48150000 48350000 OLFAC-11-A; Olfactory Receptor chr11 50100000 51300000 OLFAC-11-B; Olfactory Receptor chr11 54550000 55300000 OLFAC-11-C; Olfactory Receptor chr5 140450000 140800000 CELLCELL-5; Cell-cell interaction chr19 56900000 57050000 ECM-19; Extra cellular matrix chr21 30700000 31150000 KERATIN-21; Keratin proteins chr9 139860000 140140000 VOLTDEP-9; Voltage dep. ion channel prot. chr6 30800000 31100000 KERATIN-6; Keratin Proteins chr5 178225000 178480000 ZNF-5; Zinc Finger Proteins chr11 15550000 15950000 OLFAC11-D; Olfactory Receptors chr8 143775000 144100000 LY6K chrX 80450000 82600000 Unknown chr19 59700000 59900000 Unknown chr19 60900000 61275000 Unknown chr19 61800000 62275000 Unknown chr19 21850000 23000000 Unknown chr4 44600000 46850000 Unknown   Table 3: Lowly enriched H3K4me3 domains in LP P2. Mapped lowly enriched H3K4me3 domains in LP P2 population. Chromsome location and gene family annotation is indicated, where known. 1.2.9 Conserved quantitative H3K4me3 changes occur at key developmentally regulated genes My findings demonstrated an enrichment of H3K4me3 domains in P2 that are absent in P5. This suggested that there were other specific H3K4me3 differences in these populations, similar to H3K27me3. Therefore, I investigated quantitative differences in 56 canonical H3K4me3 at functional loci such as gene promoters. Presence of H3K4me3 is generally positively correlated to gene expression, therefore identifying these regions could result in the characterization of a unique epigenetic signature of pluripotent hESCs. I sought to determine H3K4me3 changes between P2 and P5 that are conserved between the epigenetically different LP and HP cell lines. I hypothesized that conserved differentially enriched loci between LP and HP may be functionally significant for the maintenance of pluripotency or early differentiation regardless of passage length or Xi. To this end, I identified loci that were 50% or more over-enriched for H3K4me3 between P2 and P5 in both LP and HP, not including the lowly enriched H3K4me3 domains (Appendix D and E). In order to determine passage length independent changes in the H3K4me3 between P2 and P5, I then identified loci over-enriched in P2 and P5 that were conserved between HP and LP. For P2 H3K4me3 over-enrichment, 21 loci were identified which included 14 that mapped to predicted promoter regions (data from hetergeneous H1 cells; (Ernst et al., 2011)) but with no known annotated transcripts. The 7 remaining loci were mapped to annotated genes. Among known genes that mapped to gene promoters are TDGF1 and OCT4 (Figure 8B). The presence of higher enrichment of H3K4me3 in the P2 suggests that these pluripotency-associated genes are higher expressed in the P2 compared to the P5. Higher expression of these genes suggested that the P2 population of HES3 cells display an epigenetic state that is characteristic of cells in that have not undergone early 57 differentiation compared to the P5 population. Consistent my epigenetic observations, a study found that TDGF1 and OCT4 were transcribed at higher levels in the P2 compared to the P5 (Laslett et al., 2007). The other conserved genes are PDZD4, ZSCAN2, RYR2, LRFN5 and C9ORF129. The role of these genes in embryonic stem cell maintenance remains unclear and open to investigation. Importantly, I have also identified putative transcripts (14 loci mapping to predicted promoter regions) without known function that might possibly play a critical role in the maintenance of pluripotency. At regions with P5 H3K4me3 over-enrichment, 30 loci were conserved between LP and HP and nearly all were mapped to known genes. Correlation of these loci to gene function was conducted using the GREAT program and I found the top significant biological process to be ‘Organ Development’ (OTX2, TNFRSF19, WNT8B, LHX5, FEZF1, PKDCC, DKK1, MAB21L2, GLI3, RSPO3, SOX5, HESX1, BMP5, CITED2, NRG1; p <2.237e-20). 58 Figure 8: Conserved differentially enriched H3K4me3 loci between HP and LP. (A) Conserved P2 H3K4me3 over-enriched and P5 H3K4me3 over-enriched loci between HP and LP. Loci overlapping with known genes and unknown promoter regions are indicated within square brackets. Known functionally significant regions involved in development are underlined in each category. (B) Representative differentially enriched H3K4me3 loci are shown.[POU5F1 = OCT4] 1.2.10 Independent quantitative H3K4me3 changes in LP and HP Since my earlier findings indicated that passage length can have a large impact on the epigenomic signature of H3K27me3 enrichment, I hypothesized that there would be similar large differences in H3K4me3 enrichment between P2 and P5 that are not conserved between the cell types. To this end, I conducted GO analysis on quantitatively 59 different H3K4me3 loci between P2 and P5, separately between LP and HP and looked for unique functional categories between cell types. Figure 9 shows the significant biological processes conserved in LP P2 over-enriched and similarly for HP P2 over-enriched loci. For LP P2, I found neuronal/neurogenesis- associated genes to be conserved in the 602 LP P2 over-enriched loci (that did not map to the lowly enriched H3K4me3 domain defined in the previous section), along with pluripotency-associated factors, TDGF1 and OCT4 (Figure 9). However, 161 HP P2 over-enriched loci mapped to only 7 genes: NANOG, SKIL, TEAD4, CER1, DUSP5, and OCT4. These genes are significantly (p < .05) associated with endoderm formation, blastocyst development and cell fate specification (Figure 9). As shown in the previous section on a very small subset of these loci (21), overlapped between LP P2 and HP P2 over-enriched loci. Strikingly, 150 out of 602 LP P2 H3K4me3 over-enriched and 126 out of 161 HP P2 H3K4me3 over-enriched loci mapped to predicted enhancer regions. The role of enhancer enrichment differences in H3K4me3 remains unclear. Off target effects due to promoter-enhancer indirect association cannot be ruled out. In P5 over-enriched loci, 264 LP P5 over-enriched loci were mapped to the closest genes and they were found to be involved in processes such as differentiation and organ development (Figure 9). Similarly, the 120 HP P5 over-enriched loci were mapped to genes involved in developmental functions. As I found in the previous section, only 30 loci (25%) of the 120 HP P5 over-enriched H3K4me3 loci overlapped with LP P5 over- 60 enriched H3K4me3 loci. I found a large number of LP P5 over-enriched and HP P5 over- enriched loci that mapped to predicted enhancer regions as described above; 58 (out of 264) HP P5K4 over-enriched and 105 (out of 120) LP P5K4 over-enriched loci mapped to predicted to enhancer regions. However most of these enhancer elements do not overlap between LP and HP. A summary of H3K4me3 over-enriched loci and mapped enhancer regions from both the LP and HP are summarized in Appendix D and E. My analysis of differentially methylated H3K4me3 loci between P2 and P5 showed that out of the approximately 13,000 canonical H3K4me3 enriched loci in the genome, less that 600 loci (<5% of total loci) appeared to be >50% differentially enriched between P2 and P5. Most of these changes are not conserved between LP and HP. In the previous section, I identified a core set of genes and genomic differential H3K4me3 regions that may play a key role in embryonic stem cell identity, however a large number of changes between P2 and P5 appeared to be stochastic and occur at predicted enhancer regions. Together my results indicate that H3K4me3 enrichment differences in P2 and P5 are stochastic and passage length and culture conditions may play a role non-conserved H3K4me3 changes observed in the LP and HP cells. 61 Figure 9: GREAT GO analysis of differentially H3K4me3 enriched loci in P2 and P5 from HP and LP. GREAT analysis of significant biological process categories in loci common between HP P2 and LP P2 over-enriched datasets and also HP P5 and LP P5 over-enriched loci. 1.2.11 A Subset of epigenetic changes at key regulated genes correspond with gene expression My next question was to understand the correlation of epigenetic changes in both H3K4me3 and H3K27me3 with gene expression. Epigenetic changes of active and repressive marks occur concurrent with transcriptional changes at the target genes (Ernst et al., 2011; Guenther et al., 2007). In my study, I have characterized quantitative epigenetic changes that occur between P2 and P5 in both H3K4me3 and H3K27me3 in the LP, HP and H9. Ideally, a whole genome sequencing of RNA transcripts in the P2 62 and P5 populations could use and mapped to epigenetic differences observed between P2 and P5. In my study, I did not conduct gene expression experiments. However, our collaborators have published Illumina gene expression array data of similar high passage HES2 cells in P2 and P5. To test my hypothesis that epigenetic changes observed between P2 and P5 populations correlated with gene expression differences, I combined epigenetic information the HP HES3 cells with high passage HES2 gene expression data. To begin my analysis, I manually picked genes associated with changes in H3K4me3 and/or H3K27me3. Figure 10 shows a representative list of gene expression for genes that have quantitative differences in H3K4me3 or H3K27me3 or both in the HP cells. The gene expression data are derived from previously published HES2 data (Hough et al., 2009). My results showed that gene expression correlates with the various categories of epigenetic changes observed at these genes (Figure 10). TDGF1 is an example of a gene conserved for H3K27me3 over-enrichment in the P2 population and for H3K27me3 over- enrichment in the P5 population in all the cell lines analyzed. I defined changes in both H3K4me3 and H3K27me3 as ‘epigenetic switches’. TGDF1 is therefore an example of an epigenetic switch that results in the gene repression in the P5 population. In all the datasets presented in this study, I found only 1 gene, GREB1L, where an epigenetic switch results in a correlated gene expression in the P5 population. This gene 63 is related to hormone dependent growth factor GREB1, by sequence similarity. However the functional role of GREB1L in ES cells remains unclear. One study analyzed the genes differentially regulated between mouse ESCs (grown in LIF) and cultured without LIF for 5 days until when they formed embroid bodies (EBs) (Comparison of Gene Expression Profiles of J1 ES and J1 EB Cells: GEO accession: GSE3223). This study found GREB1L to be significantly upregulated during spontaneous differentiation. In our study, I found GREB1L to be the only ‘epigenetic switch’ to positively correlate with gene over-expression in the P5 population. In the HES2 cells, I observed a 40-fold increase in gene expression in the P5 compared to the P2 (Figure 11A). 64 Figure 10: Differential expression of gene targets between P2 and P5. Representative gene expression data between P2 an P5 for different categories of quantitative differences in H3K27me3 and H3K4me3 between P2 and P5. Gene name, normalized intensity level and epigenetic change category are indicated. 65 The role of GREB1L is open for investigation in future studies. Given its similarity to GREB1 (Rae et al., 2005), I speculated that this gene may play a role in growth regulation of hESCs. I have established that the P2 and P5 populations are epigenetically heterogeneous in nature. In this section, I have correlated a selected representative set of the epigenetic changes with gene expression. To conduct an unbiased analysis to understand the overall correlation between gene expression and epigenetic changes, I conducted genome-wide analyses of all genes that were differentially expressed, and correlated their expression to the epigenetic modification status of H3K4me3 and H3K27me3. I filtered the gene expression data to isolate genes differentially expressed by 2 fold or greater between the P2 and P5, from the HES2 dataset. I found 3082 genes to be either up regulated or down regulated between P2 and P5. At the promoter (2500 bp around transcription start site [TSS]) of each of these genes in the HP population, I calculated the normalized read count between P2 and P5 for H3K4me3 and H3K27me3 (Figure 11B). Strikingly, I found epigenetic changes and switches at promoter regions that correlate with gene expression with very few exceptions. A large cluster of epigenetic changes occurs at genes previously classified as bivalent (denoted by orange bars in Figure 11B). It is important to note that regardless of bivalency status, very few gene expression differences occur in H3K27me3 without changes in H3K4me3. My analysis also reveals 66 an important observation that bivalent genes do undergo dynamic regulation in H3K4me3 and H3K27me3 and these subtle changes were missed in previous studies, because experiments were conducted using a heterogeneous population of cells. Therefore using our methodology, I established that bivalent genes in hESCs are plastic and dynamically regulated epigenetically. My results indicate that by purifying cells based on pluripotency associated factor expression, I identified loci undergoing dynamic changes in either H3K4me3 or H3K27me3 or both and consequently, target genes also undergo changes in gene expression. Therefore, many bivalent genes are not static enrichments for H3K4me3 and H3K27me3 enrichment. By purifying cells, I defined a unique highly resolved epigenetic characterization of cells not undergoing early differentiation, using H3K4me3 and H3K27me3 enrichment differences. 67 Figure 11: GREB1L enrichment data and genome-wide epigenetic changes correlating with gene expression differences. (A) Genome browser track for GREB1L showing H3K4me3 and H3K27me3 enrichment between P2 and P5 from both the HP and LP populations. The normalized intensity value of GREB1L gene transcript expression between P2 and P5 is indicated. (B) Heatmap of genes that are differentially expression 2-fold or higher between P2 and P5. Red color indicates over-expression in the P2 population and green color indicates over- expression in the P5 population. Quantile normalized read count of H3K4me3 and H3K27me3 2500 bp around TSS between P2 and P5 in the HP cell line is shown. Red indicates high enrichment and green indicates low enrichment. 68 1.3 Discussion and Future Directions In this study, I sought to understand the epigenetic signature of purified human embryonic stem cells by sorting hESC cultures into P2 and P5. My results indicated that early H3K27me3 enrichment of the X-chromosome and Xi dramatically varied by hESC cell line, passage length and culture conditions. LP and HP cells are drastically different in their pan X-chromosome H3K27me3 regardless whether cells represent the high GCTM-2/ high TG-30 (P2) or low GCTM-2 / low TG-30 (P5). However, purified cell populations in H9 hESCs appear to be differentially modified for H3K27me3 at the X- chromosome where the P2 populations have low levels of the H3K27me3 on the X- chromosome and P5 populations display H3K27me3. X-chromosome analysis was proof of principle that bulk epigenomic changes were occurring in P2 vs. P5 in different cell lines and the effects are dependent on cell-type, passage length and culture conditions. I concluded that the H9 cells represent a population, which is related to both the LP and HP phenotype. I found that the H9 P2 and LP P2, and H9 P5 and HP P5 are epigenetically similar to one another in terms of X-chromosome inactivation status. I then asked the question if the similarity observed in the X-chromosome between H9 and HES3 cells held true at genes and other functionally significant regions. I defined conserved genomic locations that are over-enriched in H3K27me3 in either the P2 or P5, in the LP, HP and H9 populations. Consistent with my observations with bulk changes in H3K27me3 in the X-chromosome, I observed that at H9 P2 and LP P2 H3K27me3 over- enriched loci overlap specifically and the genes appear to play a role in neurogenesis. I 69 also found that H9 P5 and HP P5 over-enriched loci specifically overlapped and mapped to genes that play a role in development. Importantly, I showed that in this comparison the H9 P2 H3K27me3 over-enriched loci did not overlap with the HP P5 H3K27me3 over-enriched loci and vice-versa. Therefore, my analysis provided further evidence that the epigenome of H9 may be an intermediate phenotype between LP and HP. The genes conserved between LP and H9 P2 over-enriched H3K27me3 and H9 P5 and HP P5 H3K27me3 over-enriched loci may therefore play an important role in early exit from pluripotency and formation of a heterogeneous population of cells in culture. I then analyzed the conserved changes in H3K4me3 in the LP and HP populations to find genes that are conserved for enrichment differences between P2 and P5. I defined a core set of genes H3K4me3 over-enriched in P2 in both LP and HP such as TDGF1, OCT4, etc. I also defined a set of genes H3K4me3 over-enriched in the P5 in both LP and HP such as WNT8B, BMP4, SOX5 and HESX1 among others. Some of these conserved loci were mapped to putative promoters, but their target has not been defined or annotated. Together, the conserved differential H3K4me3 methylation of genes in the P2 and P5 shed more light on the early epigenetic changes that occur when cells exit pluripotency. In low passage P2 population, I defined a unique epigenetic signature phenotype, previously undefined. In the LP P2 population, I found long contiguous domains of lowly enriched H3K4me3 found at clusters of genes belonging to various families such as epidermal growth factors, zinc-fingers, keratin proteins and olfactory receptors. It is 70 unclear what is the functional significance of these domains but the domains are clearly conserved because they enriched the entire span of clusters of various protein families. These domains are not found in any other cell type or population isolated. I speculated that these domains allow the chromatin to maintain an open confirmation, along with other unknown epigenetic and genetic factors; this may allow for specific regulation of gene families during early differentiation or the general loss of open chromatin during differentiation. Genes such as zinc-finger families of proteins are transcription factors that may play an important role in maintenance of pluripotency. Similarly, extra-cellular and cell-surface proteins may also play an important role in cell-cell communication required to maintain pluripotency. I also found that gene expression changes between P2 and P5, in general, was correlated with the epigenetic changes observed between the two populations. Lower enrichment of H3K4me3 corresponded to lower expression and vice-versa. Importantly, it is important to note, that at loci that underwent significant gene expression changes between P2 and P5 primarily contain quantitative changes in H3K4me3. At bivalent genes, I found that changes in H3K27me3 rarely occured at loci that did not undergo changes in H3K4me3. My analysis shed more light on the regulation of gene expression at bivalent genes. I demonstrated that bivalent genes undergo dynamic temporal regulation by the large number of quantitative differences in H3K4me3 and H3K27me3 observed. Also, my data strongly suggested that the quantitative change in H3K4me3 is the predominant 71 regulatory pathway that confers changes in gene expression between P2 and P5 at bivalent genes. I therefore concluded that along with transcriptional heterogeneity observed between populations of hESCs isolated from culture, I observed subtle but predicable quantitative epigenetic changes between P2 and P5. My results revealed that genomes undergo rapid, dynamic and stochastic epigenetic regulation that reflected the inherent heterogeneity of cell cultures. Importantly, isolation of pure populations of cells is critical for the study and characterization of human embryonic stem cells. Apart from conserved epigenetic changes in the H3K4me3 and H3K27me3 across cell types for either P2 or P5 discussed in this study, I observed that a large number of these changes are not conserved across the different cell lines. Some of these loci mapped to functionally relevant regions such as predicted promoters and enhancers. However, the function of these control regions has not been elucidated yet. Importantly, my results indicated that culture conditions and passage length may play a role in the largely non-conserved epigenetic plasticity observed between P2 and P5. To follow up my study on the epigenetic plasticity of human embryonic stem cells, there are many avenues to pursue further validation experiments. Low passage HES3 cells and high passage HES3 cells are epigenetically very different with respect to their X- chromosomes H3K27me3 enrichment. I initially hypothesized that P2 populations of both HP and LP cells will be characterized by no enrichment of H3K27me3 and the P5 72 populations of HP and LP will be enriched with H3K27me3, indicating early differentiation. However, I found no X-chromosome-wide H3K27me3 enrichment differences between LP P2 and LP P5 populations; the X-chromosome was devoid of H3K27me3 enrichment. In the HP population, I detected large enrichment of X- chromosome H3K27me3 in both P2 and P5, with no quantitative differences between the purified populations. Contrary to my hypothesis, I concluded that LP cells had undergone X-chromosome inactivation during repeated passaging and purifying cells based on pluripotency markers failed to isolate a population without HP H3K27me3 enrichment on the X-chromosome. My results are consistent with previous studies that showed hESCs with active X-chromosomes tend to spontaneously undergo X-chromosome inactivation during passaging by expressing XIST and accumulating H3K27me3. Given the high passage HES2 data, we know that XIST gene is highly expressed in these cells. However, the correlation between XIST clouds and H3K27me3 status will need to be validated in the LP and HP populations. I expect LP populations to lack the XIST cloud and HP populations to contain XIST clouds. Importantly, it remains unknown in H9 cells if H3K27me3 enriched P5 populations contain XIST clouds or if XIST clouds are absent in the P2 population. Also, the role of some of the genes containing quantitative epigenetic changes between the P2 and P5 remains unknown. For example, GREB1L is over-expressed in the P5 population. However due to the inherent heterogeneity of the P5 population, containing cells in different lineages, it is unclear if GREB1L is involved in the specification of 73 certain lineage or if GREB1L is an important regulator required for the exit from pluripotency. Therefore, an over-expression model of GREB1L in hESCS may shed more light regarding the role of this gene in cultured stem cells; high levels of GREB1L in hESCs may increase the propensity for spontaneous differentiation of pluripotent stem cells. Similarly, if GREB1L functions as a master regulator of early differentiation, then knocking out GREB1L in ESCs may allow cells to readily maintain their pluripotent state and prevent differentiation. This system may prove to be a useful model to study cells in their pluripotent state or maintain pluripotency in culture. Similar approaches and validation experiments should also be carried out on other genes conserved for H3K4me3 and/or H3K27me3 differences to understand their role in maintenance or exit from pluripotency. For example, NANOG and OCT4 have been shown to lowly expressed in the P2 compared to the P5. These loci also have over- enrichment of H3K4me3 consistent with gene expression changes. However, along with NANOG and POU5F1, which have been studied previously, the role of other genes such as WNT8B, OTX2, ETS1, ETV4, PKM2, undergoing epigenetic changes in both H3K4me3 and/or H3K27me3 and their transcription changes in hESCs remains unclear. PKM2 is especially interesting because it has been shown to directly interact with master pluripotency regulator OCT4. Downstream biological experiments involving the effect of the knockdown or over-expression of these genes in maintaining pluripotency or in early differentiation needs to be elucidated. It is important to consider the possibility that many of the epigenetic and gene expression changes may occur as a result of indirect effects of 74 perturbing the expression of other regulatory genes and appropriate controls will need to be considered. The presence of the unique H3K4me3 domains has not been shown in any previous study. Therefore, it is important to replicate this observation by conducting validation experiments on multiple early passage hESCs and late passage cells (as negative control) between the P2 and P5, using ChIP-seq or traditional ChIP-PCR techniques. It will be especially prudent to assay for positive regulators of enhancers such as H3K27ac and H3K4me2 at these domains to understand if they serve as regulatory elements controlling genes in the same protein family or others. If validated, these domains will be useful as biomarkers for pluripotent hESCs. Most importantly, I have characterized the epigenetic heterogeneity of hESCs grown in culture and presented a unique signature of pluripotent hESCs. The description of lowly enriched H3K4me3 domains and temporal epigenetic changes accompanying early X- chromosome inactivation is a novel finding in my study. These characterizations, along with other future epigenetic modification studies using our framework may be very important in accurately defining pluripotent hESCs in the future. Therefore, follow up studies are required to verify our epigenetic characterization in other stem cell lines. Further understanding is needed regarding the early epigenetic changes that occur during early differentiation for a variety of other important epigenetic modifications such as DNA methylation and other histone modifications. For example, in the future, lowly 75 enriched H3K4me3 markers, along with other unique signatures, maybe used as a marker to define cells in their most pluripotent state. Also, understanding early epigenetic changes accompanying early differentiation will provide a framework to define and design culture conditions to keep hESC cultures in their pluripotent state. The temporal characterization of these modifications and their changes during continual passaging remains a technical and informatics challenge that needs to be overcome to accurately define, characterize and the use of pluripotent stem cells in the clinical environment. 76 Chapter 2: Role of PR-SET7 mediated H4K20me1 in gene expression regulation 2.1 Introduction The role of PR-SET7 in gene expression regulation remains unclear. PR-SET7 and H4K20me1 has been shown to be critical components of DNA repair, cell-cycle progression and chromatin compaction. There are competing theories about the role of PR-SET7 and H4K20me1 in gene regulation. A set of studies has shown that consistent with the association of H4K20me1 in chromatin compaction, loci enriched for H4K20me1 coincide with regions of low transcriptional activity. H4K20me1 regions also exclude active transcription associated H4K16 acetylation. However, analysis studies based on chromatin immuno-precipitation (ChIP) and high-throughput ChIP followed by sequencing (ChIP-seq) at active and repressed loci have shown that H4K20me1 is found at the 5’ end of highly expressed genes and co-localized with hyperacetylated histones, suggesting its role in active gene transcription. The goal of my study was to therefore elucidate the role of H4K20me1 in the transcriptional regulation of H4K20me1 enriched genes in-vivo. I used high-throughput ChIP-on-chip analysis to identify targets of H4K20me1 on chromosomes 21 and 22. After genes were found, we conducted gene expression experiments to assay for perturbation of gene expression in the absence of PR-SET7 and H4K20me1. My analysis revealed that H4K20me1 is predominantly found at gene loci compared to control 77 H4K20me3. We found that loss of PR-SET7 and H4K20me1 resulted in the over- expression of H4K20me1 target genes, suggesting that H4K20me1 acts as a transcriptional repressor at its target genes, regardless of their control transcription levels. I also investigated the role of H4K20me1 in embryonic stem cells and their differentiation. I investigated the correlation between gene expression, PR-SET7 and H4K20me1 enrichment in Drosophila S2 embryonic stem cells. I also investigated the correlation between H4K20me1 and acetylated loci and characterized H4K20me1 inhibitors to be used in future studies. 2.2 Results 2.2.1 H4K20me1 and H4K20me3 enriched regions on chromosomes 21 and 22 In order to determine the location of H4K20me1 and H4K20me3 in chromosomes 21 and 22, we used modification specific antibodies in Hela cells. ChIPs were conducted to isolate regions of the genome containing the histone modifications. The pulled down material was then used to isolate the DNA, processed and hybridized to a Nimblegen genome tiling array, which spanned chromosome 21 and 22 at the 100 base pair resolution and input DNA was used as the control (ChIPs conducted by Sabrina Houston). In order to determine regions of enrichment on the array, the publically available MA2C program was used to determine peaks of significant enrichment over input signal (Song et al., 2007). Visual inspection to individually validate peak calls from the program was 78 performed. At p<0.005 significant level, I identified 359 peaks for H4K20me1 and 162 at p<.001 significance level. Similarly, in a separate experiment, we found 426 and 195 H4K20me3 peaks at p <.005 and p <.001 significance level respectively (Table 4). The summary of all mapped genes is found in Table 5 (H4K20me1) and Table 6 (H4K20me3). Modification Significance Total no. of peaks No. of genes with peaks No. of peaks within genes H4K20me1 P<.005 359 156 275 P<.001 162 83 124 H4K20me3 P<.005 426 142 197 P<.001 195 73 89 Table 4: H4K20me1/me3 peak and gene target summary. MA2C and gene mapping summary of H4K20me1 and H4K20me3 ChIP-chip on HELA cells in Chromsome 21 and 22. 79 Chromosome Gene Gene length Peak Density # Peaks chr22 RPL3 6783 0.921273305 4 chr22 ATF4 2122 0.734715298 4 chr22 HTF9C 5371 0.423144145 4 chr22 HIC2 34058 0.251504121 3 chr21 SNF1LK 12602 0.240636394 1 chr22 PDXP 8203 0.224987229 1 chr21 PTTG1IP 24229 0.217901712 3 chr22 UBE2L3 56367 0.215462201 8 chr21 ADARB1 151960 0.211482281 20 chr22 CSNK1E 27392 0.166393044 4 chr22 LOC164714 39504 0.156652742 3 chr22 RANBP1 9681 0.151505171 2 chr22 PISD 12330 0.140213124 2 chr22 MED15 80033 0.13662563 6 chr21 TCP10L 8982 0.132302168 1 chr22 BRD1 51515 0.128913444 3 chr21 PRED57 3899 0.122353442 1 chr21 ETS2 19028 0.118150685 2 chr22 KIAA1666 5793 0.114849907 1 chr21 PDXK 43211 0.108804993 4 chr21 RRP1 14566 0.107146417 1 chr22 POLR3H 18660 0.103476821 2 chr22 DGCR8 31565 0.103035208 2 chr21 C21orf56 23232 0.102672978 1 chr22 CECR5 21759 0.100884112 1 chr22 NDUFA6 5358 0.096702892 1 chr22 PLXNB2 19802 0.094854162 2 chr22 SHANK3 58572 0.088931202 2 chr22 POLDIP3 31235 0.084388186 2 chr22 CDC42EP1 8909 0.079811229 1 chr22 MAFF 14515 0.077441919 1 chr21 SON 34463 0.076320597 1 chr22 SBF1 28219 0.074349773 1 chr22 HMOX1 13108 0.072549441 1 chr22 TUBGCP6 27283 0.071683494 1 chr22 TOB2 13536 0.07091826 1 chr22 MYH9 106656 0.070437605 6 chr21 PFKL 27326 0.070066411 2 chr21 MRAP 20474 0.063525064 1 chr21 WDR4 36473 0.057822886 1 chr21 PCNT 121627 0.057029585 3 chr22 MCM5 24380 0.055220884 1 chr22 POLR2F 14089 0.053601511 1 chr22 CHKB 14104 0.053560498 1 80 Chromosome Gene Gene length Peak Density # Peaks chr22 CPT1B 14104 0.053560498 1 chr22 BCR 137672 0.053083005 4 chr22 C22orf13 14866 0.051556516 1 chr22 SNRPD3 16892 0.046891747 1 chr22 FLJ41993 18114 0.044465148 1 chr21 C21orf70 36933 0.043598143 1 chrX ACRC 35173 0.043026086 1 chr22 SULT4A1 37992 0.042536558 1 chr21 LSS 40331 0.038183762 1 chrX PGK1 22554 0.037427818 1 chr22 CRKL 36176 0.037191669 1 chr21 C21orf58 22736 0.03718657 1 chr22 DGCR2 86173 0.036401121 2 chr22 PACSIN2 76111 0.035705971 2 chr22 TXNRD2 66320 0.032720691 1 chr22 CERK 53846 0.03117312 1 chr22 A4GALT 28749 0.030657829 1 chr22 MN1 53221 0.03004036 1 chr21 TMEM50B 30833 0.028899348 1 chr22 PRR5 68969 0.028199654 2 chr21 RRP1B 36527 0.027363362 1 chr22 DIP 53030 0.025679139 1 chr21 COL18A1 58210 0.024328991 1 chr21 TRPM2 89481 0.021583264 1 chr22 MICAL3 44351 0.021062767 1 chr22 EMID1 53633 0.017756583 1 chr21 AGPAT3 122359 0.016424342 2 chr22 CELSR1 176337 0.014848463 2 chr21 PCBP3 92493 0.014796975 1 chr22 HIRA 100996 0.014141376 1 chr21 RUNX1 261497 0.01298891 2 chr22 TBC1D22A 411174 0.012719776 4 chr22 EFCAB6 283465 0.012112194 2 chr22 CACNG2 138800 0.011871102 1 chr22 CABIN1 166832 0.01043335 1 chr22 PARVB 169929 0.008835483 1 chr21 HSF2BP 130302 0.008468211 1 chr21 KCNJ6 291910 0.006892842 1 chr22 OSBP2 213019 0.005720326 1 Table 5: H4K20me1 enriched genes. Identification and characterization of statistically significant (p<0.001) H4K20me1-associated genes using MA2C on the human chromosome 21 and 22 ChIP-chip data sets. Genes are ranked according to their peak density (total length of H4K20me1 peak(s) divided by the gene length). 81 Chromosome Gene Gene Length Peak Density # Peaks chr22 TUG1 7131 0.604861056 4 chr22 RPL3 6783 0.317512008 2 chr22 CENPM 8400 0.208633094 2 chr21 CBR1 3178 0.190135976 1 chr22 LOC388910 3111 0.180002323 1 chr21 LOC441956 1839 0.156697098 1 chr22 LOC164714 39504 0.149986668 3 chr21 CRYAA 3773 0.145583953 1 chrX CXorf26 5264 0.143998514 1 chr22 NDUFA6 5358 0.132989501 1 chr21 TCP10L 8982 0.125397045 1 chr22 MORC2 41588 0.122961264 3 chr21 ADARB1 151960 0.114949829 12 chr21 RRP1 14566 0.107146417 1 chr21 C21orf56 23232 0.099192538 1 chr21 DNMT3L 15877 0.098236422 2 chr21 KCNJ15 29316 0.091911765 2 chr22 SHANK3 58572 0.087214384 2 chr21 SON 34463 0.086329855 1 chr22 APOBEC3B 10379 0.072422697 1 chr21 MRAP 20474 0.063525064 1 chr22 MB 16590 0.061113626 1 chr22 TCN2 19887 0.053728286 1 chr22 ACTBL1 31606 0.052552148 1 chr22 FLJ42953 18769 0.051506037 1 chr21 ETS2 19028 0.050962166 1 chr21 SETD4 29867 0.04665366 1 chr21 PCNT 121627 0.046410283 2 chr21 PCBP3 92493 0.045921647 2 chr22 CECR5 21759 0.045856414 1 chrX ACRC 35173 0.04548472 1 chr21 WDR4 36473 0.043527982 1 chr21 UBASH3A 43754 0.042636131 2 chr21 TRPM2 89481 0.040008002 2 chr21 LSS 40331 0.038183762 1 chr22 TXNRD2 66320 0.036201615 2 chr21 FLJ45139 61139 0.033013701 2 chr22 PLA2G6 70259 0.03299938 2 chr22 COMT 27220 0.032090465 1 chr21 TMEM50B 30833 0.031651667 1 chr21 SLC19A1 27723 0.031604611 1 chrX ERCC6L 34352 0.031366054 1 82 Chromosome Gene Gene Length Peak Density # Peaks chr22 TMEM184B 53719 0.031239974 1 chr22 ZBED4 36230 0.029954469 1 chr22 BRD1 51515 0.027185828 1 chr22 ELFN2 59506 0.026920592 1 chr22 HIC2 34058 0.026543304 1 chr22 MN1 53221 0.025272049 1 chr22 MED15 80033 0.024551927 2 chr21 KCNE1 65585 0.023211648 1 chr22 SYN3 494144 0.022740191 8 chr22 FBLN1 62863 0.022673083 1 chr22 MICAL3 44351 0.021062767 1 chr21 BACH1 46731 0.020103004 1 chr22 CELSR1 176337 0.020072922 1 chr21 PKNOX1 59046 0.019366034 1 chr22 BCR 137672 0.017461515 2 chr22 ACO2 59864 0.016063888 1 chr22 OSBP2 213019 0.014401494 2 chr22 EFCAB6 283465 0.013496444 2 chr22 RBM9 289803 0.010497692 2 chr21 RUNX1 261497 0.009363401 2 chr21 CYYR1 107053 0.009328938 1 chr22 CLTCL1 112251 0.008917122 1 chr21 DOPEY2 129733 0.008503842 1 chr21 HSF2BP 130302 0.008468211 1 chr21 HUNK 130749 0.008440429 1 chr22 SCUBE1 140126 0.007896941 1 chr22 CACNG2 138800 0.007276507 1 chr21 KCNJ6 291910 0.006892842 1 chr21 ITSN1 246826 0.005972432 1 chr22 TBC1D22A 411174 0.002759951 1 chr21 DSCAM 834697 0.001249707 1   Table 6: H4K20me3 enriched genes. Identification and characterization of statistically significant (p<0.001) H4K20me3-associated genes using MA2C on the human chromosome 21 and 22 ChIP-chip data sets. Genes are ranked according to their peak density (total length of H4K20me1 peak(s) divided by the gene length). After I determined peaks of enrichment of H4K20me1 and H4K20me3, they were mapped to gene loci on chromosomes 21 and 22. My analysis, mapping peaks to gene promoters, gene bodies and intergeneic regions, revealed that H4K20me1 was enriched in 83 gene bodies (75% of all peaks). In contrast, 54% of all H4K20me3 peaks were found at intergenic regions. At the promoter regions (defined by 5 kb upstream and 500 bp downstream of transcription start site), 6% percent of all H4K20me1 peaks were found here compared to only 2% of all H4K20me3, three times as many H4K20me1 in promoter regions compared to H4K20me3. Together, I showed that H4K20me3 is predominantly found at gene promoters and gene bodies compared to H4K20me1 (Figure 12A). The ChIP-chip experiment excluded repetitive sequences from the array because they are uninformative and the location of a given enrichment at a repetitive cannot be determined. In order to determine the distribution of H4K20me1 and H4K20me3 at repetitive regions, we conducted ChIP-cloning on the material. Briefly, DNA isolated from the pulled down material was used to create bacterial libraries and these libraries were then sent for sequencing. ChIP-cloning was conducted to understand the distribution of H4K20me1 and H4K20me3 at low-complexity DNA sequences and repeats on chromosomes 21 and 22. Results indicated that approximately 42% of H4K20me1 clones corresponded to non-repetitive regions, compared to 77% of H4K20me3. Together, H4K20me1 is preferentially enriched at gene bodies compared to H4K20me3 that is preferentially found at repetitive elements, 43% of which are SINE elements (Figure 12B). My results indicated that the locations of H4K20me1 and H4K20me3 are physically distinct. As summarized in Table 4, I found that at p<.005, we were able to determine 156 genes enriched for H4K20me1 (83 genes at p<.001) and similarly, for H4K20me3, 142 84 genes were mapped at p <.005 and 73 genes at p<.001. A large majority of peaks mapped to genes from the H4K20me1 compared to the H4K20me3 (>76% of peaks versus <47% of peaks, at p<.001). Among all genes either enriched for H4K20me1 or H4K20me3, only 34 genes were co-modified with H4K20me1 and H4K20me3. Therefore, most genes were only enriched for H4K20me1 or H4K20me3, but not both (Figure 12). Our results suggested that the two histone modifications are physically distinct and therefore may also function in distinct roles. 85 Figure 12: H4K20me1 is preferentially enriched within genes while H4K20me3 is typically located within repetitive elements. (A) Chromatin immunoprecipitations (ChIPs) were performed in HeLa cells to isolate H4K20me1- and H4K20me3-associated DNA. The ChIPed material was hybridized to a NimbleGen oligonucleotide genome tiling microarray containing the entire length of human chromosomes 21 and 22. Statistically significant peaks (P < 0.005) of enrichment were identified using MA2C software. The percentages of peaks located in specific genomic regions are represented. Proximal is defined as the regions 5 kb upstream and 500 bp downstream of the gene body. (B) ChIPed material was cloned and 86 insert positive H4K20me1- associated clones and 57 H4K20me3-associated clones were directly sequenced. The general distribution of the sequences and the different classes of corresponding repetitive elements are shown 2.2.2 Profile of H4K20me1 and H4K20me3 at target genes In Figure 13, at representative genes, I observed that H4K20me1 is preferentially enriched on gene bodies and H4K20me3 is enriched at the proximal 5’ region. However, to understand the overall distribution of each modification on their target genes, I calculated the cumulative peak density in a meta-analysis across the 5 kb upstream region 86 to the normalized gene body length and to the 5000 downstream region from the gene body, using all p<.005 peaks for H4K20me1 and H4K20me3. In order to correct for differences in gene length, I calculated the density based on gene bodies based on percentage of total length. The proximal and distal density values were plotted based on single base-pair values. Using this approach, I visualized the overall distribution of H4K20me1 and H4K20me3 at their target genes (Figure 14). To determine regions of significant density enrichment in my meta analysis, I used the poisson distribution model to determine significant regions at p<.05 (black bars in Figure 14). Consistent with my observations in Figure 13, I found that for H4K20me1 the region of largest enrichment of H4K20me1 occurs between first 5-20% of the gene body. Also, the enrichment levels gradually decrease through the first 50% of the gene body. However, we found only 1 region of significant enrichment for H4K20me3. We found a region at the promoter region, as expected, between -1300 and -800 relative to transcription start site. Overall, H4K20me1 occupied a broad region with the highest levels of H4K20me1 occupancy at the 5` end of the gene and gradually decreases across the body of the gene. However, we observed a relatively punctate enrichment pattern at the promoter region for H4K20me3. These findings further indicated that H4K0me1 and H4K20me3 occupied distinct regions at gene loci and may perhaps have distinct functions in gene transcription regulation. 87 Figure 13: Identification of H4K20me1- and H4K20me3-associated genes. Three representative H4K20me1-associated genes. (A) and H4K20me3-associated genes (B) identified by ChIP-chip are shown. The log 2 ratios of the H4K20me1 and H4K20me3 ChIP-chip data (y-axis) were mapped to human chromosomes 21 and 22 (x-axis) using the UCSC genome browser. Enrichment of H4K20me1 (top track) and H4K20me3 (bottom track) ChIP DNA compared to input DNA is shown. The large black boxes above the tracks indicate statistically significant (P < 0.001) peaks for H4K20me1 or H4K20me3. 88 Figure 14: Meta-analysis of H4K20me1/me3 enrichment at gene loci. H4K20me1 is enriched in the first half of gene bodies whereas H4K20me3 is targeted ∼1 kb upstream of transcription start. Peak densities of either statistically significant (P < 0.005) H4K20me1- or H4K20me3- associated genes were summed (y-axis) and plotted as a percentage of the gene body or nucleotide position within the 5 kb proximal regions (x-axis). A Poisson distribution was used to determine the average peak density (gray line) and statistically significant (P < 0.05) peaks of H4K20me1 and H4K20me3 enrichment (black bars). 2.2.3 H4K20me1 is needed for transcriptional repression of specific target genes The previous section suggested that H4K20me1 is present at gene promoters and gene bodies proximal to the promoter and therefore may play a role in the transcriptional regulation. After I had determined genes enriched for H4K20me1 and H4K30me3, we then determined the role of PR-SET7, its catalytic activity and possibly the role of H4K20me3 in gene regulation. Since PR-SET7 is the only known methyltransferase that 89 catalyzes H4K20me1, we decided to knockdown PR-SET7. We reasoned that a knockdown of PR-SET7 would result in the loss of H4K20me1 and affect gene expression regulation. We transfected Hela cells with control shRNA and a PR-SET7 shRNA plasmid that depletes PR-SET7. We showed that loss of PR-SET7 results in the loss of H4K20me1 but no global changes in H4K20me3/me3 were observed (Figure 15A). We also showed that knockdown also resulted in the depletion of H4K20me1 at representative gene BRD1, which we determined by ChIP-chip to contain H4K20me1, using ChIP followed by qPCR. After both global and local loss of H4K20me1 was verified in PR-SET7 shRNA, we collected RNA from cells transfected with either control shRNA or PR-SET7 shRNA. We used selected MTMR3 from a panel of H4K20me1 and H4K20me3 negative peaks as a negative control gene that is not directly regulated by H4K20me1 or H4K20me3. We determined that MTMR3 does not change gene expression with PR-SET7 knockdown. However, we determined that H4K20me1 target genes were generally upregulated by approximately 2-fold in the absence of PR-SET7 (Figure 15B). Importantly, we did not detect significant changes in gene expression of H4K20me3 target genes upon PR-SET7 knockdown and global H4K20me1 decrease (Figure 15B), again suggesting that these modifications are non-overlapping in terms of gene regulation. 90 Figure 15: Depletion of the PR-Set7 H4K20 monomethyltransferase results in the selective derepression of H4K20me1-associated genes. HeLa cells were transfected with a control shRNA plasmid or a PR-Set7 shRNA plasmid. (A) Western analysis of whole cell lysates using the indicated antibodies. (B) qRT-PCR analysis was performed to analyze the expression levels of seven H4K20me1-associated genes or four H4K20me3-associated genes in cells containing the control shRNA (white) or PR-Set7 shRNA (gray) plasmid. Results are plotted relative to MTMR3 expression (y-axis). Three independent biological replicates were performed to generate the standard deviation in each experiment. The Student's t-test was used to determine statistically significant changes at *P < 0.01 and **P < 0.05. 91 In order to determine if PR-SET7 and/or H4K20me1 is responsible for the repressive phenotypes observed at H4K20me1 target genes, we repeated the experiment with a catalytically dead point mutant of PR-SET7 (PR-SET7CD). The expression of plasmid with PR-SET7 has been shown to result in a dominant negative phenotype with reduction of global levels of H4K20me1 and no changes in PR-SET7 levels (Houston et al., 2008). The experiments were repeated with PR-SET7CD transfection in a similar manner to PR- SET7 shRNA. Figure 16A confirms the global reduction of H4K20me1 and Figure 16B shows that PR-SET7CD results in the local reduction of H4K20me1 at the BRD1 gene. We found a similar 2-fold increase in gene expression at H4K20me1 targets in the PR- SET7CD samples compared to control. As observed earlier, we found no change in the gene expression of H4K20me3 targets (Figure 16D). Together my study shows that PR-SET7 catalyzed H4K20me1 are targeted to specific genes and loss of PR-SET7 and H4K20me1 resulted in the 2-fold over-expression of target genes. Importantly, using a catalytically dead dominant negative point mutant of PR-SET7, we showed that the H4K20me1 mark itself is important for gene repression. This transcriptional repression by H4K20me1 is distinct from H4K20me3 enriched genes. 92 Figure 16: H4K20me1 is essential for repression of endogenous H4K20me1-associated genes. HeLa cells were transfected with a control eukaryotic plasmid or a plasmid expressing a catalytically dead (CD) dominant negative PR-Set7 point mutant. (A) Western analysis of whole cell lysates using the indicated antibodies. (B) ChIPs were performed on the HeLa cells transfected with a control (top), a PR-Set7 CD (middle), or a PR-Set7 shRNA expression plasmid (bottom) using either the H4K20me1-specific antibody, a histone H3 general antibody (positive control), or rabbit IgG (negative control). Thirty cycles of PCR amplifications were performed each time for the two H4K20me1 positive regions of BRD1 using 0.1, 1, or 3 µl of ChIPed DNA from each sample (triangle). (C) qRT-PCR analysis was performed to analyze the expression levels of seven H4K20me1-associated genes or four H4K20me3-associated genes (D) in cells expressing the control (white) or PR-Set7 CD (gray) plasmid. Results are plotted relative to MTMR3 expression (y-axis). Three independent biological replicates were performed to generate the standard deviation in each experiment. The Student's t-test was used to determine statistically significant changes at *P < 0.01. 93 2.2.4 Global Role of PR-SET7 and H4K20me1 in gene expression repression In the previous section, I determined that H4K20me1 functions in gene transcription repression. My previous studies used ChIP-chip analyses on chromosomes 21 and 22. One of my major goals of this study was to determine if genome-wide H4K20me1 mediated gene expression plays a critical role in the function and identity of cells. Therefore, I expanded my study to include genome-wide analyses of the role of H4K20me1 in gene expression regulation. First, I studied the genes regulated by PR-SET7 on a genome-wide level. To this effect, HELA cells were transfected with PR-SET7shRNA, puromycin selected and were collected four days post transfection (work done by Lauren Congdon). The experiment was conducted in biological duplicates and hybridized to the Illumina Human WG-6 3.0 array. Probes significantly altered in intensity value by 2-fold or more between experiment and control (p <.005) were filtered and annotated to known genes. I determined 43 genes to be 2-fold or more altered by the loss of PR-SET7 and global reduction of H4K20me1. 41 genes were found to be up-regulated and only 2 genes were down regulated; consistent with our previous H4K20me1 at genes mainly functions as a repressor. One of the down-regulated genes is PR-SET7 and the other is HERPUD1 (Table 7). 94 Table 7: Genes with expression change >2-fold in HELA cells lacking PR-Set7 and global loss of H4K20me1 95 I then conducted a gene ontology analysis on the 41 up-regulated genes to analyze if there are significant functional conservation categories among these genes. My analysis revealed no significant functional categories among the up-regulated genes. In the next step of my analysis, I took advantage of recent studies that have ChIP-sequenced H4K20me1 in many different mammalian cell lines. In order to further understand the distribution of H4K20me1 enriched genes, I took advantage of the data available in multiple datasets to determine if there were sets of H4K20me1 positive genes conserved in all cell lines. A recently study conducted ChIP-seq analysis for H4K20me1 in nine different human cell lines (embryonic stem cells (H1), erythrocytic leukaemia cells (K562), B-lymphoblastoid cells (GM12878), hepatocellular carcinoma cells (HepG2), umbilical vein endothelial cells (HUVEC), skeletal muscle myoblasts (HSMM), normal lung fibroblasts (NHLF), normal epidermal keratinocytes (NHEK) and mammary epithelial cells (HMEC)), among many other histone modifications and transcription factors. In this study, the authors concluded that H4K20me1 is found at actively transcribed gene regions by the association of H4K20me1 to other active histone modifications and their correlation with gene expression. However, the authors did not conduct a H4K20me1 specific analysis on the genes that are unique to specific cell-lines and also conserved between them (Ernst et al., 2011). To determine this, I downloaded the H4K20me1 ChIP-seq aligned data from each of the nine cell types. For each cell type, peaks of significant enrichment were determined using SICER. For my subsequent analyses, I used cell lines from different origins with the top 96 three total number of H4K20me1 peaks (H1embryonic stem cells, HMEC mammary epithelial cells and HSMM muscle myoblast cells). H4K20me1 positive peaks in each cell type were mapped to known genes in the genome and I conducted a three-way analysis to understand the distribution of peaks between the different cell lines. Figure 17A shows the meta-analysis on the enrichment profile of H4K20me1 in H1 cells with H3 enrichment as a control. As I observed with the low-resolution meta-analysis of ChIP- chip H4K20me1 peak distribution, the high resolution ChIP-seq enrichment read profile of all H4K20me1 genes in H1 reveals a profile with the highest levels just downstream from TSS and a gradual decrease towards the 3’ end of the gene. I conducted a three-way gene overlap analysis between H1, HSMM and HMEC to investigate the distribution of H4K20me1 positive genes in the 3 cell lines. My analysis revealed that approximately 5-10% of all genes in the genome are enriched for H4K20me1 (Figure 17B). However, the distribution of a majority of these genes appears to be cell line specific. In the H1 cells, genes specifically enriched for H4K20me1 are highly functionally conserved in embryonic development and differentiation; these genes were not highly transcribed genes in hESCs (Bernstein et al., 2006), further suggesting a role of H4K20me1 at repressed loci, in embryonic stem cells. I then analyzed the genes that are conserved in all three cell lines and enriched for H4K20me1. 560 genes were conserved in all three cell types; a gene ontology analysis on these genes revealed functionally conserved categories of genes involved in translational 97 regulation and RNA binding (Figure 17C). My observations suggested that H4K20me1 may play a role in both actively transcribing genes (discussed in the following sections) and also play a role in repressed genes as observed in H1 cells. Therefore, H4K20me1 appears to play a cell-type specific role in embryonic stem cells. In order to understand the dynamics of H4K20me1 regulation of embryonic stem cells, I used mouse embryonic stem cells as a model system. I conducted a pilot experiment to determine the global role of H4K20me1 along with other histone modifications. I utilized mouse embryonic stem cells and differentiated them into predominantly the neuronal lineage using retinoic acid, similar to previously conducted studies (Ward et al., 2004). During differentiation, developmentally regulated genes are up-regulated. If H4K20me1 represses these genes, during differentiation they may lose H4K20me1 and become de-repressed. However, for my pilot study, I began by determining bulk changes in H4K20me1 and other modifications during differentiation. 98 Figure 17: Cell type specific distribution of H4K20me1. (A) Meta-analysis of all genes enriched for H4K20me1 in H1 cells. H4K20me1 and control H3 profiles are drawn in blue and red, respectively. Transcriptions start site is indicated with arrow pointing towards 3` end of genes; 5kb upstream and 25kb downstream of TSS is plotted. (B) Three-way comparison of H4K20me1 positive genes between H1, HSMM and HMEC. (C) DAVID GO analysis conducted on genes specific to H1 and H4K20me1 genes conserved in all 3 cell types. To further investigate the role of H4K20me1 in mESCs, I conducted an experiment to differentiate mouse ESCs into neuronal progenitors (NPCs) using retinoic acid and used differentiated mouse embryonic stem cell (MEFs) as a control. Briefly, mESCs were treated with 100nM retinoic acid. The undifferentiated cells were plated on gelatin with culture media containing DMEM, 15% FBS, 10e-4 2-mercaptoethanol and 1000U of leukemia inhibitory factor (mLIF). To induce differentiation, the cells were supplemented with 100nM all-trans-retinoic acid and mLIF was removed. Media was replaced daily and 99 Figure 18: Histone modification changes during mESC differentiation mESCs treated with 100nM retinoic acid. Day 0 (a), Day 6(b), MEFs (c) shown in figure. The undifferentiated cells were plated on gelatin with culture media containing DMEM, 15% FBS, 10e-4 2- mercaptoethanol and 1000 U of leukemia inhibitory factor (mLIF). To induce differentiation, the cells were supplemented with 100nM all-trans-retinoic acid (Sigma) and mLIF was removed. Media was replaced daily and cells were collected at Day 6 post-differentiation. cells were collected at Day 6 post-differentiation. Whole cell lysates were made using these samples and western blot analyses were conducted for a panel of all H4K20 methylation degrees and other related modifications. Consistent with my hypothesis, I observed a global loss of H4K20me1 by day 6 post-differentiation and also lower levels of PR-SET7 (Figure 18). The loss of H4K20me1 is consistent with previous studies that have shown that during differentiation, progenitor cells lose the capacity to self-renew. H4K20me1 and PR-SET7 are present in high levels at the G2/M phase of the cell cycle (Karachentsev et al., 2005; Rice et al., 2002; Wu et al., 2010) and lowly cycling cells display lower level of H4K20me1 and PR-SET7. Interestingly, I observed no increase in 100 H4K20me2 and an increase in H4K20me3. The functional significance of higher levels of H4K20me2 in differentiating cells remains unclear. However the observation of increased H4K20me3 in differentiating cells is consistent with previous studies that have shown that H4K20me3 is induced during differentiation to possibly compact chromatin (Wongtawan et al., 2011). Consistent with this study, I found differentiated MEFs were highly enriched for H4K20me3. I used H3K9me3 as a positive control; H3K9me3 is found in low levels in mESCs and increases in enrichment during differentiation to decrease the amount of transcriptionally permissive chromatin in the genome (Loh, Zhang et al., 2007). The concomitant increase of H4K20me3 and H3K9me3 is consistent with a previous study, which demonstrated that H4K20me3 and H3K9me3 are largely colocalized to the same regions of the genome and loss of H4K20me3 resulted in the loss of H3K9me3 (Pannetier et al., 2008). Similar to H3K9me3, I found a concomitant decrease of H3K9me1 along with H4K20me1. The role of the relationship between H4K20me1 and H3K9me1 is unclear. A previous study indicated that knockdown of H4K20me1 de-methylase, PHF8, increased H3K9me1 levels in addition to H4K20me1 (Liu et al., 2010). My observations of concomitant decrease of H4K20me1 and H3K9me1 during differentiation suggested a common regulatory pathway for H4K20me1 and H3K9me1. Overall my pilot study suggested that H4K20me1 may play a role in maintaining the epigenomic identity and in the repression of genes required for maintaining pluripotency. Loss of H4K20me1 may be required for differentiation. 101 In other cell types analyzed, H4K20me1 appears to play a role in active genes, contrary to my observations in embryonic stem cells. I have identified genes enriched with H4K20me1 and these are involved in house keeping activities of the cell and are expressed at high levels. However, my work in Hela clearly demonstrated a role of H4K20me1 in gene repression. To further understand the relationship of H4K20me1 with active genes, I sought to understand the relationship of H4K20me1 with active gene associated H4 acetylation marks. Histone acetylation at the 5’ end of genes is a hallmark of active transcription (Kurdistani et al., 2004; Z. Wang et al., 2008). One study has shown that K20 methylated H4 histones inhibited the acetylation of K16 on histone H4 (H4K16ac), which is mark of active gene transcription (Nishioka et al., 2002); this study is consistent with my work that have demonstrated that H4K20me1 plays a role in gene repression. However, another study, on the contrary, showed that at gene promoter regions, H4K20me1 and H4 acetylation is positively correlated (Z. Wang et al., 2008) . A recent study demonstrated that H4K20me1 and H4K16 are present in the same nucleosome but on opposite H4 tails (Voigt et al., 2012). The effect of nucleosome co-modification of active H4K16ac and potentially repressive H4K20me1 in gene expression regulation remains unclear and open to investigation. We have shown in our studies that PR-SET7 has been shown to be involved in gene repression. H4K20me1 has also been shown to be associated with actively transcribing 102 genes. Given the opposite role the two opposing roles of H4K20me1, I hypothesized that H4K20me1 is involved in gene expression modulation, by acting as a modulator to repress the expression of active genes, enriched with H4K16ac and other markers, to control overall transcription levels. Therefore I predicted that H4K16ac enrichment would negatively correlate with H4K20me1 in genes. Alternatively, PR-SET7 and H4K20me1 may have functionally opposing roles depending on other transcription factors. To answer these questions, I adopted the data from a previously conducted study in which the authors conducted ChIP-seq analyses on histone modifications, which included H4K20me1 and Histone H4 acetyl marks in CD4+ T cells (Z. Wang et al., 2008). My goal was to determine the correlation between H4K20me1 and H4K16ac and H3K9ac levels in a quantitative manner. To this end, I mapped H4K20me1 positive peaks to the 5’ ends of genes. I then filtered all genes, which contained any significant enrichment of H4K20me1. I then calculated the enrichment levels of H4K16ac and H3K9ac at each of the H4K20me1 genes, by calculating the number of reads in 400 base pair windows across 6 kb covering 1000 bp of the promoter region and the first 5000 bp of the gene body. I used this information to construct a heatmap using k-means clustering with three expected clusters (k = 3; high, medium and low H4K20me1 enriched genes). It is also important to note that this study has shown that H4K20me1 positive genes are correlated with active gene transcription. Consistent with my hypothesis, my results show an inverse relationship between the levels of H4K20me1 and H4K16/H3K9 acetylation. Genes with the highest levels of 103 H4K16ac/H3K9ac enrichment at H4K20me1 positive genes correlates with the lowest level of H4K20me1 enrichment, and vice-versa (Figure 19). Therefore my results indicate that although H4K20me1 and H416ac are co-modified on the same gene, the quantitative enrichment of H4K20me1 and H416ac are negatively correlated. My results suggested that in expressed genes, H4K20me1 might act as a modulator where the presence of high H4K20me1 levels on the gene is correlated with the low levels of active gene transcription markers H416ac and H3K9ac and reduced expression. Further studies are required to determine the consequence of the loss of H4K20me1 at these genes. I have investigated the direct role of H4K20me1 in gene expression regulation. However, I assumed that since PR-SET7 is the only known methyltransferase of H4K20me1, the protein would track with H4K20me1 at gene regions. These studies suggested that PR- SET7 will be at H4K20me1 regions since it is the sole H4K20me1 methyltransferase. To verify the presence of PR-SET7 in mouse or humans, our lab has attempted to ChIP- seq PR-SET7 in Hela and mouse ESCs. However, we have not been able to conduct successful ChIPs for PR-SET7 at gene loci or perform high-throughput sequencing to verify our assumption. Although currently available antibodies for PR-SET7 perform well for western blot analyses, these antibodies failed in high-throughput sequencing experiments in both mouse and human cells. The failure of the antibodies to ChIP may be due to the protein kinetics and access to the epitope. We have conducted multiple ChIP- seq experiments for PR-SET7 in both mouse and humans and only obtained signal which was similar to background H3 or input data (data not shown). 104 Figure 19: K-means clustering heatmap of H4K20me1 positive genes. Levels of H4K16ac and H3K9ac are also included and clustered together with the H4K20me1data. However, recently, a study was published which analyzed a compendium of transcription and histone modifications in Drosophila S2 embryonic stem cell lines. In this study, 105 using ChIP-chip analyses the authors successfully conducted experiments to define regions of PR-SET7 and H4K20me1, among many other modifications. The PR-SET7 ChIPs may have been successful since the antibodies were developed in a different non- mammalian model system. I took advantage of this study and downloaded and processed the data to ascertain the role of PR-SET7, H4K20me1 in gene transcription regulation in fly ESCs. I used peaks identified in this study to map PR-SET7 and H4K20me1 to gene promoters (-5000 bp from TSS) and gene bodies. I was able to identify 1005 genes enriched for H4K20me1 and 3272 genes enriched for PR-SET7. Interestingly, only ~50% of H4K20me1 positive genes (359) were co-modified with PR-SET7. The results suggested that PR-SET7 does not always track with H4K20me1 enrichment. My initial results indicated that PR-SET7 and H4K20me1 may have functionally distinct roles and PR-SET7 may function in an H4K20me1 independent fashion. To understand the physical interaction between PR-SET7 and H4K20me1, I conducted a meta-analysis to visualize the enrichment profiles of PR-SET7 and H4K20me1 at co- modified genes. My results indicated that co-modified genes do not have similar enrichment profiles; PR-SET7 appears to be promoter bound and H4K20me1 follows the canonical enrichment profile as shown in previous sections (Figure 20A). I speculated that PR-SET7 maybe promoter bound due to its interaction with other proteins or complexes, which are involved in transcription regulation. 106 Given that there is low overlap between PR-SET7 and H4K20me1 within genes and also the presence of genes specifically modified with either PR-SET7 or H4K20me1, I investigated the relationship between PR-SET7 and H4K20me1 and gene expression data. I divided gene expression in 15 categories based on the expression levels in S2 cells and calculated the distribution of PR-SET7 specific, H4K20me1 specific and co-modified genes in the 15 categories. My analysis revealed that genes with the lowest levels are predominantly modified with only H4K20me1 (blue bars); genes modified with only PR- SET7 are mostly represented at medium expression categories (red bars) and genes co- modified with PR-SET7 and H4K20me1 (green bars) are predominantly represented at high expression categories (Figure 20B). For reference, the genome wide gene frequency of expression categories (purple bars) is also shown. Together, my analyses have revealed PR-SET7 and H4K20me1 may have distinct roles in gene expression regulation and the co-modification of PR-SET7 and H4K20me1 is found mostly in highly expressed genes, compared to H4K20me1 only genes, which are lowly expressed in the genome. These results suggested that PR-SET7 may play a role in active genes by its protein-protein interactions with transcription factors and regulators at the 5’ end of the gene. Consistent with this hypothesis, the loss of PR-SET7 on H4K20me1 positive genes resulted in lower gene expression levels because of the loss of interaction with the transcriptional machinery. Therefore, PR-SET7 may play a role in gene expression regulation outside its catalytic activity. 107 Figure 20: PR-SET7 and H4K20me1 distribution in Drosophila. (A) Meta-analysis of ChIP- chip data of PR-SET7 and H4K20me1 at 359 co-modified genes. Transcription start site (TSS) indicated with arrow; Density of sample signal over input is plotted 5kb upstream and 7.5kb downstream of TSS. (B) Frequency of PRSET7only (blue), H4K20me1only (red), PR- SET+H4K20me1 (green) enriched in 15 gene expression categories from low (1) to high (15). Whole genome gene distribution frequency is plotted in purple. 108 2.2.5 Small Molecule Inhibitors of PR-SET7 Small molecules inhibitors are very useful methods to regulate protein-protein interactions and catalytic activities of target proteins. Small molecule inhibitors are effective alternatives to genetic approaches of protein knockdown and/or catalytic dead mutant protein replacement, because their doses can be moderated in a gradient manner, they do not rely on gene expression systems, their effects can be reversed and can be delivered readily into cells and tissues. The successful design of specific and potent small molecular inhibitors targeting proteins of interest remains a challenge in the field of therapeutics and drug design. In our work, in order to study the catalytic activity of PR- SET7, we ectopically expressed a catalytically dead mutant of PR-SET7 to create a dominant negative phenotype. Although protein expression vectors are effective tools for studying protein activity, the system relies on perturbing the endogenous levels of the target protein and is prone to off target effects and mutational consequences due to random integration of the expression vector in the genome. Small molecule inhibitors, therefore, are an effective alternative to study the catalytic activity by designing specific molecule that target the catalytic domain and inhibit the activity of the target protein. In collaboration with Dr. Minkui Luo and his colleagues, I characterized small molecules that were designed to be potent, specific inhibitors of PR-SET7. These inhibitors were designed to mimic the co-factor of PR-SET7, S-adenosyl-L-methionine (SAM). These inhibitors are designed to bind PR-SET7 and block its enzymatic activity. I assayed the potency and specificity of drugs using a gradient of doses and their effect on H4K20me1 109 and other histone modifications. I predicted that at an ideal non-toxic dosage of drug will inhibit the catalytic function of PR-SET7 and over time will lead to a depletion of H4K20me1 in treated cells, compared to a non-drug control. Three drugs were tested on 293T cells; name coded SP, FH and FM. I used NIH 293T cells for my drug treatments. DMSO was used as the control and equal volumes of DMSO diluted 10mM stock solution of drug was mixed with media and treated on cells. On day 1 of the experiment, 10,000 HELA cells were plated in a 6-well format and 6 hours later, cells were treated with appropriate drug concentrations (Figure 21). It is important to note that several drug doses were initially tested for toxicity. I started with 1 µM and increased dosage in a logarithmic increment to 100 µM. In all drugs tested, 100 µM was found to be toxic and 90-100% of cells died within 24 hours. At 10 µM, I was able to observe some cell death and 30-50% of cells appeared to be viable after 24 hours. Therefore, I chose a range between 1 µM (no visible phenotype compared to DMSO) and 10 µM concentration. The cells were treated with drugs and were then harvested 72 hours later and whole cell lysates were made. Western blot analyses were then conducted to determine the bulk levels of H4K20me1. For both SP and FH inhibitor (Figure 21A), I was not able to detect any appreciable decrease in H4K20me1 regardless of concentration compared to DMSO control. 110 Figure 21: NIH293T cells treated with SP, FH and FM drugs. (A) The negative results of SP and FH treated cells at Day 3. Western blots of H3 general and H4K20me1 are shown. (B) Western blot of FM drug treatment at various concentrations collected at Day 3 shown. (C) Growth curve of viable cells at Day 3,4 and 5 of FM treated cells. However, I was able to detect a reduction in the level of H420me1 at low concentrations of 3 and 6 µM compared to DMSO control, 72 hours after FM treatment (Figure 21B). As expected, no changes in PR-SET7 protein levels were detected since the drug was not designed to not reduce the protein levels of PR-SET7. The drug was also found to be specific to only the H4K20me1 methyltransferase, PR-SET7, as it did not affect activity of the related H4K20 methyltransferase, Suv4-20 and H4K20me3. I also observed a slow growth phenotype in the 6 µM concentration compared to DMSO, consistent with requirement of PR-SET7 catalytic activity for proper cell-cycle progression. 111 Since H4K20me1 is required for proper progression of cell-cycle through mitosis, I hypothesized that cells treated with FM will move through the cell-cycle slowly and will begin to accumulate at the G2/Mitosis phase (G2/M) of the cell-cycle. To gain further understanding of the growth kinetics of HELA cells under drug treatment, I repeated my experiments with 10,000 cells seeded and treated my cells with 6 µM and 10 µM FM. I collected cells at days 3, 4 and 5 and counted the number of viable cells by trypan blue staining. The grown curve of HELA cells under drug treatment had very low cell number and underwent a linear growth trajectory compared to the exponential growth observed when cells were treated with DMSO (Figure 21C). Although it is important to note that at Day 3, when I observed a loss of H4K20me1, the cell numbers were comparable. However, subsequent cell divisions were severely affected for drug treated cells at Days 4 and 5. It has been shown that loss or over expression of PR-SET7 results in the loss of H4K20me1 and cells arrest in the G2/M phase of the cell cycle, unable to complete mitosis (Houston et al., 2008; Jørgensen et al., 2007; Karachentsev et al., 2005). I sought to further understand if the slow growth phenotype observed with FM was due to cells arresting at G2/M. I repeated my experiments and collected cells at Days 3, 4 and 5, which were then fixed with 70% ethanol and stained with propidium iodide for subsequent cell-cyle analysis. One difference between the growth curve experiment and the cell-cycle experiment was that for the latter experiment, the cells were drug treated again on Day 3 (Figure 22). The cells were then analyzed using the CyAn analyzer 112 machine. The results showed that on Day 3 the cell cycle profile between 6 µM and DMSO were comparable. However, a day after re-treating the cells with 6 µM and 10 µM, I observed significant increase of cells at the G2/M phase compared to DMSO. However, 24 hours later the cells appear to have a comparable profile to DMSO with the exception of a large amount of debris for the drug treated cells, suggesting cell death. My results indicated that the loss of H4K20me1 by the inhibition of the catalytic activity of PR-SET7 using FM, results in slow growth phenotype because of cells piling up at the G2/M phase and by possibly undergoing cell death. FM therefore with further validation may be a potent and specific inhibitor of PR-SET7 in-vivo. 113 Figure 22: Propidium Iodide stained cell-cycle analyses of FM drug treated NIH293T cells. The debris, G1, S and G2M phases of the cell cylce are labeled. A summary of the experiment is specified at the top of the figure. DMSO, 6µM and 10µM treatments at Day 3,4 and 5 are shown. I was able to successfully characterize a specific and potent small molecule inhibitor (FM) that inhibited the catalytic activity of PR-SET7 and depleted the treated cells of H4K20me1 within 72 hours. The loss of H4K20me1 resulted in a slow growth phenotype because of the inability of the cells to efficiently complete mitosis. Therefore FM appears to be a specific inhibitor of PR-SET7 and in the future can be used to knockdown levels of H4K20me1 in cells instead of shRNA or siRNA based approaches. 114 2.3 Discussion and Future Directions In this chapter, I investigated the role played by PR-SET7 mediated H4K20me1 in gene expression regulation. I defined the exact genomic locations of H4K20me1 and H4K20me3 enrichment in chromosome 21 and 22 and identified genes enriched for either modification. My results revealed that H4K20me1 is preferentially enriched at the 5’ end of genes and H4K20me3 is preferentially enriched at repetitive regions with a small subset of peaks enriched in the proximal promoter region of genes (Congdon et al., 2010). These finds are consistent with previously conduced studies (Barski et al., 2007).We identified a panel of genes that contained peaks of H4K20me3 that have not been previously observed and their role in gene transcription regulation has not yet been defined. Using our panel of H4K20me1 enriched genes, we sought to find the role of H4K20me1 in gene expression regulation. RNAi against PR-SET7 and catalytically dead dominant negative PR-SET7 constructs were used to deplete levels of H4K20me1 in cells and also at target genes. I observed a consistent ~2 fold over-expression of target genes upon depletion of PR-SET7 or ablation of its catalytic activity by the dominant negative catalytically dead point mutant. A consistent 2-fold over-enrichment suggested that H4K20me1 maybe allelically regulated in gene repression and more studies are needed to verify if H4K20me1 is imprinted or allelically excluded in one copy of a gene versus the other. Overall, my study demonstrated a role of H4K20me1 in gene repression in at least a subset of genes. 115 Using publically available data, I investigated the functional role of H4K20me1 enriched genes. I used the published ENCODE data for H4K20me1 in various cell lines. Consistent with my previous findings, H420me1 is found the 5’ end of genes and tapers off in enrichment towards the 3’ end of genes. Genes common in all cell-lines investigated appear to be functionally significant in housekeeping and RNA processing and are generally highly expressed genes. It has been shown that H4K20me1 is cell cycle regulated with the highest levels of the mark at the G2/M stage (Houston et al., 2008; Wu et al., 2010). Therefore for future studies, I propose to investigate if a set of genes regulated by H4K20me1 is also cell-cycle regulated by assaying their expression levels during the various stages of the cell-cycle and assay for depletion of H4K20me1 at these target genes after exit from M and G1/S when the levels of H4K20me1 are the lowest. Also, given that H4K20me1 is universally conserved in expressed housekeeping genes involved in RNA binding and translation, it is unclear if knockdown of H4K20me1 increases the levels of translation associated proteins and increase the rate of translation. H4K20me1 enriched genes appear to be cell-line specific, especially in H1 hESCs. These cells appear to have a significantly higher number of H4K20me1 enriched loci compared to any other cell-line. Genes specific for H4K20me1 in H1 cells are significantly associated with genes involved in development and pattern specification functions. These genes are generally repressed and my observations suggested a role of H4K20me1 in gene repression. Given that H4K20me1 is specifically enriched at developmental genes in 116 hESCs, the role of H4K20me1 in stem cells remains unclear. To further understand genome-wide regulation of H4K20me1, I used mouse ESCs (mESCs) to determine bulk levels of H4K20me1 by Western blots. The mESCs were then differentiated into the neuronal lineage using retinoic acid. My analysis revealed a global loss of H4K20me1 in differentiating cells. My results are consistent with my previous observation of H4K20me1 specific genes present on developmentally regulated genes; during differentiation these genes are generally de-repressed. I reasoned that during differentiation these genes are de-repressed and lose H4K20me1, which contributes to the global loss of H4K20me1 during differentiation. Future studies must include experiments to understand the specific role of H4K20me1 during differentiation. I propose to conduct ChIP analyses at H4K20me1 target genes before and after differentiation to assay the up- regulation of developmental genes during differentiation. Also, in order to determine the role of H4K20me1 in embryonic stem cells, I propose an experiment of knocking down H4K20me1 using the FM small molecule inhibitor and determining if loss of H4K20me1 causes spontaneous differentiation. In the next steps of understanding the role of H4K20me1 in gene expression, I investigated the contradictory results regarding the association of H4K20 me1 with active gene histone acetylation marks. One study showed that H4K20me1 and active H4K16 acetylation are competitive in-vitro (Nishioka et al., 2002). However, another study showed that H4K20me1 enrichment correlated with histone hyperacetylation (Talasz et al., 2005), at genes in vivo. In order to further understand correlation between 117 H4K20me1 enrichment and histone acetylation (ac), I mapped H4K20me1 to genes in CD4+ T cells and found that genes with the lowest levels of H4K20me1 are correlated with the highest levels of H4K16 and H3K9ac. However, it remains to be investigated if H4K20me1 genes associated with relatively high levels of lysine acetylation are differentially expressed compared to H4K20me1 genes devoid of acetylation. I expect the latter set of genes to be lowly expressed. I also propose to assay the change of expression in H4K20me1 depleted cells in the context of acetylated and non-acetylated gene targets. It is unclear if the ~2-fold up-regulation upon PR-SET7 knockdown is conserved in differentially acetylated genes. Also in future studies, it remains to be investigated what the role of PR-SET7 in gene expression regulation is? My analysis of the Drosophila S2 cells revealed that only a subset of genes were co-modified with PR-SET7 and H4K20me1 and were associated with highly expressed genes. However, the physical location of PR-SET7 and H4K20me1 on gene bodies appears to be distinct in the ChIP- chip data. These results need to be independently verified by conventional ChIPs and/or an independent model system. Importantly, there were subsets of genes in the Drosophila genome that were enriched with only H4K20me1 and these genes correlated with lowly expressed genes. Genes enriched with only PR-SET7 enrichment were predominantly found between the high and low end of gene expression. Therefore, the functional role of the 3 subsets genes remains unclear. I propose to combine PR-SET7, H4K20me1 and gene expression data and analyze the data in a quantitative manner in the form of a cluster analysis and determine the functional correlation of the various cluster 118 combinations. For example, I expect genes with the lowest levels of H4K20me1, PR- SET7 and expression to be developmentally regulated in the S2 embryonic stem cells. Another useful tool in studying the role of PR-SET7 and H4K20me1 is the use of enzyme inhibitors. My characterization of the FM small molecule inhibitor of PR-SET7 has shown that H4K20me1 levels can be specifically depleted. In order to comprehensively test the specificity of the inhibitor, I propose to investigate a larger panel of epigenetic marks to verify the specificity of this drug. I also propose to investigate the cell-cycle arrest phenotype associated with slow growth observed with the FM drug. My initial results indicated that 24 hours after cells were re-treated with drug and split at Day 3 (Figure 21), I observed an increase in the number of cells in the G2/M phase of the cell cycle with the drugs compared to DMSO control and an increase in cell debris. Future validation studies may include TUNEL assays to investigate if there will be an increase in apoptosis and increases in G2/M phase markers such as H3 serine 10 phosphorylation in drug treated cells. After validation of the FM drug, the inhibitor can then be used to investigate the role of PR-SET7 catalyzed H4K20me1 in chromatin regulation and gene expression. In my previous studies, I used an ectopic over expression PR-SET7 catalytically dead mutant or shRNA knockdown vectors to study the role of H4K20me1 in gene expression; using an inhibitor will allow for future studies to be conducted without perturbing endogenous expression of the PR-SET7 protein itself. 119 My results demonstrated that PR-SET7-mediated H4K20me1 functions to repress certain genes in a cell-type specific manner, regardless of basal levels of expression. However, my study also suggested that H4K20me1 together with localization of PR-SET7 might have a distinct function associated with highly expressed genes, compared with genes modified with only H4K20me1 or PR-SET7, which are expressed at lower levels. PR-SET7 mediated H4K20me1 is implicated in many important biological functions in the cell. PR-SET7 is involved in functions such as cell-cycle progression, chromatin condensation, DNA damage repair and DNA replication. Aberrant regulation of PR- SET7 has been shown to be involved in cell-cycle progression defects, DNA damage repair defects and chromosomal instability, along with deregulation of H4K20me1 targeted genes. Such defects typically lead to abnormal growth of cells and may eventually lead to cancer. Therefore, deregulation of PR-SET7 and its targets maybe a potential driver of carcinogenesis. PR-SET7 is highly expressed in some cancers and participates in epithelial to mesenchymal transition, a key step to cancer metastasis. Therefore, understanding the role of PR-SET7 is critical to understanding carcinogenesis. Little is known regarding the role of PR-SET7 and H4K20me1 in gene regulation. My study has provided initial insights regarding the involvement of PR-SET7 in gene expression regulation and also characterized a potent inhibitor of PR-SET7. Along with its role in chromatin regulation, understanding the role and specific function of PR-SET7 in transcriptional regulation is indispensible in designing targeted therapies in PR-SET7 driven cancer cell types. A small molecule inhibitor of PR-SET7 has shown positive and 120 encouraging initial results in arresting cell cycle and promoting apoptosis. Such drugs are potentially promising therapeutic measures that can be utilized to arrest and kill fast growing PR-SET7 positive cells and advance the field of cancer medicine. 121 Chapter 3: Material and Methods 3.1 Materials and Methods: Chapter 1 3.1.1 Cell culture, passaging and cell sorting HES3 cells were cultured on irradiated mouse embryonic fibroblasts (MEFs). The media used was formulated using DMEM, 20% fetal bovine serum, 1% L-glutamine, .5% penicillin/streptomycin, 1% non essential amino acids, 1% insulin/transferring/selenium and .18% beta-mercaptoethanol. Cells were passaged using manual dissection every 6 days. H9 cells were grown in F12-DMEM with 20% KSR, FGF-2, penicillin/stremptomycin and l-glutamine. The media was grown in the presence of MEFs and then transferred to H9 cultured as MEF conditioned media. Low passage HES3 cells (LP) were collected between passages 10 and 25 and high passage HES3 cells were collected between passages 60-75 (HP). We used high passage H9 cells collected between passages 60-70 (H9). For FACS analysis, HES3 cell colonies grown on 35mm plates were used to isolate cells. The cells were washed with Trypsin and then incubated in .5ml of Trypsin at 37 degrees C for 3 min. After MEFs dislodged from the plate, the cells were removed and the hESCs were scrapped, homogenized by pipette and collected. The H9 cells were directly 122 trypsinized and collected since these cells were grown without direct contact with MEF cells. The cells were then formaldehyde fixed and washed with PBS.(+ 2.5% FBS). For every 100000 cells, we used 500ul of GCTM-2 (mouse IgM), 500ul TG30 (mouse IgG2a) and 1 ul of human embryonic stem cell marker TRA-181 (Abcam cat# 16289). The cells were incubated at room temperature for 30 minutes. Cells were then washed with PBS (+ 2.5% FBS). Secondary antibodies were diluted in PBS in the following manner. For every million cells we prepared 1ml of secondary antibody with Alexa Fluor 647 goat antimouse IgM (Invitrogen cat# A21238) at dilution 1:2000; Alexa Fluor 488 goat antimouse IgG2a (Invitrogen cat# A21131) at dilution 1:1000; and R-phycoerythrin antimouse IgG1 (Jackson Immunoresearch cat# 115-115-205) at dilution 1:500. 100ul of antibody was used to stain non-primary antibody stained cells to be used as a negative control. The secondary antibody was incubated with the primary antibody stained cells for 30 minutes in the dark. The cells were washed with PBS (+2.5% FBS) and then FACS analyzed using the BD Bioscience FACSAria cell sorter. The cell were first gated to isolate TRA-181 positive cells and then the top ~20% and bottom ~20% of the cells staining for GCTM-2 and TG30 were isolated as the P2 and P5 populations respectively. 3.1.2 Chromatin immunoprecipitation and sequencing The ChIPs, library construction and sequencing were carried using a previously established protocol (Frietze et al., 2012). Approximately 2 million cells were used for 123 each ChIP. The fixed cells were dounce homogenized and sonicated using the Bioruptor (Diagenode) to between chromatin lengths between 200-500 bp and quantified. Approximately 10µg of chromatin was mixed with 10µl of antibody and incubated overnight. In this study we used the Cell Signaling H3K27me3 and H3K4me3 antibodies. The samples were then precipitated using 15µl of equal volume Dynabeads Protein A and G. The samples were then reverse crosslinked and the DNA was purified using the Qiagen PCR clean up kit. The libraries were constructed using the standard Illumina Sample Preparation Kit for Genomic DNA protocol and sent for sequencing on the Illumina Solexa GAII sequencer. 3.1.3 Sequence alignment and processing All libraries were sequenced as single end reads and mapped to the Hg18 Human Genome using bowtie (Langmead, Trapnell, Pop, & Salzberg, 2009). We used the default settings of bowtie to align the reads to the genome. We then used reads uniquely aligning to the genome for the next stem of our analysis. The genome was then tilled into 400 bp windows. The reads were offset by 75 bp and the total read count in each window was measured and normalized to number of reads per 10 million reads sequenced. The read count information was used to make enrichment plots on the Integrated Genome Viewer (Nicol et al., 2009). Quantile normalization was conducted for every P2 and P5 for a given modification (H3K4m3 or H3K27me3) for a given cell type (LP, HP or H9). The R programming 124 environment was used to normalize the 400 bp read count file. We used the library preprocessCore library and normalize.quantiles function to conduct the quantile normalization. The normalized files were then used for downstream peak calling and quantitative analysis. 3.1.4 Peak calling and quantitative analysis The program SICER was used to call peaks of significant enrichment. We used a fixed window value of 400 bp and gap length of 800 bp for the diffused H3K27me3 and a gap length of 400 bp for the punctate H3K4me3 mark. A stringent E-value cutoff of .0005 was used to filter peaks. Since the libraries were modified by quantile normalization, in order to call peaks, we directly used the program find_islands_in_pr.py in the SICER package. The peaks were then annotated using the Homer package (Heinz et al., 2010b). Homer was also used to compute peak overlap between libraries and also with other datasets of interest at default settings. The statistical significance values for overlap between two datasets were calculated using the hypergeometric distribution in R. Gene ontology was conducted using the GREAT tool (Valouev et al., 2008). I used 5 kb upstream and 1 kb downstream from transcription start site and a distal distance of 10 KB to map loci of interest to gene. To find significant quantitative difference between two libraries, we used the package ChIPDiff to call regions of quantitative differences between two libraries. Since this package accepts only aligned bed files (chromosome, start position and read orientation) 125 as input, we converted the quantile normalized read count data from 400 bp windows into bed files by using a random number caller to synthetically recreate as bed file with the total number of recreated reads equal to the number of quantile normalized reads in the 400 bp window. We then used the following parameters to find significant fold differences between the P2 and P5 libraries: maxIterationNum 5000, minP .995, maxTrainingSeqNum 1000000, minFoldChange 1.5, minRegionDist 2500. The significantly enriched loci were then annotated using the Homer package as described above. Also for any given peak or quantitatively different loci we calculated the number of quantile normalized reads in each significant window using the Perl language (see Perl code section below). 3.1.5 Gene expression and heatmap For the gene expression analysis, we used previously RMA normalized HES2 data from the Illumina Sentrix Human 6 arrays (GEO accession: GSE13201). Each population was analyzed in triplicates. For gene expression level differences, we averaged the three experiments for each population and calculated standard error. To construct the heatmap dataset for analyzing gene expression differences between P2 and P5 and histone modification differences, the log average intensity value of P2 and P5 were calculated and 2-fold or greater probes were used for downstream analysis. The probes were then mapped to annotated gene bodies. For each gene, we calculated the 126 number of quantile normalized reads in P2 and P5 for each modification within 2500 bp around transcription start site (TSS). The data was then K-means clustered (20 clusters) using the Cluster3 software (de Hoon et al., 2004) and the visualized using JavaTreeView (Saldanha, 2004). 3.2 Materials and Methods: Chapter 2 3.2.1 Chromatin immunoprecipitation HeLa cells were fixed in 1% formaldehyde for 10 min before quenching with 0.125 M glycine for 5 min. 10 8 nuclei per ml nuclei were isolated using nuclear isolation buffer (150 mM NaCl, 10 mM HEPES, pH 7.5, 1.5 mM MgCl 2 , 10 mM KCl, 0.5% NP-40, 0.5 mM DTT) and resuspended in nuclear lysis buffer (50 mM Tris–HCl, pH 8.1, 10 mM EDTA, 1% SDS) . Nuclei were sonicated between 200 to 600 bp. For each ChIP, 5x10^6 sonicated material was used for immunoprecipitation with either rabbit IgG, a Histone H3 general antibody (Abcam, Ab1791 Lot #172452), a histone H4K20me1-specific (Active Motif, #39175), a histone H4K20me3-specific antibody (Active Motif, #39180), or a FLAG antibody (Sigma, #F7425 Lot #096K4803). Immunoprecipitation was carried out with Protein-A-conjugated sepharose (Amersham, GE Biosciences) and washed with RIPA buffer (50 mM HEPES, pH 7.4, 1 mM EDTA, 1% NP-40, 0.7% sodium deoxycholate, 500 mM LiCl). DNA was eluted using 10% Chelex (Bio-Rad) solution. 127 3.2.2 ChIP-Chip and ChIP-Cloning The ChIPed material above was linker-ligated and PCR amplified using the oligoJW102 and oligoJW103 primers (Table 4 below). Four micrograms of PCR amplified DNA was sent to NimbleGen Corporation for Cy3/Cy5 labeling, array hybridization, and data collection. Name T m (°C) Forward (5’-3’) Reverse (5’-3’) GAPDH (expression) 82 CAGCCGAGCCACATCGCTC AGACA TGAGGCTGTTGTCATA CTTCTC PTTG1IP (expression) 81 ACCGGCTTCCCTTTGTAAA T CTGGCCTTCTCCTCAC TCCT PFKL (expression) 87 CAGAAAGCCATGGATGAC AA TATACTGTGTGTCCAT GGGAGAT PDXK (expression) 87 TGAATTCAGATCAGCTCCA G AAACTGGTTGGGCGT GATAA LOC220686 (expression) 87 CTACAACTTCATCCGAAGC A CATCTCCATGAACCAC TTGAA BRD1 (expression) 87 ATGAAGGCTGCCAAAGAG AA TTTCCTCATTGTGGCA AAGTC ADARB1 (expression) 87 TCGTGGATGGTCAGTTCTT T TTGGCATCTTTAACAT CTGTGC COL18A1 (expression) 87 TCAGTGCCACCACCATCTT GGATGTGGAACAGCA GTGAG CBR1 (expression) 82 TGATCCCACACCCTTTCAT A AGCTTTTAAGGGCTCT GACG TUG1 (expression) 80 ACCAAGGAGTCCCCTTACC T GCCTTTGGAAAACCA ATGAT MORC2 (expression) 80 AGCCCTCCACTTCCGAATG CCGTAAACAATTCCG GAGGAT CENPM (expression) 81 CTGAAGGTCCACTTGGCAA AGT ACAAACACGATCAGG TCAATTCG PTTG1IP H4K20me1 positive region 1 60 ATCAGCAGGCACTCTTTTG G CAGGTTGGTACAGGG AGCAT PTTG1IP H4K20me1 positive region 2 60 CACACTGTCCACTCCACTC G TTTTGGCCCAGATTGA GTTC PTTG1IP H4K20me1 negative region 60 GGATGAGCAGCAGGAGCA CTTTTTGAGGCTGCGC ACT 128 Name T m (°C) Forward (5’-3’) Reverse (5’-3’) BRD1 H4K20me1 positive region 1 60 TACCTGGTCCTATCGGATG C AAGACTGTCCCCTCAG ACCA BRD1 H4K20me1 positive region 2 60 CTCCAGGTACCCACTCAAC C TAGGGCGACTCAGGA CTTTG BRD1 H4K20me1 negative region 60 CTGTGTGTGCTTCCCTAAC G CCGTTTAAGGCCCCTT TAGT pTK-luc2 ChIP 63 CTAGCAAAATAGGCTGTCC C TCGCTCGGTGTTCGAG GCCA PTTG1IP pTK- luc2 cloning 55 AAAAAACTCGAGGACACA CCGAATGGCAGTT AAAAAAGAATTCCCT GCCTCCAAGTGTGCTT A RUNX1 pTK-luc2 cloning 55 AAAAAACTCGAGGGGAAA GGTCTCAGTAAATGTCA AAAAAAGAATTCCAG GAAGACTCACCCCTTG A CECR5 pTK-luc2 cloning 65 AAAAAACTCGAGCCCATT CCCAACCTCCTACT AAAAAAGAATTCTCA TGTCTGGTGGTTTCCA A 21R pTK-luc2 cloning 65 AAAAAACTCGAGACACAC GCTCATCTCTCACC AAAAAAGAATTCGTG TCTGCATGGACAGAT GC 22R pTK-luc2 cloning 65 AAAAAACTCGAGCTCCAC TGTGTGTTGGCTCT AAAAAAGAATTCAGA CACACCAGAGCCTCG AA RUNX1 H4K20me1 negative pTK-luc2 cloning 55 AAAAAACTCGAGGCCTGT GCCTCTCTCATCTT AAAAAAGAATTCTCTT GCCTGTTTGGTCTGTG OligoJW102 63 GCGGTGACCCGGGAGATC TGAATTC OligoJW103 30 GAATTCAGATC Table 8: List of primers used for ChIP-PCR/cloning and gene expression Three independent biological replicates were performed for both monomethylated and trimethylated H4K20. This material was also ligated into the pGEM-T-Easy TA cloning vector (Promega), transformed into E. coli, and insert-positive clones were directly sequenced. 129 3.2.3 Peak detection and gene identification The .gff files generated by NimbleGen were used to determine sites of H4K20me1 and H4K20me3 enrichment using the freely available software, Model-based Analysis of 2- Color arrays (MA2C). Two channel data from the input and ChIP sample were loaded into the program. Default parameters were used to determine peaks with a P-value threshold of 0.005 or 0.001. The .gff files were converted to .bed files and, along with the peaks detected by MA2C, were visualized using the Hg18 version of the human genome on the UCSC genome browser. Gene annotation files were downloaded from the NimbleGen website and a panel of genes containing at least one statistically significant peak (p< 0.005) for H4K20me1 or H4K20me3 within the gene body, or within a 5 kb region 5′ upstream or a 500 bp region 3′ downstream of the gene, were identified. 3.2.4 Peak distribution analysis To characterize the average distribution of H4K20me1 and H4K20me3 peaks in the promoter and 3′ flanking region of modification positive genes, the cumulative number of peaks per nucleotide position was first determined. To analyze the peak distribution within the genes, each H4K20me-positive gene body was divided into 1/10,000th in order to normalize for differences in gene lengths; the number of peaks within each fraction was summed. The peak numbers were converted to tag density values by dividing the cumulative peak count for each nucleotide or gene body fraction by the total peak numbers from all H4K20me1 or H4K20me3-positive genes (y-axis). Tag density values were analyzed using a Poisson distribution with λ equal to the average tag density value 130 (gray line) in order to determine statistically significant (P < 0.05) regions of H4K20me1 or H4K20me3 enrichment (black bars). 3.2.5 Gene expression analysis Lipofectamine 2000 (Invitrogen) was used to transfect HeLa cells with pSUPERIOR.retro.puro vectors (OligoEngine) producing either a functional or non- functional shRNA for PR-Set7 (Houston et al., 2008). Positive cells were selected by adding 4 µg/ml of puromycin 24 h following transfection. A Qiagen RNeasy Kit was used to isolate total RNA from cells 6 days post-transfection when PR-Set7 depletion was observed by Western analysis. RNA was converted to cDNA using the ABI Reverse Transcription Kit according to the manufacturer's instructions. Quantitative real-time PCR was performed with SYBR Green (Bio-Rad) using a Bio-Rad I-Cycler. Expression was normalized to MTMR3 expression levels which were unaltered in all samples . Lipofectamine 2000 was also used to transfect HeLa cells with either the pQCXIP empty vector (Clontech) or the vector expressing a full-length PR-Set7 R265G point mutant (Houston et al., 2008). Following puromycin selection, total RNA was isolated for analysis 4 days post-transfection when depletion of monomethylated H4K20 was observed by Western analysis. Three independent biological replicates were performed to generate standard deviation. The Student's t-test was used to determine statistical significance. All primer sets and PCR conditions are detailed in Table 4 and were verified to be in the linear range of amplification. 131 3.2.6 PR-SET7 small molecule inhibitor, cell growth and cell-cycle characterization Six hours prior to drug treatment, 50,000 NIH293T cells were plated in the 6-well plates. Stock drugs are 10mM in DMSO concentration. The drug was diluted to equal volumes of the various concentrations in DMSO and added to media. The media on the 6-well plates was then replaced with new media containing the drug. After 72 hours, cells were harvested and whole cell lysates were made for western blot analysis using 2x Laemmli buffer and syringe homogenized after boiling for 5 minutes. For cell count analysis, 20 µl of harvest cells were stained with .5% final concentration of trypan blue. The cells were manually counted for trypan blue exclusion to tabulate viable cells for control and drug treated at Days 3, 4 and 5. For the cell-cycle analysis, the experiments were repeated in 10 cm format with 250,000 seeded cells. The cells were collected on Day 3, split and re-drugged for collection on Day 4 and Day 5. The harvested cells were washed with PBS and re-suspended in cold 70% ethanol to fix them. The fixed cells were then washed with PBS and stained with propidium iodide (PI) with the following recipe; 500 µl 1mg/ml PI, 100 20mg/ml RNAase A, 100 µl 10% Triton, 9.2 ml PBS. The stained cells were then analyzed in the CyAn Analyzer (Beckman Coulter). 3.3 Perl Code 3.3.1 Background subtraction #!/usr/local/bin/perl # Input bed file (Chr start stop windowCount): Output: Normalized count per 10000000 base #pairs in each window 132 $graph = $ARGV[0]; #$bg = $ARGV[1]; open (FILE, $graph); $i = 0; $count = 0; while($line1 = <FILE>){ @split = split('\t',$line1); $count = $count+$split[3]; $i = $i + 1; } #print "Count: $count"; $bg = $count/$i; close(FILE); #print "track type=wiggle_0 name=$name description=$name visibility=full color=0,0,255\n"; open (FILE, $graph); while ($line = <FILE>) { @split = split('\t',$line); #$split[3] = $split[3]-$bg; $normalized = ($split[3]*10000000)/$count; #print "$split[0]\t$split[1]\t$split[2]\t$normalized\n"; } 3.3.2 Comparing between multiple gene sets #input: gene sets[format: chr start stop geneName]. In order of input gene sets are labeled #0,1,10,100,1000 and combinations of overlapping are determined by adding the label values #!/usc/local/bin/perl $allgenefile = $ARGV[0]; $file1 = $ARGV[1]; $file2 = $ARGV[2]; $file3 = $ARGV[3]; $file4 = $ARGV[4]; $file5 = $ARGV[5]; $i = 1; $outfile = "output"; open (OUT,">$outfile"); open (GENE,$allgenefile); while ($geneline = <GENE>) { $totalval=0; chomp $geneline; @chops = split('\t',$geneline); $cmd = "grep -U $chops[3] $file1"; 133 if (system($cmd)==0) { $totalval = $totalval+1; } $cmd = "grep -U $chops[3] $file2"; if (system($cmd)==0) { $totalval = $totalval+10; } $cmd = "grep -U $chops[3] $file3"; if (system($cmd)==0) { $totalval =$totalval+100; } $cmd = "grep -U $chops[3] $file4"; if (system($cmd)==0) { $totalval = $totalval+1000; } print OUT "$chops[0]\t$chops[1]\t$chops[2]\t$chops[3]\t"; print OUT $totalval."\n"; } 3.3.3 Counting number of tags in a given window from a database //database is ordered by chromsome and start site and split into separate files for each chromsome in a folder. Peak file is also ordered by chromosome and start site and split into separate files in a folder. As input, Peak file folder path and Db folder path are specified// #!/usr/bin/perl @files = <$ARGV[0]/*>; foreach $file1(@files){ open(FILE1,$file1); @names = split('/',$file1); $index = scalar @names; $file2 = $ARGV[1].$names[$index-1]; #print "opening:".$file1." ".$file2."\n"; open(FILE2,$file2); 134 while($line1=<FILE1>){ chomp $line1; my @value1 = split('\t',$line1); $start1 = $value1[1]; $stop1 = $value1[2]; $count = 0; $bool = 0; $count = 0; do{ if((($start1<=$stop2)&&(($start1+($stop1-$start1))>=$start2))) { #print $line2."\n"; $line2 = <FILE2>; last unless defined $line2; my @value2 = split('\t',$line2); $start2 = $value2[1]; $stop2 = $value2[2]; $val = $value2[3]; $count = $count + $val; } else{ $line2 = <FILE2>; last unless defined $line2; my @value2 = split('\t',$line2); $start2 = $value2[1]; $stop2 = $value2[2]; $val = $value2[3]; } }while($stop2<=$stop1); print $line1."\t".$count."\n"; } } 3.3.4 Creating equivalent database files // In order to make a head to head comparison between two given chip-seq experiments, the chromosome start and end windows which contain read count must be the same between libraries. Given a Arg#1 window length Arg# 2 Chromosome length data base (chromosome 135 length), the program calculates a standardized library with the same window start and end locations// #!/usr/local/bin/perl #print "in program"; $window = $ARGV[0]; $chrlen = $ARGV[1]; $range =$window; #%len = (); #%count = (); #@split1; #print $chrlen; $previous = "0"; open(LEN, $chrlen) or die "what the"; while ($line = <LEN>) { #print "in ".$line."\n"; @split = split(' ',$line); $len{$split[0]} = $split[1]; $filename = $split[0].".bed"; $window = $range; open(FILE,$filename); $wigline = <FILE>; while($window < $split[1]){ chomp $wigline; @wigsplit = split('\t',$wigline); $wigcomp = $wigsplit[0].$wigsplit[1].$wigsplit[2]; $chr = $split[0]; $start = $window-$range; $end = $window-1; $comp = $chr.$start.$end; #print $wigcomp."\t".$wigcomp."\n"; #print "comparing".$comp." ".$wigcomp." ".$window."\n"; if($comp eq $wigcomp){ print $wigline."\n"; $window = $window + $range; $wigline = <FILE>; } else{ $out = $chr."\t".$start."\t".$end."\t"."0\n"; 136 print $out; $window = $window +$range; } } } 3.3.5 Graph Meta-plot from ChIP-Chip Peak data //Program accepts peak file(chromosome start end) and genefile (sample format: chr1 RNG Genes 58953 59871 +1 + . ID=3;Accession=NM_001005484;Name=OR4F5;color=9400D3;url=http://genome.ucs c.edu/cgi-bin/hgTracks?&clade=vertebrate&org=Human&db=hg18&position=chr1:58953- 59871&pix620&Submit=submit;) Outputs a meta plot with 5KB up and 5KB downstream of gene body; genebody is divided into 10,000 fractions; each value represents 200bp // #!/usr/local/bin/perl use POSIX qw(ceil floor); $peakfile =$ARGV[0]; $genefile = $ARGV[1]; $off_index; $gene_index; @up; @ds; @body; sub match{ my ($chr,$start,$end,$genechr,$genestart, $geneend) = (@_[0],@_[1],@_[2],@_[3],@_[4],@_[5]); $boolean = x; if (($chr eq $genechr)&&($start<=$geneend)&&(($start+($end-$start))>=($genestart))) { $boolean =1; return $boolean; } else { $boolean = 0; return $boolean; } } sub smooth{ my (@array) = @_; 137 $inarraysize = @array; $forloop = $inarraysize - 200; my $array_index; my @outarray; for($j = 0;$j<$forloop;$j++) { my $total=0; my $offset = $j; for($k = 0;$k<200;$k++) { $total = $total+$array[$offset]; $offset++; } $avg = $total/200; $outarray[$j] = $avg; } return @outarray; } $peaknumber = 0; open(PEAKFILE,$peakfile); while ($line1=<PEAKFILE>) { next if ($line1 =~/^#/); if ($line1 =~/^chr/) { chomp $line1; my @value1 = split ('\t',$line1); $chr1 = $value1[0]; $start1 = $value1[1]; $stop1 = $value1[2]; open(GENEFILE,$genefile); while($line2 = <GENEFILE>) { next if ($line1 =~/^#/); if($line2 =~ /^chr/) { chomp $line2; my @value2 = split ('\t',$line2); $genechr1 = $value2[0]; $genestart1 = $value2[3]; $genestop1 = $value2[4]; $genedir = $value2[6]; #print "test $genedir"; 138 $result = &match($chr1,$start1,$stop1,$genechr1,($genestart1- 5000),($genestop1+5000)); if($result==1) { $peaknumber++; $off_index = 0; $gene_index = 0; for($i = 1;$i<=5000;$i++) { $up_change = $i-5001; if($genedir eq '+') { $my_start = $genestart1+$up_change; $my_stop = $genestop1 + $i; } if($genedir eq '-') { $my_start = $genestop1+5000-$i; $my_stop = $genestart1- $i; } $up[$off_index] = $up[$off_index]+ &match($chr1,$start1,$stop1,$genechr1,$my_start,$my_start); $ds[$off_index] = $ds[$off_index]+ &match($chr1,$start1,$stop1,$genechr1,$my_stop,$my_stop); $off_index++; } for($i = 0;$i<=10000;$i++) { $gene_length = $genestop1-$genestart1; $gene_change = .0001*$i*$gene_length; $gene_point = $genestart1+$gene_change; $gene_point_minus = $genestop1-$gene_change; if($genedir eq '+'){ $body[$gene_index] = $body[$gene_index]+&match($chr1,$start1,$stop1,$genechr1,$gene_point,$gene_point); $gene_index++; } if($genedir eq '-'){ $body[$gene_index] = $body[$gene_index]+&match($chr1,$start1,$stop1,$genechr1,$gene_point_minus,$gene_point_ minus); $gene_index++; } } 139 } } } } } @all; $alloff = 0; foreach (@up) { $all[$alloff] = $_; $alloff++; } foreach (@body) { $all[$alloff] = $_; $alloff++; } foreach (@ds) { $all[$alloff] = $_; $alloff++; } $size = @all; @out = &smooth(@all); $size1 = @out; open (EXCEL,">$peakfile.xls"); open (SMOOTHEXCEL,">$peakfile.smooth.xls"); foreach(@all) { print EXCEL "$_\n"; } foreach(@out) { print SMOOTHEXCEL "$_\n"; } $tot=0; foreach(@all) { $tot = $tot +$_; } print $tot."\n"; $avgwindow = ($size/20000); print "avg window $size = $avgwindow\n"; 140 3.3.6 Plot meta-analysis of ChIP-seq count/ChIP-chip intensity data //Program is executed in the folder with database ordered and split into individual chromosome files. Program accepts accepts gene file as 3.5.5 and outputs meta plot 5Kb up stream and 25Kb down stream of transcription start site// #!/usr/local/bin/perl $genefile = $ARGV[0]; $test = "chr1 522200 522399 1"; @array = (); %peakhash; $chr = "chr"; @names = ("1","2","3","4","5","6","7","8","9","10","11","12","13","14","15","16","17","18","19","20","21 ","22","M","X","Y"); for($i=0;$i<25;$i++) { $counter = 1; $file = $chr."$names[$i].bed"; $chrhash=$chr."$names[$i]"; #print $file; %$chrhash = (); $hash = \%$chrhash; open(FILE,$file); while ($line = <FILE>) { chomp $line; $hash->{$counter} = $line; # print $hash->{$line}; $counter++; } } while (($key, $value) = each %chrM) { #print $value." $key\n"; } open(GENES,$genefile) or die "No GENE file in argument 2"; #print $geneline = <GENES>; while($geneline = <GENES>){ chomp $geneline; my @genevalue = split(' ',$geneline); $genechr = $genevalue[0]; $second = $genevalue[1]; $type = $genevalue[2]; $genestart = $genevalue[3]; 141 $geneend = $genevalue[4]; $geneframe = $genevalue[5]; $genestrand = $genevalue[6]; $genedot = $genevalue[7]; $genename = $genevalue[8]; #println $chr." ".$genename; if($genestrand eq '+') { $point = $genestart; } else { $point = $geneend; } $ref = \%$genechr; foreach (keys(%$genechr)){ $key = $_; @split =split('\t', $ref->{$key}); #print "$ref->{$key}\n"; #print "start $split[1] $split[2]\n"; #print $point; if(($point>=($split[1]-100))&&($point<=($split[2]+100))) { if($genestrand eq '+'){ $index = $key-26; for ($i = 0; $i<125; $i++){ @temp = split('\t',$ref->{$index}); $array[$i]=$array[$i]+$temp[3]; $index ++; } } else{ $index = $key+26; for ($i = 0; $i<125; $i++){ @temp = split('\t',$ref->{$index}); $array[$i]=$array[$i]+$temp[3]; $index = $index-1; } } } } 142 } foreach (@array){ print "$_\n"; } 3.3.7 Peak file annotation to gene //Program maps input peak file(arg 1) to genes(arg 2; format as 3.5.5) given an offset upstream and downstream of gene body (arg 3 and arg 4, respectively. Output format is : chr start stop name geneLength peakDensity peakID// #!/usr/local/bin/perl $ma2cfile = $ARGV[0]; $genefile = $ARGV[1]; $up_off = $ARGV[2]; $down_off = $ARGV[3]; %peaksingene =(); %genetest=(); %genecount = (); open(MA2CFILE, $ma2cfile) or die "No MA2C file in argument 1"; #print $line= <MA2CFILE>; %peaklength = (); while($line = <MA2CFILE>){ chomp $line; my @value = split(' ',$line); $chr = $value[0]; $start = $value[1]; $stop = $value[2]; $peakid = $value[3]; $score = $value[4]; open(GENES,$genefile) or die "No GENE file in argument 2"; #print $geneline = <GENES>; while($geneline = <GENES>){ chomp $geneline; my @genevalue = split(' ',$geneline); $genechr = $genevalue[0]; $second = $genevalue[1]; $type = $genevalue[2]; $genestart = $genevalue[3]; $geneend = $genevalue[4]; $geneframe = $genevalue[5]; $genestrand = $genevalue[6]; $genedot = $genevalue[7]; $genename = $genevalue[8]; #println $chr." ".$genename; if($genestrand eq '+') { 143 $upoff = $up_off; $downoff =$down_off; } else { $upoff =$down_off; $downoff = $up_off; } #print "$upoff $downoff\n"; $genelength = ($geneend-$genestart)+($up_off+$down_off); $print_genelength = $geneend-$genestart; $peaklength = $stop-$start; if(($chr eq $genechr)&&($start<=($geneend+$downoff))&&(($start+($stop- $start))>=($genestart-$upoff))) { $peaksingene{$line}=$line; $effective_genestart = $genestart-$upoff; $effective_geneend = $geneend+$downoff; my @mygeneinfo = split(';',$genename); my @name = split('=', $mygeneinfo[2]); $index = $name[1]."\t".$genelength; $genecount{$index} = $genecount{$index}+$peaklength; $density = $genecount{$index}/$genelength; $peaklength{$index}=$peaklength{$index}+($stop-$start); $density = $peaklength{$index}/($downoff+$upoff+$print_genelength); $genetest{$index}=$genetest{$index} +1; $genedensity{$index}= "$chr\t$genestart\t$geneend\t$name[1]\t$print_genelength\t$density\t$peakid\t$genetest{$index} "; } } } close(MA2CFILE); $hashcount = scalar keys %genecount; @out = values %genedensity; foreach(@out){ print "$_\n"; } $peakingeneout=$ma2cfile.".peaksingene"; open(OUT,">$peakingeneout"); @peakout = values %peaksingene; foreach (@peakout){ print OUT "$_\n"; } system( "echo Non-redundant gene count in $ma2cfile is: $hashcount\n"); 144 3.3.8 Splitting bed file to chromosome files Input 1: bed file of tag count data (format: Chr start end tagcount) Output: Individual file for each chromosome of input file #!/usr/local/bin/perl $bedfile = $ARGV[0]; %chroms; open (BED,$bedfile) or die "No BED File"; while ($line = <BED>){ @cols = split('\t',$line); chomp $line; if (exists $chroms{$cols[0]}){ open (OUT, ">>$chroms{$cols[0]}.bed"); print OUT $line."\n"; } else { $name = $cols[0].".bed"; open (OUT, ">$name"); print OUT $line."\n"; $chroms{$cols[0]}=$cols[0]; } close (OUT); 145 Bibliography Andrews, P. W., Banting, G., Damjanov, I., Arnaud, D., & Avner, P. (1984). Three Monoclonal Antibodies Defining Distinct Differentiation Antigens Associated with Different High Molecular Weight Polypeptides on the Surface of Human Embryonal Carcinoma Cells. Hybridoma, 3(4), 347–361. doi:10.1089/hyb.1984.3.347 Abbas, T., Shibata, E., Park, J., Jha, S., Karnani, N., & Dutta, A. (2010). CRL4(Cdt2) regulates cell proliferation and histone gene expression by targeting PR-Set7/Set8 for degradation. Molecular cell, 40(1), 9–21. doi:10.1016/j.molcel.2010.09.014 Adewumi, O., Aflatoonian, B., Ahrlund-Richter, L., Amit, M., Andrews, P. W., Beighton, G., Bello, P. A., et al. (2007). Characterization of human embryonic stem cell lines by the International Stem Cell Initiative. Nature biotechnology, 25(7), 803–16. doi:10.1038/nbt1318 Akopian, V., Andrews, P. W., Beil, S., Benvenisty, N., Brehm, J., Christie, M., Ford, A., et al. (2010). Comparison of defined culture systems for feeder cell free propagation of human embryonic stem cells. In vitro cellular & developmental biology. Animal, 46(3-4), 247–58. doi:10.1007/s11626-010-9297-z Amit, M., Shariki, C., Margulets, V., & Itskovitz-Eldor, J. (2004). Feeder layer- and serum-free culture of human embryonic stem cells. Biology of reproduction, 70(3), 837–45. doi:10.1095/biolreprod.103.021147 Azuara, V., Perry, P., Sauer, S., Spivakov, M., Jørgensen, H. F., John, R. M., Gouti, M., et al. (2006). Chromatin signatures of pluripotent cell lines. Nature cell biology, 8(5), 532–8. doi:10.1038/ncb1403 Bannister, A. J., Schneider, R., Myers, F. A., Thorne, A. W., Crane-Robinson, C., & Kouzarides, T. (2005). Spatial distribution of di- and tri-methyl lysine 36 of histone H3 at active genes. The Journal of biological chemistry, 280(18), 17732–6. doi:10.1074/jbc.M500796200 Barski, A., Cuddapah, S., Cui, K., Roh, T.-Y., Schones, D. E., Wang, Z., Wei, G., et al. (2007). High-resolution profiling of histone methylations in the human genome. Cell, 129(4), 823–37. doi:10.1016/j.cell.2007.05.009 Beck, B., & Blanpain, C. (2012). Mechanisms regulating epidermal stem cells. The EMBO journal, 31(9), 2067–75. doi:10.1038/emboj.2012.67 146 Beck, D. B., Oda, H., Shen, S. S., & Reinberg, D. (2012). PR-Set7 and H4K20me1: at the crossroads of genome integrity, cell cycle, chromosome condensation, and transcription. Genes & development, 26(4), 325–37. doi:10.1101/gad.177444.111 Bernstein, B. E., Mikkelsen, T. S., Xie, X., Kamal, M., Huebert, D. J., Cuff, J., Fry, B., et al. (2006). A bivalent chromatin structure marks key developmental genes in embryonic stem cells. Cell, 125(2), 315–26. doi:10.1016/j.cell.2006.02.041 Bird, A. (2002). DNA methylation patterns and epigenetic memory. Genes & development, 16(1), 6–21. doi:10.1101/gad.947102 Bolstad, B. M., Irizarry, R. ., Astrand, M., & Speed, T. P. (2003). A comparison of normalization methods for high density oligonucleotide array data based on variance and bias. Bioinformatics, 19(2), 185–193. doi:10.1093/bioinformatics/19.2.185 Boyer, L. A., Lee, T. I., Cole, M. F., Johnstone, S. E., Levine, S. S., Zucker, J. P., Guenther, M. G., et al. (2005a). Core transcriptional regulatory circuitry in human embryonic stem cells. Cell, 122(6), 947–56. doi:10.1016/j.cell.2005.08.020 Boyer, L. A., Lee, T. I., Cole, M. F., Johnstone, S. E., Levine, S. S., Zucker, J. P., Guenther, M. G., et al. (2005b). Core transcriptional regulatory circuitry in human embryonic stem cells. Cell, 122(6), 947–56. doi:10.1016/j.cell.2005.08.020 Brown, C. J., Ballabio, A., Rupert, J. L., Lafreniere, R. G., Grompe, M., Tonlorenzi, R., & Willard, H. F. (1991). A gene from the region of the human X inactivation centre is expressed exclusively from the inactive X chromosome. Nature, 349(6304), 38– 44. doi:10.1038/349038a0 Cao, R., Wang, L., Wang, H., Xia, L., Erdjument-Bromage, H., Tempst, P., Jones, R. S., et al. (2002). Role of histone H3 lysine 27 methylation in Polycomb-group silencing. Science (New York, N.Y.), 298(5595), 1039–43. doi:10.1126/science.1076997 Centore, R. C., Havens, C. G., Manning, A. L., Li, J.-M., Flynn, R. L., Tse, A., Jin, J., et al. (2010). CRL4(Cdt2)-mediated destruction of the histone methyltransferase Set8 prevents premature chromatin compaction in S phase. Molecular cell, 40(1), 22–33. doi:10.1016/j.molcel.2010.09.015 Chadwick, B. P., & Willard, H. F. (2004). Multiple spatially distinct types of facultative heterochromatin on the human inactive X chromosome. Proceedings of the National Academy of Sciences of the United States of America, 101(50), 17450–5. doi:10.1073/pnas.0408021101 147 Chambers, I., Colby, D., Robertson, M., Nichols, J., Lee, S., Tweedie, S., & Smith, A. (2003). Functional Expression Cloning of Nanog, a Pluripotency Sustaining Factor in Embryonic Stem Cells. Cell, 113(5), 643–655. doi:10.1016/S0092- 8674(03)00392-1 Congdon, L. M., Houston, S. I., Veerappan, C. S., Spektor, T. M., & Rice, J. C. (2010). PR-Set7-mediated monomethylation of histone H4 lysine 20 at specific genomic regions induces transcriptional repression. Journal of cellular biochemistry, 110(3), 609–19. doi:10.1002/jcb.22570 Daniels, R., Zuccotti, M., Kinis, T., Serhal, P., & Monk, M. (1997). XIST expression in human oocytes and preimplantation embryos. American journal of human genetics, 61(1), 33–9. doi:10.1086/513892 de Hoon, M. J. L., Imoto, S., Nolan, J., & Miyano, S. (2004). Open source clustering software. Bioinformatics (Oxford, England), 20(9), 1453–4. doi:10.1093/bioinformatics/bth078 DeLange, R. J., Fambrough, D. M., Smith, E. L., & Bonner, J. (1969). Calf and Pea Histone IV. II. THE COMPLETE AMINO ACID SEQUENCE OF CALF THYMUS HISTONE IV; PRESENCE OF egr-N-ACETYLLYSINE. J. Biol. Chem., 244(2), 319–334. Retrieved from http://www.jbc.org/content/244/2/319.abstract?ijkey=790a2047d679e08cbdfb79cf64 c8ea5ef84ae759&keytype2=tf_ipsecsha Dvash, T., Lavon, N., & Fan, G. (2010). Variations of X chromosome inactivation occur in early passages of female human embryonic stem cells. (G. J. C. Veenstra, Ed.)PloS one, 5(6), e11330. doi:10.1371/journal.pone.0011330 Epstein, C. J., Smith, S., Travis, B., & Tucker, G. (1978). Both X chromosomes function before visible X-chromosome inactivation in female mouse embryos. Nature, 274(5670), 500–503. doi:10.1038/274500a0 Ernst, J., Kheradpour, P., Mikkelsen, T. S., Shoresh, N., Ward, L. D., Epstein, C. B., Zhang, X., et al. (2011). Mapping and analysis of chromatin state dynamics in nine human cell types. Nature, 473(7345), 43–9. doi:10.1038/nature09906 Frietze, S., Wang, R., Yao, L., Tak, Y., Ye, Z., Gaddis, M., Witt, H., et al. (2012). Cell type-specific binding patterns reveal that TCF7L2 can be tethered to the genome by association with GATA3. Genome Biology, 13(9), R52. doi:10.1186/gb-2012-13-9- r52 148 Guenther, M. G., Levine, S. S., Boyer, L. A., Jaenisch, R., & Young, R. A. (2007). A chromatin landmark and transcription initiation at most promoters in human cells. Cell, 130(1), 77–88. doi:10.1016/j.cell.2007.05.042 Heard, E. (2005). Delving into the diversity of facultative heterochromatin: the epigenetics of the inactive X chromosome. Current opinion in genetics & development, 15(5), 482–9. doi:10.1016/j.gde.2005.08.009 Heard, E., Rougeulle, C., Arnaud, D., Avner, P., Allis, C. D., & Spector, D. L. (2001). Methylation of Histone H3 at Lys-9 Is an Early Mark on the X Chromosome during X Inactivation. Cell, 107(6), 727–738. doi:10.1016/S0092-8674(01)00598-0 Heinz, S., Benner, C., Spann, N., Bertolino, E., Lin, Y. C., Laslo, P., Cheng, J. X., et al. (2010a). Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities. Molecular cell, 38(4), 576–89. doi:10.1016/j.molcel.2010.05.004 Heinz, S., Benner, C., Spann, N., Bertolino, E., Lin, Y. C., Laslo, P., Cheng, J. X., et al. (2010b). Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities. Molecular cell, 38(4), 576–89. doi:10.1016/j.molcel.2010.05.004 Hoffman, L. M., & Carpenter, M. K. (2005). Characterization and culture of human embryonic stem cells. Nature biotechnology, 23(6), 699–708. doi:10.1038/nbt1102 Hough, S. R., Laslett, A. L., Grimmond, S. B., Kolle, G., & Pera, M. F. (2009). A continuum of cell states spans pluripotency and lineage commitment in human embryonic stem cells. (T. A. Reh, Ed.)PloS one, 4(11), e7708. doi:10.1371/journal.pone.0007708 Houston, S. I., McManus, K. J., Adams, M. M., Sims, J. K., Carpenter, P. B., Hendzel, M. J., & Rice, J. C. (2008). Catalytic function of the PR-Set7 histone H4 lysine 20 monomethyltransferase is essential for mitotic entry and genomic stability. The Journal of biological chemistry, 283(28), 19478–88. doi:10.1074/jbc.M710579200 Jørgensen, S., Elvers, I., Trelle, M. B., Menzel, T., Eskildsen, M., Jensen, O. N., Helleday, T., et al. (2007). The histone methyltransferase SET8 is required for S- phase progression. The Journal of cell biology, 179(7), 1337–45. doi:10.1083/jcb.200706150 Karachentsev, D., Sarma, K., Reinberg, D., & Steward, R. (2005). PR-Set7-dependent methylation of histone H4 Lys 20 functions in repression of gene expression and is essential for mitosis. Genes & development, 19(4), 431–5. doi:10.1101/gad.1263005 149 Kashyap, V., Rezende, N. C., Scotland, K. B., Shaffer, S. M., Persson, J. L., Gudas, L. J., & Mongan, N. P. (2009). Regulation of stem cell pluripotency and differentiation involves a mutual regulatory circuit of the NANOG, OCT4, and SOX2 pluripotency transcription factors with polycomb repressive complexes and stem cell microRNAs. Stem cells and development, 18(7), 1093–108. doi:10.1089/scd.2009.0113 Kolle, G., Ho, M., Zhou, Q., Chy, H. S., Krishnan, K., Cloonan, N., Bertoncello, I., et al. (2009). Identification of human embryonic stem cell surface markers by combined membrane-polysome translation state array analysis and immunotranscriptional profiling. Stem cells (Dayton, Ohio), 27(10), 2446–56. doi:10.1002/stem.182 Kouzarides, T. (2007). Chromatin modifications and their function. Cell, 128(4), 693– 705. doi:10.1016/j.cell.2007.02.005 Krietsch, W. K., Fehlau, M., Renner, P., Bücher, T., & Fundele, R. (1986). Expression of X-linked phosphoglycerate kinase in early mouse embryos homozygous at the Xce locus. Differentiation; research in biological diversity, 31(1), 50–4. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/3732658 Kurdistani, S. K., Tavazoie, S., & Grunstein, M. (2004). Mapping global histone acetylation patterns to gene expression. Cell, 117(6), 721–33. doi:10.1016/j.cell.2004.05.023 Lyon, M. F. (1961). Gene Action in the X-chromosome of the Mouse (Mus musculus L.). Nature, 190(4773), 372–373. doi:10.1038/190372a0 Langmead, B., Trapnell, C., Pop, M., & Salzberg, S. L. (2009). Ultrafast and memory- efficient alignment of short DNA sequences to the human genome. Genome biology, 10(3), R25. doi:10.1186/gb-2009-10-3-r25 Laslett, A. L., Grimmond, S., Gardiner, B., Stamp, L., Lin, A., Hawes, S. M., Wormald, S., et al. (2007). Transcriptional analysis of early lineage commitment in human embryonic stem cells. BMC developmental biology, 7(1), 12. doi:10.1186/1471- 213X-7-12 Lee, J., Kim, H. K., Han, Y.-M., & Kim, J. (2008). Pyruvate kinase isozyme type M2 (PKM2) interacts and cooperates with Oct-4 in regulating transcription. The international journal of biochemistry & cell biology, 40(5), 1043–54. doi:10.1016/j.biocel.2007.11.009 Li, Y., Powell, S., Brunette, E., Lebkowski, J., & Mandalam, R. (2005). Expansion of human embryonic stem cells in defined serum-free medium devoid of animal- derived products. Biotechnology and bioengineering, 91(6), 688–98. doi:10.1002/bit.20536 150 Li, Z., Nie, F., Wang, S., & Li, L. (2011). Histone H4 Lys 20 monomethylation by histone methylase SET8 mediates Wnt target gene activation. Proceedings of the National Academy of Sciences of the United States of America, 108(8), 3116–23. doi:10.1073/pnas.1009353108 Liu, W., Tanasa, B., Tyurina, O. V., Zhou, T. Y., Gassmann, R., Liu, W. T., Ohgi, K. A., et al. (2010). PHF8 mediates histone H4 lysine 20 demethylation events involved in cell cycle progression. Nature, 466(7305), 508–12. doi:10.1038/nature09272 Loh, Y.-H., Zhang, W., Chen, X., George, J., & Ng, H.-H. (2007). Jmjd1a and Jmjd2c histone H3 Lys 9 demethylases regulate self-renewal in embryonic stem cells. Genes & development, 21(20), 2545–57. doi:10.1101/gad.1588207 Lu, J., Hou, R., Booth, C. J., Yang, S.-H., & Snyder, M. (2006). Defined culture conditions of human embryonic stem cells. Proceedings of the National Academy of Sciences of the United States of America, 103(15), 5688–93. doi:10.1073/pnas.0601383103 Ludwig, T. E., Bergendahl, V., Levenstein, M. E., Yu, J., Probasco, M. D., & Thomson, J. A. (2006). Feeder-independent culture of human embryonic stem cells. Nature methods, 3(8), 637–46. doi:10.1038/nmeth902 Luger, K. (2003). Structure and dynamic behavior of nucleosomes. Current Opinion in Genetics & Development, 13(2), 127–135. doi:10.1016/S0959-437X(03)00026-1 Manenti, G., Trincucci, G., Pettinicchio, A., Amendola, E., Scarfò, M., & Dragani, T. A. (2008). Cis-acting genomic elements of the Pas1 locus control Kras mutability in lung tumors. Oncogene, 27(43), 5753–8. doi:10.1038/onc.2008.194 Margueron, R., & Reinberg, D. (2010). Chromatin structure and the inheritance of epigenetic information. Nature reviews. Genetics, 11(4), 285–96. doi:10.1038/nrg2752 Martin, G. R. (1981). Isolation of a Pluripotent Cell Line from Early Mouse Embryos Cultured in Medium Conditioned by Teratocarcinoma Stem Cells. Proceedings of the National Academy of Sciences, 78(12), 7634–7638. doi:10.1073/pnas.78.12.7634 Mills, A. A. (2010). Throwing the cancer switch: reciprocal roles of polycomb and trithorax proteins. Nature reviews. Cancer, 10(10), 669–82. doi:10.1038/nrc2931 151 Milne, T. A., Dou, Y., Martin, M. E., Brock, H. W., Roeder, R. G., & Hess, J. L. (2005). MLL associates specifically with a subset of transcriptionally active target genes. Proceedings of the National Academy of Sciences of the United States of America, 102(41), 14765–70. doi:10.1073/pnas.0503630102 Monk, M., & Harper, M. I. (1979). Sequential X chromosome inactivation coupled with cellular differentiation in early mouse embryos. Nature, 281(5729), 311–313. doi:10.1038/281311a0 Narsinh, K. H., Sun, N., Sanchez-Freire, V., Lee, A. S., Almeida, P., Hu, S., Jan, T., et al. (2011). Single cell transcriptional profiling reveals heterogeneity of human induced pluripotent stem cells. The Journal of clinical investigation, 121(3), 1217–21. doi:10.1172/JCI44635 Nesterova, T. B., Senner, C. E., Schneider, J., Alcayna-Stevens, T., Tattermusch, A., Hemberger, M., & Brockdorff, N. (2011). Pluripotency factor binding and Tsix expression act synergistically to repress Xist in undifferentiated embryonic stem cells. Epigenetics & chromatin, 4(1), 17. doi:10.1186/1756-8935-4-17 Nichols, J., Zevnik, B., Anastassiadis, K., Niwa, H., Klewe-Nebenius, D., Chambers, I., Schöler, H., et al. (1998). Formation of Pluripotent Stem Cells in the Mammalian Embryo Depends on the POU Transcription Factor Oct4. Cell, 95(3), 379–391. doi:10.1016/S0092-8674(00)81769-9 Nicol, J. W., Helt, G. A., Blanchard, S. G., Raja, A., & Loraine, A. E. (2009). The Integrated Genome Browser: free software for distribution and exploration of genome-scale datasets. Bioinformatics (Oxford, England), 25(20), 2730–1. doi:10.1093/bioinformatics/btp472 Nishioka, K., Rice, J. C., Sarma, K., Erdjument-Bromage, H., Werner, J., Wang, Y., Chuikov, S., et al. (2002). PR-Set7 is a nucleosome-specific methyltransferase that modifies lysine 20 of histone H4 and is associated with silent chromatin. Molecular cell, 9(6), 1201–13. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/12086618 Nusslein-Volhard, C., Kluding, H., & Jurgens, G. (1985). Genes Affecting the Segmental Subdivision of the Drosophila Embryo. Cold Spring Harbor Symposia on Quantitative Biology, 50(0), 145–154. doi:10.1101/SQB.1985.050.01.020 Oda, H., Okamoto, I., Murphy, N., Chu, J., Price, S. M., Shen, M. M., Torres-Padilla, M. E., et al. (2009). Monomethylation of histone H4-lysine 20 is involved in chromosome structure and stability and is essential for mouse development. Molecular and cellular biology, 29(8), 2278–95. doi:10.1128/MCB.01768-08 152 Oh, S. (2012). Stem Cells and Cancer Stem Cells,Volume 3. (M. A. Hayat, Ed.)Stem Cells, 3. doi:10.1007/978-94-007-2415-0 Okamoto, I., Patrat, C., Thépot, D., Peynot, N., Fauque, P., Daniel, N., Diabangouaya, P., et al. (2011). Eutherian mammals use diverse strategies to initiate X-chromosome inactivation during development. Nature, 472(7343), 370–4. doi:10.1038/nature09872 Pan, G., & Thomson, J. A. (2007). Nanog and transcriptional networks in embryonic stem cell pluripotency. Cell research, 17(1), 42–9. doi:10.1038/sj.cr.7310125 Pan, G., Tian, S., Nie, J., Yang, C., Ruotti, V., Wei, H., Jonsdottir, G. A., et al. (2007). Whole-genome analysis of histone H3 lysine 4 and lysine 27 methylation in human embryonic stem cells. Cell stem cell, 1(3), 299–312. doi:10.1016/j.stem.2007.08.003 Pannetier, M., Julien, E., Schotta, G., Tardat, M., Sardet, C., Jenuwein, T., & Feil, R. (2008). PR-SET7 and SUV4-20H regulate H4 lysine-20 methylation at imprinting control regions in the mouse. EMBO reports, 9(10), 998–1005. doi:10.1038/embor.2008.147 Penny, G. D., Kay, G. F., Sheardown, S. A., Rastan, S., & Brockdorff, N. (1996). Requirement for Xist in X chromosome inactivation. Nature, 379(6561), 131–7. doi:10.1038/379131a0 Plath, K., Fang, J., Mlynarczyk-Evans, S. K., Cao, R., Worringer, K. A., Wang, H., de la Cruz, C. C., et al. (2003). Role of histone H3 lysine 27 methylation in X inactivation. Science (New York, N.Y.), 300(5616), 131–5. doi:10.1126/science.1084274 Pomp, O., Dreesen, O., Leong, D. F. M., Meller-Pomp, O., Tan, T. T., Zhou, F., & Colman, A. (2011). Unexpected X chromosome skewing during culture and reprogramming of human somatic cells can be alleviated by exogenous telomerase. Cell stem cell, 9(2), 156–65. doi:10.1016/j.stem.2011.06.004 Rae, J. M., Johnson, M. D., Scheys, J. O., Cordero, K. E., Larios, J. M., & Lippman, M. E. (2005). GREB 1 is a critical regulator of hormone dependent breast cancer growth. Breast cancer research and treatment, 92(2), 141–9. doi:10.1007/s10549- 005-1483-4 Ramirez, J.-M., Gerbal-Chaloin, S., Milhavet, O., Qiang, B., Becker, F., Assou, S., Lemaître, J.-M., et al. (2011). Brief report: benchmarking human pluripotent stem cell markers during differentiation into the three germ layers unveils a striking heterogeneity: all markers are not equal. Stem cells (Dayton, Ohio), 29(9), 1469–74. doi:10.1002/stem.681 153 Rho, J.-Y., Yu, K., Han, J.-S., Chae, J.-I., Koo, D.-B., Yoon, H.-S., Moon, S.-Y., et al. (2006). Transcriptional profiling of the developmentally important signalling pathways in human embryonic stem cells. Human reproduction (Oxford, England), 21(2), 405–12. doi:10.1093/humrep/dei328 Rice, J. C., Nishioka, K., Sarma, K., Steward, R., Reinberg, D., & Allis, C. D. (2002). Mitotic-specific methylation of histone H4 Lys 20 follows increased PR-Set7 expression and its localization to mitotic chromosomes. Genes & development, 16(17), 2225–30. doi:10.1101/gad.1014902 Saitoh, T., Mine, T., & Katoh, M. (2002). Expression and regulation of WNT8A and WNT8B mRNAs in human tumor cell lines: up-regulation of WNT8B mRNA by beta-estradiol in MCF-7 cells, and down-regulation of WNT8A and WNT8B mRNAs by retinoic acid in NT2 cells. International journal of oncology, 20(5), 999–1003. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/11956596 Sakaguchi, A., Karachentsev, D., Seth-Pasricha, M., Druzhinina, M., & Steward, R. (2008). Functional characterization of the Drosophila Hmt4-20/Suv4-20 histone methyltransferase. Genetics, 179(1), 317–22. doi:10.1534/genetics.108.087650 Sakaguchi, A., & Steward, R. (2007). Aberrant monomethylation of histone H4 lysine 20 activates the DNA damage checkpoint in Drosophila melanogaster. The Journal of cell biology, 176(2), 155–62. doi:10.1083/jcb.200607178 Saldanha, A. J. (2004). Java Treeview--extensible visualization of microarray data. Bioinformatics (Oxford, England), 20(17), 3246–8. doi:10.1093/bioinformatics/bth349 Sasaki, N., Okishio, K., Ui-Tei, K., Saigo, K., Kinoshita-Toyoda, A., Toyoda, H., Nishimura, T., et al. (2008). Heparan sulfate regulates self-renewal and pluripotency of embryonic stem cells. The Journal of biological chemistry, 283(6), 3594–606. doi:10.1074/jbc.M705621200 Schotta, G., Lachner, M., Sarma, K., Ebert, A., Sengupta, R., Reuter, G., Reinberg, D., et al. (2004). A silencing pathway to induce H3-K9 and H4-K20 trimethylation at constitutive heterochromatin. Genes & development, 18(11), 1251–62. doi:10.1101/gad.300704 Silva, J., Mak, W., Zvetkova, I., Appanah, R., Nesterova, T. B., Webster, Z., Peters, A. H. F. ., et al. (2003). Establishment of Histone H3 Methylation on the Inactive X Chromosome Requires Transient Recruitment of Eed-Enx1 Polycomb Group Complexes. Developmental Cell, 4(4), 481–495. doi:10.1016/S1534- 5807(03)00068-6 154 Silva, S. S., Rowntree, R. K., Mekhoubad, S., & Lee, J. T. (2008). X-chromosome inactivation and epigenetic fluidity in human embryonic stem cells. Proceedings of the National Academy of Sciences of the United States of America, 105(12), 4820–5. doi:10.1073/pnas.0712136105 Simon, J. A., & Kingston, R. E. (2009). Mechanisms of polycomb gene silencing: knowns and unknowns. Nature reviews. Molecular cell biology, 10(10), 697–708. doi:10.1038/nrm2763 Singh, A. M., Hamazaki, T., Hankowski, K. E., & Terada, N. (2007). A heterogeneous expression pattern for Nanog in embryonic stem cells. Stem cells (Dayton, Ohio), 25(10), 2534–42. doi:10.1634/stemcells.2007-0126 Song, J. S., Johnson, W. E., Zhu, X., Zhang, X., Li, W., Manrai, A. K., Liu, J. S., et al. (2007). Model-based analysis of two-color arrays (MA2C). Genome biology, 8(8), R178. doi:10.1186/gb-2007-8-8-r178 Spektor, T. M., & Rice, J. C. (2009). Identification and characterization of posttranslational modification-specific binding proteins in vivo by mammalian tethered catalysis. Proceedings of the National Academy of Sciences of the United States of America, 106(35), 14808–13. doi:10.1073/pnas.0907799106 Steger, D. J., Lefterova, M. I., Ying, L., Stonestrom, A. J., Schupp, M., Zhuo, D., Vakoc, A. L., et al. (2008). DOT1L/KMT4 recruitment and H3K79 methylation are ubiquitously coupled with gene transcription in mammalian cells. Molecular and cellular biology, 28(8), 2825–39. doi:10.1128/MCB.02076-07 Stewart, M. H., Bossé, M., Chadwick, K., Menendez, P., Bendall, S. C., & Bhatia, M. (2006). Clonal isolation of hESCs reveals heterogeneity within the pluripotent stem cell compartment. Nature methods, 3(10), 807–15. doi:10.1038/nmeth939 Takahashi, K., Tanabe, K., Ohnuki, M., Narita, M., Ichisaka, T., Tomoda, K., & Yamanaka, S. (2007). Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell, 131(5), 861–72. doi:10.1016/j.cell.2007.11.019 Takahashi, K., & Yamanaka, S. (2006). Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell, 126(4), 663–76. doi:10.1016/j.cell.2006.07.024 Takawa, M., Cho, H.-S., Hayami, S., Toyokawa, G., Kogure, M., Yamane, Y., Iwai, Y., et al. (2012). Histone Lysine Methyltransferase SETD8 Promotes Carcinogenesis by Deregulating PCNA Expression. Cancer research, 72(13), 3217–3227. doi:10.1158/0008-5472.CAN-11-3701 155 Talasz, H., Lindner, H. H., Sarg, B., & Helliger, W. (2005). Histone H4-lysine 20 monomethylation is increased in promoter and coding regions of active genes and correlates with hyperacetylation. The Journal of biological chemistry, 280(46), 38814–22. doi:10.1074/jbc.M505563200 Tardat, M., Murr, R., Herceg, Z., Sardet, C., & Julien, E. (2007). PR-Set7-dependent lysine methylation ensures genome replication and stability through S phase. The Journal of cell biology, 179(7), 1413–26. doi:10.1083/jcb.200706179 Tchieu, J., Kuoy, E., Chin, M. H., Trinh, H., Patterson, M., Sherman, S. P., Aimiuwu, O., et al. (2010). Female human iPSCs retain an inactive X chromosome. Cell stem cell, 7(3), 329–42. doi:10.1016/j.stem.2010.06.024 Thomson, J. A. (1998). Embryonic Stem Cell Lines Derived from Human Blastocysts. Science, 282(5391), 1145–1147. doi:10.1126/science.282.5391.1145 Trojer, P., Li, G., Sims, R. J., Vaquero, A., Kalakonda, N., Boccuni, P., Lee, D., et al. (2007). L3MBTL1, a histone-methylation-dependent chromatin lock. Cell, 129(5), 915–28. doi:10.1016/j.cell.2007.03.048 van den Berg, I. M., Laven, J. S. E., Stevens, M., Jonkers, I., Galjaard, R.-J., Gribnau, J., & van Doorninck, J. H. (2009). X chromosome inactivation is initiated in human preimplantation embryos. American journal of human genetics, 84(6), 771–9. doi:10.1016/j.ajhg.2009.05.003 Valouev, A., Johnson, D. S., Sundquist, A., Medina, C., Anton, E., Batzoglou, S., Myers, R. M., et al. (2008). Genome-wide analysis of transcription factor binding sites based on ChIP-Seq data. Nature methods, 5(9), 829–34. doi:10.1038/nmeth.1246 Voigt, P., Leroy, G., Drury, W. J., Zee, B. M., Son, J., Beck, D. B., Young, N. L., et al. (2012). Asymmetrically modified nucleosomes. Cell, 151(1), 181–93. doi:10.1016/j.cell.2012.09.002 Walsh, J., & Andrews, P. W. (2003). Expression of Wnt and Notch pathway genes in a pluripotent human embryonal carcinoma cell line and embryonic stem cells. APMIS, 111(1), 197–211. doi:10.1034/j.1600-0463.2003.1110124.x Wang, L., Schulz, T. C., Sherrer, E. S., Dauphin, D. S., Shin, S., Nelson, A. M., Ware, C. B., et al. (2007). Self-renewal of human embryonic stem cells requires insulin-like growth factor-1 receptor and ERBB2 receptor signaling. Blood, 110(12), 4111–9. doi:10.1182/blood-2007-03-082586 156 Wang, Z., Zang, C., Rosenfeld, J. A., Schones, D. E., Barski, A., Cuddapah, S., Cui, K., et al. (2008). Combinatorial patterns of histone acetylations and methylations in the human genome. Nature genetics, 40(7), 897–903. doi:10.1038/ng.154 Ward, C. M., Barrow, K. M., & Stern, P. L. (2004). Significant variations in differentiation properties between independent mouse ES cell lines cultured under defined conditions. Experimental Cell Research, 293(2), 229–238. doi:10.1016/j.yexcr.2003.10.017 Wongtawan, T., Taylor, J. E., Lawson, K. A., Wilmut, I., & Pennings, S. (2011). Histone H4K20me3 and HP1α are late heterochromatin markers in development, but present in undifferentiated embryonic stem cells. Journal of cell science, 124(Pt 11), 1878– 90. doi:10.1242/jcs.080721 Wu, S., Wang, W., Kong, X., Congdon, L. M., Yokomori, K., Kirschner, M. W., & Rice, J. C. (2010). Dynamic regulation of the PR-Set7 histone methyltransferase is required for normal cell cycle progression. Genes & development, 24(22), 2531–42. doi:10.1101/gad.1984210 Xu, C., Inokuma, M. S., Denham, J., Golds, K., Kundu, P., Gold, J. D., & Carpenter, M. K. (2001). Feeder-free growth of undifferentiated human embryonic stem cells. Nature biotechnology, 19(10), 971–4. doi:10.1038/nbt1001-971 Xu, H., Wei, C.-L., Lin, F., & Sung, W.-K. (2008). An HMM approach to genome-wide identification of differential histone modification sites from ChIP-seq data. Bioinformatics (Oxford, England), 24(20), 2344–9. doi:10.1093/bioinformatics/btn402 Yang, F., Sun, L., Li, Q., Han, X., Lei, L., Zhang, H., & Shang, Y. (2012). SET8 promotes epithelial-mesenchymal transition and confers TWIST dual transcriptional activities. The EMBO journal, 31(1), 110–23. doi:10.1038/emboj.2011.364 Zang, C., Schones, D. E., Zeng, C., Cui, K., Zhao, K., & Peng, W. (2009). A clustering approach for identification of enriched domains from histone modification ChIP-Seq data. Bioinformatics (Oxford, England), 25(15), 1952–8. doi:10.1093/bioinformatics/btp340 Zhao, X. D., Han, X., Chew, J. L., Liu, J., Chiu, K. P., Choo, A., Orlov, Y. L., et al. (2007). Whole-genome mapping of histone H3 Lys4 and 27 trimethylations reveals distinct genomic compartments in human embryonic stem cells. Cell stem cell, 1(3), 286–98. doi:10.1016/j.stem.2007.08.004 157 Appendix A: H3K27me3 Quantitative Changes in LP LP P2K27 over-enriched loci (>=50%) compared to LP P5K27, within 5000bp around TSS. Chromosome start, end, distance to TSS, gene name, quantile normalized tag count in LPP2 and LPP5 shown Chr Start End Dist to TSS Gene Name #LPP2 #LPP5 chr1 33108000 33110000 1 FNDC5 137.5 49 chr2 219453000 219454000 1 WNT10A 364.5 168.5 chr16 2461000 2462000 -1 NTN3 26.5 4.5 chr17 76063000 76067000 -1 NPTX1 1002 374 chr1 204747000 204748000 -2 RASSF5 140.5 65 chr11 113436000 113437000 2 ZBTB16 159.5 52.5 chr20 57612000 57614000 2 PHACTR3 291.5 110 chr15 30797000 30798000 3 GREM1 271.5 95.5 chr19 411000 413000 -4 SHC2 54.5 10.5 chr4 147779000 147780000 5 POU4F2 152.5 91.5 chr5 107034000 107035000 -5 EFNA5 14.5 4 chr8 54955000 54957000 5 RGS20 151 37 chrX 151991000 151992000 5 PNMA6A 20.5 9.5 chr5 131375000 131376000 6 ACSL6 89.5 26 chr16 1322000 1327000 -6 BAIAP3 440 140.5 chr10 23043000 23044000 9 PIP4K2A 40 21 chr1 3558000 3560000 11 TP73 336 150.5 chr3 185430000 185432000 -11 VWA5B2 469.5 238 chr4 24641000 24642000 12 LGI2 32 5 chr14 101096000 101097000 12 MIR1247 54.5 9 chr7 150385000 150387000 -15 CDK5 31 4 chr1 849000 853000 16 SAMD11 855.5 350 chr19 60579000 60581000 -16 TMEM190 22.5 4.5 chr18 70275000 70276000 -17 FAM69C 138 36.5 chr19 52215000 52217000 17 NPAS1 387.5 136.5 chr4 2012000 2015000 -18 C4orf48 213 58.5 chr18 46340000 46341000 18 MAPK4 53 9 chr10 76829000 76834000 19 ZNF503 935 444 chr6 147871000 147872000 -21 SAMD5 164.5 47.5 chr1 1557000 1559000 -22 MMP23A 51.5 7 chr6 41856000 41857000 22 PRICKLE4 51.5 11.5 chr11 128266000 128267000 -23 KCNJ5 112.5 52 chr1 53300000 53301000 27 PODN 81.5 26.5 chr2 26923000 26926000 27 DPYSL5 347 112.5 chr4 140696000 140698000 27 SETD7 171 83 chr20 41251000 41253000 -29 PTPRT 251.5 90 chr9 94986000 94988000 -33 WNK2 74.5 16.5 158 chr17 35077000 35079000 -33 PNMT 80 19 chr1 4614000 4616000 35 AJAP1 164 41 chr17 11085000 11086000 35 SHISA6 122.5 44.5 chr5 149526000 149527000 -37 CDX1 125.5 33 chr2 111593000 111597000 38 BCL2L11 403 156.5 chrX 50230000 50231000 -39 DGKK 15 3 chr2 94900000 94902000 41 TEKT4 33.5 10.5 chr4 164484000 164485000 -41 NPY5R 44 7 chr14 72773000 72775000 42 PAPLN 138.5 34 chr9 139182000 139185000 43 MIR3621 129 22 chr1 2450000 2453000 44 HES5 438.5 188 chr3 16901000 16902000 44 PLCL2 29.5 6 chr10 122206000 122207000 44 PPAPDC1A 41.5 7.5 chr10 7493000 7494000 -46 SFMBT2 267 110 chr1 19844000 19845000 47 NBL1 53.5 14.5 chr3 184628000 184629000 49 MCF2L2 31.5 4.5 chr11 118692000 118694000 50 MCAM 104 38 chr16 57054000 57056000 -50 NDRG4 223 69.5 chr10 46413000 46414000 -52 GPRIN2 22.5 4 chr7 28964000 28965000 54 TRIL 54.5 10 chr17 41328000 41330000 55 LOC100130148 738.5 348.5 chr1 21868000 21869000 -57 RAP1GAP 23 2.5 chr17 62391000 62392000 58 CACNG4 43.5 11.5 chr9 139041000 139044000 61 ABCA2 108 26.5 chrX 71443000 71444000 62 CITED1 218.5 147 chr3 129689000 129691000 63 GATA2 350.5 135.5 chr8 41873000 41874000 -63 ANK1 55 9 chr15 32662000 32664000 63 GOLGA8B 126 30 chr20 61573000 61576000 -63 KCNQ2 75 14.5 chr5 10617000 10618000 65 ANKRD33B 106 23.5 chr1 6401000 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1063 GRIN3B 51.5 5.5 chr5 76285000 76286000 1064 CRHBP 208.5 85.5 chr8 10625000 10628000 -1068 SOX7 771.5 401 chr7 107882000 107884000 1077 NRCAM 103 19 chr19 59619000 59620000 1083 TTYH1 36.5 4.5 chr3 195888000 195890000 1089 FAM43A 187 48 chr20 61201000 61203000 -1089 HAR1A 119 25 chr3 106568000 106571000 1097 ALCAM 106.5 22 chr19 18761000 18763000 1114 COMP 451.5 192.5 chr20 55273000 55275000 1114 BMP7 73.5 14 chr4 4439000 4441000 1116 D4S234E 150 46.5 chr15 73427000 73428000 1116 NEIL1 35 5.5 chr19 45796000 45800000 -1117 LTBP4 181 43.5 chr9 77696000 77697000 1120 PCSK5 55 16 chr6 166500000 166502000 1121 T 322.5 136.5 chr19 60571000 60574000 1126 IL11 225 90.5 chr11 69341000 69343000 1129 FGF3 446.5 193.5 chr20 42248000 42249000 1132 JPH2 14.5 3.5 chr18 72334000 72336000 1134 ZNF516 32 6 chr11 72766000 72767000 1139 RELT 16.5 3 chr21 35183000 35185000 -1143 RUNX1 213 51 chr16 2463000 2465000 -1148 TBC1D24 39.5 7 chr4 1154000 1157000 1157 SPON2 307 122 chr19 868000 871000 1158 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CYP1B1 85 28.5 chr16 416000 418000 1331 RAB11FIP3 65 14 chr2 42129000 42131000 1335 PKDCC 102.5 25 173 chr19 10261000 10265000 1345 ICAM5 604.5 237.5 chr2 128793000 128795000 -1359 HS6ST1 94.5 27 chr9 130978000 130980000 1361 IER5L 303 74.5 chr4 30331000 30334000 1365 PCDH7 118 29 chr16 85102000 85104000 1366 FOXF1 851.5 440 chr9 109289000 109292000 1368 KLF4 321 90 chr10 64247000 64248000 -1368 EGR2 105.5 42.5 chr10 43080000 43082000 1373 RASGEF1A 161 41 chr8 143757000 143758000 -1376 PSCA 14 2 chr1 29436000 29438000 1385 PTPRU 203 79.5 chr11 36355000 36356000 1389 PRR5L 65 26 chr16 2015000 2016000 -1389 SLC9A3R2 37 17 chr11 67164000 67166000 -1393 TBX10 81.5 15.5 chr1 2973000 2981000 1398 PRDM16 1450 548.5 chr17 37694000 37695000 1409 STAT5A 49 12.5 chr11 62446000 62448000 -1412 CHRM1 59.5 14 chr5 526000 529000 -1420 PP7080 247 66 chr19 19511000 19512000 1426 CILP2 33 8.5 chr1 16222000 16223000 1427 CLCNKA 18.5 3.5 chrX 99550000 99551000 1427 PCDH19 62.5 18 chr14 56345000 56346000 1437 OTX2 111 32.5 chr20 61598000 61601000 1449 EEF1A2 217.5 45 chr16 153000 156000 -1473 HBM 101 19.5 chr4 54791000 54792000 1479 PDGFRA 448.5 212.5 chr16 87047000 87051000 1485 ZFPM1 559.5 219.5 chr1 224991000 224993000 1499 ITPKB 152.5 35 chr14 105064000 105067000 1502 TMEM121 135 37.5 chr11 305000 306000 1509 IFITM1 30.5 4.5 chr5 743000 747000 1510 TPPP 217 80 chr9 20610000 20612000 1514 MLLT3 327 122 chr1 153319000 153320000 1528 EFNA3 103.5 21.5 chr6 36755000 36757000 1535 CDKN1A 225.5 77.5 chr8 37673000 37675000 1541 ZNF703 217.5 75.5 chr11 63629000 63630000 1562 FLRT1 23.5 4.5 chr12 6752000 6755000 1569 LAG3 144 17.5 chr11 1727000 1728000 -1575 IFITM10 42 12.5 chr18 44320000 44322000 1575 CTIF 110.5 34 chr2 241040000 241045000 -1591 MIR149 209.5 54.5 chrX 152558000 152561000 -1591 DUSP9 91.5 28 chr4 3735000 3738000 -1594 ADRA2C 105 20 chr4 993000 995000 -1610 FGFRL1 215.5 69.5 chr19 55758000 55765000 1614 LRRC4B 315 92 chr1 210846000 210848000 -1617 ATF3 325.5 96 chr4 122211000 122212000 1623 C4orf31 92.5 27.5 chr1 946000 948000 1634 AGRN 42.5 11.5 chr17 7965000 7968000 1635 HES7 451 202.5 chr3 127380000 127381000 1675 ALDH1L1 66.5 23 174 chr1 842000 844000 1680 FLJ39609 79.5 25.5 chr17 35015000 35017000 1701 NEUROD2 544 248 chr14 104704000 104705000 1706 JAG2 30.5 4.5 chr17 21221000 21223000 1708 KCNJ12 187.5 65.5 chr16 65755000 65758000 1711 HSF4 1272.5 632.5 chr1 151919000 151920000 1712 NPR1 50.5 17.5 chr2 241024000 241027000 1712 GPC1 224.5 61 chr17 34081000 34085000 1713 C17orf96 387.5 127.5 chr3 61523000 61525000 1717 PTPRG 95 17.5 chr16 30583000 30587000 1721 FBRS 184 35.5 chr11 124540000 124543000 1731 PKNOX2 334 105.5 chr8 144581000 144583000 1745 MAFA 518.5 260 chr11 2246000 2248000 1758 ASCL2 134 53.5 chr15 64783000 64784000 1772 SMAD6 95 46 chr3 8784000 8785000 1800 OXTR 27.5 5 chr14 103671000 103675000 -1813 KIF26A 414.5 163 chr12 3472000 3473000 1814 PRMT8 199.5 98 chr19 1402000 1404000 1852 APC2 53 12.5 chr7 101716000 101718000 1875 SH2B2 29 4 chr8 145519000 145520000 1875 DGAT1 24 9 chr17 63704000 63705000 -1896 LOC440461 216 86.5 chr22 37968000 37970000 1903 PDGFB 223 111.5 chrX 152562000 152564000 1909 DUSP9 191 73 chr1 44645000 44646000 1953 RNF220 282 118 chr2 68724000 68725000 -1958 PROKR1 80 27 chr17 35771000 35774000 1971 GJD3 147.5 36 chr1 153364000 153366000 -1973 EFNA1 238.5 113.5 chr9 139062000 139063000 -2003 NPDC1 25 8 chr15 65906000 65908000 2005 SKOR1 666.5 347.5 chr19 449000 450000 2010 MADCAM1 85 17.5 chr2 217266000 217267000 2017 IGFBP5 49 9 chr18 18005000 18006000 2086 GATA6 309.5 120.5 chr4 14615000 14616000 2104 CPEB2 54 25.5 chr5 170670000 170672000 2107 TLX3 698.5 315.5 chr9 2007000 2008000 2158 SMARCA2 107.5 61.5 chr18 897000 898000 2203 ADCYAP1 386.5 142.5 chr1 20929000 20930000 2220 SH2D5 54.5 18 chr1 47676000 47677000 2224 FOXD2 122 58.5 chr21 44487000 44488000 -2238 ICOSLG 51 11.5 chr11 2441000 2442000 2240 KCNQ1 27 8 chr11 1809000 1811000 -2250 SYT8 38 3.5 chr14 99693000 99694000 2265 DEGS2 51 32.5 chr5 2802000 2803000 2269 IRX2 19.5 2.5 chr12 131579000 131580000 2270 FBRSL1 14.5 2 chr7 128259000 128261000 2281 FLNC 58 19 chr20 61275000 61281000 -2297 MIR124-3 1046 414.5 chr19 54214000 54215000 -2341 LHB 13 2.5 175 chr19 56015000 56018000 2355 KLK1 99 34.5 chr20 3010000 3012000 2370 AVP 185 62 chr14 104335000 104336000 -2375 AKT1 87.5 31.5 chr4 149585000 149586000 -2378 NR3C2 46 3.5 chr1 26360000 26361000 2402 GRRP1 62 20.5 chr11 819000 821000 2415 EFCAB4A 72.5 12 chr17 55584000 55585000 2416 CA4 11.5 1 chr9 93223000 93224000 2465 NFIL3 64.5 49 chr8 22045000 22049000 -2490 HR 377 115 chr19 6484000 6485000 2490 TNFSF9 14.5 3 chr7 523000 524000 2507 PDGFA 36 7.5 chr15 26015000 26016000 2553 OCA2 252 114 chr3 4993000 4994000 -2597 BHLHE40 72.5 20 chr11 2403000 2404000 -2649 TRPM5 18 2 chr2 233095000 233098000 -2666 CHRND 527 204.5 chr22 31529000 31530000 2698 TIMP3 65.5 15.5 chr3 188943000 188944000 2707 BCL6 46.5 9.5 chr22 18119000 18124000 -2726 TBX1 878.5 391 chr3 46891000 46892000 -2740 PTH1R 29 9.5 chr11 75597000 75599000 -2778 WNT11 393 147.5 chr8 22142000 22144000 2796 PHYHIP 41 4 chr16 968000 969000 2819 LMF1 24.5 4.5 chr17 77509000 77510000 2847 NOTUM 167 73 chr11 1977000 1980000 -2859 H19 61.5 14.5 chr9 139476000 139477000 -2893 NELF 41.5 15 chr11 2123000 2126000 2909 IGF2 604 269.5 chr19 55914000 55917000 -2917 CLEC11A 66 17 chr19 47620000 47621000 2918 LIPE 38.5 7.5 chr22 18093000 18095000 2934 GP1BB 88 11 chr7 150379000 150380000 2962 ACCN3 39 11.5 chr22 41229000 41230000 2971 SERHL 30.5 6.5 chr1 226333000 226335000 -2984 ARF1 29 5 chr11 117986000 117987000 2984 PHLDB1 155 47.5 chr2 10972000 10973000 2986 KCNF1 12.5 0 chr19 60576000 60578000 -3016 TMEM190 53.5 8.5 chr2 220083000 220085000 -3136 ACCN4 73 19 chr6 44078000 44081000 3185 C6orf223 117.5 32 chr17 75428000 75434000 -3192 CBX4 2282 1076 chr2 74579000 74581000 -3252 LOC151534 107.5 44.5 chr1 1280000 1281000 3278 MXRA8 15 1.5 chr9 114910000 114911000 3278 NCRNA00256B 19.5 1 chr10 28071000 28072000 3284 MKX 88 38.5 chr13 21146000 21147000 3285 FGF9 89 30 chr9 132526000 132527000 -3302 PRDM12 287.5 147.5 chr17 75423000 75426000 3308 CBX4 472.5 182 chr7 1242000 1243000 3320 UNCX 145 43.5 chr9 123098000 123099000 -3400 GSN 14 2 176 chr9 138563000 138564000 -3441 NOTCH1 18 2.5 chr1 155160000 155161000 3452 LRRC71 167 71 chr10 8130000 8134000 3453 FLJ45983 828 338.5 chr2 72224000 72226000 3471 CYP26B1 393.5 176.5 chr1 153321000 153322000 3528 EFNA3 398 196.5 chr2 206258000 206260000 3531 NRP2 66.5 10 chr2 45018000 45020000 -3541 SIX3 255.5 108 chr2 94903000 94906000 3541 TEKT4 77.5 21.5 chr9 128412000 128414000 -3543 LMX1B 317.5 163.5 chr15 88091000 88093000 3544 MESP1 219.5 90 chr19 60364000 60365000 -3588 TNNI3 16 1 chr19 1396000 1399000 -3648 APC2 242.5 88.5 chr5 1058000 1059000 -3668 NKD2 62 26.5 chr4 3438000 3439000 3669 DOK7 10.5 0 chr5 176763000 176765000 -3757 PFN3 258 98 chr7 128297000 128298000 3800 LOC100130705 120 49.5 chr21 45176000 45178000 -3819 ITGB2 182 56.5 chr9 139064000 139065000 3826 ENTPD2 67.5 31 chr2 128796000 128797000 -3859 HS6ST1 10 0 chr7 129205000 129207000 -3910 MIR183 240.5 86.5 chr2 144990000 144991000 3928 ZEB2 53.5 19.5 chrX 67969000 67970000 3935 EFNB1 81.5 15 chr20 30633000 30637000 -3942 LOC149950 114.5 13.5 chr19 7643000 7645000 -3943 C19orf59 37 7.5 chr1 1002000 1005000 -3950 RNF223 80.5 13.5 chr11 2873000 2874000 -4027 SLC22A18 36.5 12.5 chr7 44151000 44152000 -4059 MYL7 70.5 17 chr11 2859000 2860000 4071 CDKN1C 67 21.5 chr16 684000 685000 -4099 WDR24 104.5 40 chr1 2969000 2971000 4149 FLJ42875 425 169 chr16 88300000 88303000 -4265 LOC100128881 56 14.5 chr22 18128000 18129000 4274 TBX1 114.5 45 chr11 545000 548000 4279 C11orf35 167.5 44.5 chr22 49046000 49047000 -4284 MAPK12 13.5 3.5 chr9 138230000 138231000 4325 LHX3 142.5 70.5 chr8 145516000 145518000 4375 DGAT1 28 3.5 chr1 16225000 16226000 4427 CLCNKA 15.5 2 chr17 17625000 17631000 -4432 SMCR5 164.5 38 chr1 1127000 1128000 4452 TNFRSF18 20 2 chr22 48628000 48630000 -4501 ZBED4 558.5 231 chr16 31406000 31407000 4560 SLC5A2 39 4 chrX 500000 501000 -4579 SHOX 181.5 81.5 chrX 131917000 131920000 4593 HS6ST2 404.5 135 chr5 1939000 1942000 -4620 IRX4 620 275.5 chr4 1151000 1153000 4657 SPON2 87 23.5 chr1 6231000 6232000 4661 HES3 31 6 chr11 2427000 2428000 4703 KCNQ1 50.5 15.5 177 chr7 155291000 155295000 4728 SHH 1329 644.5 chr12 112389000 112390000 4760 LHX5 438.5 209.5 chr7 155302000 155303000 -4772 SHH 19.5 6 chr4 980000 984000 -4776 SLC26A1 181.5 57 chr16 30578000 30579000 -4779 FBRS 60.5 8 chr2 120824000 120826000 4811 INHBB 82 31.5 chr11 46368000 46371000 -4817 CHRM4 168 67.5 chr7 150581000 150582000 -4818 SMARCD3 41 6.5 chr14 105014000 105020000 4824 CRIP2 788 340 chr19 1708000 1711000 4838 ONECUT3 1449.5 711.5 chr10 103980000 103982000 4867 ELOVL3 235 72.5 chr9 139478000 139479000 -4893 NELF 10 1.5 chr11 62450000 62451000 -4912 CHRM1 95 39 chr17 71642000 71646000 4975 FOXJ1 544.5 230.5 chr11 1669000 1671000 -5001 KRTAP5-6 29.5 3 chr17 75379000 75382000 5010 CBX8 1424.5 777.5 chr1 993000 996000 5050 RNF223 98 23.5 chr6 144649000 144650000 -5066 UTRN 38.5 9.5 chr9 138816000 138818000 -5195 C9orf86 63.5 16 chr6 33289000 33290000 5236 RING1 142.5 69.5 chr16 1331000 1332000 5301 BAIAP3 27 4.5 chr16 690000 691000 5326 FBXL16 49.5 11.5 chr20 62140000 62143000 5359 NCRNA00176 114.5 27.5 chr8 11604000 11605000 5374 GATA4 142 56 chr12 48647000 48648000 -5387 AQP6 28 9 chr3 38015000 38016000 5418 VILL 17.5 1.5 chr17 74511000 74513000 5494 CANT1 75 16.5 chrX 39915000 39917000 5526 BCOR 77.5 17 chr14 100268000 100269000 5545 DLK1 12 1.5 chr20 60489000 60491000 -5579 GATA5 54.5 16 chr17 60202000 60203000 5584 LOC146880 89 20 chr1 3591000 3592000 -5596 TP73 33.5 7 chr8 29256000 29257000 5741 DUSP4 19 2 chr1 154860000 154863000 5790 HAPLN2 321.5 100.5 chr6 170447000 170448000 -5878 DLL1 59.5 9.5 chr19 60289000 60290000 5928 EPS8L1 25 5 chr9 139309000 139312000 6024 NRARP 82 18 chr16 686000 687000 -6099 WDR24 15 1.5 chr2 233449000 233450000 6262 C2orf82 98 27 chr12 123345000 123347000 6337 FAM101A 78.5 17 chr5 134391000 134392000 6363 PITX1 423 178.5 chr7 129208000 129209000 -6410 MIR183 85 35.5 chr11 2869000 2871000 -6429 CDKN1C 35 5.5 chr1 1125000 1126000 6452 TNFRSF18 29.5 7.5 chr17 7677000 7678000 -6460 KDM6B 28 1.5 chr14 100440000 100441000 6490 SNORD112 26.5 4 chr19 51684000 51685000 6509 PNMAL2 17 4 178 chr17 69862000 69864000 6553 BTBD17 34.5 9.5 chr4 2433000 2435000 -6593 RNF4 46.5 6 chr19 1695000 1701000 -6662 ONECUT3 1221 558 chr22 44743000 44747000 6672 WNT7B 583 251 chr14 102463000 102468000 6754 AMN 362 102 chr8 677000 679000 -6774 ERICH1 296 102 chr5 1928000 1930000 6880 IRX4 538 276.5 chr19 15429000 15430000 6882 RASAL3 197.5 72 chr20 33651000 33653000 6898 FER1L4 113 16.5 chr19 3324000 3325000 6927 NFIC 28.5 4 chr1 46782000 46783000 6974 KNCN 159.5 74.5 chr9 37017000 37018000 6976 PAX5 69.5 20 chr8 22037000 22038000 7010 HR 21.5 2.5 chr5 139148000 139149000 -7090 PSD2 31 6 chr12 131689000 131691000 7110 LOC645277 54 13.5 chr1 951000 954000 7134 AGRN 58.5 9 chr1 2561000 2562000 -7159 MMEL1 25 4.5 chr9 134454000 134456000 7186 BARHL1 362.5 109 chr16 66244000 66246000 7219 ACD 44.5 7 chr1 898000 900000 7260 PLEKHN1 33 7 chr22 17397000 17398000 7363 DGCR10 115.5 43.5 chr17 55458000 55459000 -7382 DHX40P1 31.5 2.5 chr15 26024000 26027000 -7447 OCA2 238 80 chr2 45014000 45016000 -7541 SIX3 467.5 193.5 chr17 24964000 24966000 7567 CORO6 52 12 chr7 1231000 1232000 -7680 UNCX 495.5 217.5 chrX 39849000 39850000 -7837 BCOR 21.5 2 chr8 11242000 11243000 7944 TDH 49.5 15 chr19 13066000 13068000 7974 LYL1 172 57.5 chr2 71024000 71025000 7994 ATP6V1B1 30 5 chr7 154935000 154936000 -8085 EN2 523 263 chr15 39581000 39582000 8086 ITPKA 393.5 195 chr17 35009000 35010000 8201 NEUROD2 798.75 467.5 chr14 104948000 104950000 -8231 MTA1 108.5 30 chr14 104470000 104471000 8268 PLD4 16 1.5 chr7 1247000 1248000 8320 UNCX 309 145 chr13 99439000 99442000 8473 ZIC2 208.5 78 chr15 80774000 80776000 8482 LOC440297 64.5 11 chr5 737000 739000 8510 TPPP 32.5 5 chr17 7953000 7955000 8585 ALOXE3 98 22 chr21 37002000 37003000 8639 SIM2 358 202 chr19 8304000 8307000 8646 KANK3 385.5 140.5 chr1 834000 838000 8680 FLJ39609 75 21.5 chr17 33829000 33830000 -8746 ARHGAP23 79 31.5 chr12 112385000 112386000 8760 LHX5 376.5 186 chr10 22665000 22666000 -8905 SPAG6 143.5 101.5 chr9 139144000 139145000 -8930 GRIN1 15.5 0 179 chr19 60654000 60656000 -8958 SHISA7 245 84.5 chr9 34565000 34569000 8990 LOC415056 324 115.5 chr4 985000 988000 -9110 FGFRL1 126 34.5 chr19 18753000 18755000 9114 COMP 94 27 chr4 3442000 3446000 9169 DOK7 81 19 chr8 144573000 144576000 9245 MAFA 110.5 26.5 chrX 153270000 153271000 -9265 RPL10 20 2.5 chr22 17399000 17400000 9363 DGCR10 18.5 5.5 chr17 71638000 71641000 9475 FOXJ1 78.5 19 chr11 64237000 64238000 9736 NRXN2 99.5 38.5 chr6 1565000 1566000 9820 FOXC1 203.5 82 chr9 138224000 138226000 9825 LHX3 398 132 chr6 85540000 85541000 -9882 TBX18 171 57 chrX 152804000 152805000 -9907 L1CAM 3.5 0 chr10 102890000 102892000 9949 TLX1 757.5 400.5 chr20 61589000 61593000 9949 EEF1A2 178.5 46.5 chr9 128426000 128427000 9957 LMX1B 338 173 LPP2K27 over-enriched loci (>=50%) mapping to predicted enhancer regions (Ernst et al., 2011) chr14 100248800 100249800 chr5 60663600 60664600 chr14 101317800 101319800 chr6 107917700 107918700 chr14 102645741 102646741 chr7 524266 528266 chr14 104381150 104383150 chr7 2523500 2526500 chr14 104624250 104629250 chr7 4964050 4967050 chr14 104628400 104631400 chr7 5425050 5435050 chr14 104851000 104852000 chr7 51351300 51352300 chr15 80774500 80776500 chr7 72676400 72677400 chr16 415450 417450 chr7 73079575 73081575 chr16 789050 793050 chr7 75750300 75751300 chr16 848000 849000 chr7 127459100 127460100 chr16 2463550 2465550 chr7 128259550 128261550 chr16 21926800 21927800 chr7 148787150 148789150 chr19 4533433 4535433 chr7 149042150 149043150 chr19 4766600 4767600 chr7 149100200 149103200 chr19 5755050 5757050 chr7 151203550 151205550 chr19 6149900 6150900 chr8 11319200 11320200 chr19 13073900 13076900 chr8 22141500 22143500 chr19 19198700 19199700 chr8 22465800 22468800 chr19 38246750 38248750 chr8 41282716 41291716 chr19 45794950 45798950 chr8 143589933 143591933 chr19 55744350 55747350 chr9 2611800 2612800 chr19 55913300 55916300 chr9 34563500 34567500 chr19 55918550 55920550 chr9 128467775 128469775 180 chr19 59185900 59186900 chr9 128473150 128475150 chr19 60654650 60656650 chr9 128523750 128524750 chr20 30632050 30636050 chr9 131206100 131207100 chr20 42248050 42249050 chr9 131260725 131261725 chr20 42678610 42679610 chr9 131411600 131414600 chr20 45847100 45848100 chr9 132066350 132068350 chr20 60493900 60495900 chr9 138563390 138564390 chr20 60907300 60908300 chr9 138815350 138817350 chr20 61573550 61576550 chr9 139309800 139312800 chr20 61588050 61592050 chr10 23043000 23044000 chr20 61753700 61754700 chr10 31647633 31649633 chr21 43670200 43672200 chr10 46413100 46414100 chr22 17337700 17338700 chr10 135086600 135087600 chr22 28431383 28432383 chr11 1083100 1084100 chr22 44845650 44846650 chr11 1669475 1671475 chr22 47263650 47264650 chr11 1809350 1811350 chr22 49049050 49054050 chr11 1976300 1979300 chr22 49267200 49268200 chr11 2783950 2784950 chrX 39840200 39842200 chr11 2811700 2813700 chrX 39896700 39898700 chr11 2823450 2825450 chrX 128913700 128914700 chr11 2868575 2870575 chrX 128942000 128947000 chr11 62445550 62447550 chrX 151995150 151998150 chr11 63973300 63974300 chrX 152663100 152664100 chr16 66243600 66245600 chrX 152804150 152805150 chr16 78189733 78192733 chrX 154264150 154265150 chr16 86460250 86461250 chr1 945766 947766 chr16 87785966 87787966 chr1 957250 964250 chr16 88299400 88302400 chr1 3435700 3438700 chr16 88303875 88306875 chr1 5973650 5977650 chr16 88514450 88520450 chr1 20928700 20929700 chr17 1558850 1560850 chr2 25417133 25418133 chr17 4383333 4386333 chr2 42129066 42131066 chr17 17624350 17630350 chr2 71023733 71024733 chr17 17634700 17635700 chr2 94903400 94906400 chr17 35770300 35773300 chr2 111592950 111596950 chr17 45992550 45994550 chr2 220083125 220085125 chr11 124539400 124542400 chr2 233095900 233098900 chr12 1800400 1802400 chr2 236067700 236068700 chr12 6751250 6754250 chr2 241597100 241598100 chr12 6800800 6801800 chr3 13010100 13011100 chr12 130877350 130880350 chr3 16901000 16902000 chr12 131610783 131615783 chr3 32407900 32408900 chr13 97592700 97594700 chr3 38015300 38016300 chr14 33489300 33490300 chr3 46710433 46712433 chr14 72772350 72774350 chr3 50216850 50217850 chr17 55584000 55585000 chr3 129876400 129877400 chr17 58059200 58060200 181 chr3 144320650 144322650 chr17 69960150 69963150 chr3 185795075 185796075 chr17 71638672 71641672 chr3 188942866 188943866 chr17 75516550 75518550 chr3 196236900 196237900 chr17 76043900 76044900 chr4 37132300 37133300 chr17 76981650 76990650 chr4 105632200 105633200 chr17 78199650 78200650 chr4 183966300 183967300 chr17 78629000 78633000 chr5 525400 528400 chr18 72334066 72336066 chr5 1778400 1779400 chr19 410750 412750 chr19 60577960 60579960 chr19 1463300 1465300 LP P5K27 over-enriched loci (>=50%) compared to LP P2K27, within 10,000bp around TSS. Chromosome start, end, distance to TSS, gene name, quantile normalized tag count in LPP2 and LPP5 shown Chr Start End Dist. To TSS Gene Name #LPP2 #LPP5 chr21 46225000 46226000 -591 COL6A1 7.5 43 chr7 27126000 27127000 -761 HOXA3 165.5 358.5 chr7 27133000 27135000 -836 HOXA3 456 977 chr17 37099000 37100000 847 EIF1 1.5 31.5 chr10 43604000 43606000 871 HNRNPA3P1 10.5 57 chr1 84716000 84717000 -1008 RPF1 37 78 chr17 38980000 38981000 -1212 ETV4 36 110 chr17 44021000 44022000 -1322 LOC404266 325.5 526.5 chr17 44153000 44154000 1381 PRAC 197 390 chr10 22672000 22674000 -1405 SPAG6 336.5 551.5 chr3 46595000 46596000 1420 TDGF1 0 21.5 chr11 16379000 16380000 1489 SOX6 112 217 chrX 503000 504000 -1579 SHOX 48 155.5 chr5 50716000 50717000 1785 ISL1 65 138.5 chr4 111765000 111766000 -1797 PITX2 197.5 372 chr2 104832000 104833000 1866 LOC100506421 57 119 chr16 71651000 71653000 -1965 ZFHX3 108.5 229.5 chr5 54560000 54562000 -2100 LOC345643 519.5 896.75 chr18 55089000 55090000 2105 RAX 220 477.5 chr9 66235000 66238000 2411 LOC442421 180.5 467.5 chrX 507000 508000 2421 SHOX 78 187.5 chr11 16381000 16386000 -2511 SOX6 421 754.5 chr7 27138000 27141000 -2576 HOXA4 581.5 1127 chr7 27099000 27100000 2650 HOXA1 250.5 529.5 chr1 39808000 39809000 -2731 SNORA55 3 4 chr10 92673000 92675000 -2988 ANKRD1 348.5 694.5 chr7 27202000 27204000 3250 HOXA13 307 665.5 182 chr1 39802000 39803000 3269 SNORA55 2.5 11 chr2 176662000 176663000 -3278 HOXD13 246 420.5 chr9 67947000 67948000 -3305 LOC642236 37.5 78.5 chr9 99780000 99784000 -3310 ANP32B 602 1171.5 chr4 174690000 174693000 3316 NBLA00301 719 1240 chr12 113602000 113604000 3352 TBX3 346.5 648 chr10 22653000 22654000 3355 BMI1 139.5 244.5 chr7 150061000 150063000 -3369 GIMAP5 346.5 661.5 chr17 44164000 44165000 -3390 HOXB13 118.5 256 chr2 176740000 176741000 3449 HOXD3 140 289 chr10 15945000 15947000 -3475 FAM188A 62.5 144 chr9 21953000 21954000 -3638 C9orf53 84.5 197 chr9 21988000 21989000 3710 CDKN2B-AS1 27.5 55.5 chr5 92978000 92979000 3750 MIR2277 105.5 224 chr2 176704000 176709000 3786 HOXD8 997 1868.5 chr14 60049000 60050000 3809 SIX6 126.5 267.5 chr6 108589000 108591000 -3908 NR2E1 515.5 938.5 chr2 174911000 174913000 3933 SP9 150 338 chr2 176764000 176768000 -4068 LOC401022 277 549 chr12 88274000 88275000 -4073 DUSP6 36.5 88 chr2 101985000 101987000 4109 IL1R2 9.5 70 chr10 101278000 101279000 -4180 NKX2-3 120.5 315.5 chr4 81332000 81335000 -4181 PRDM8 533 882 chr8 103498000 103499000 -4407 UBR5 0 13.5 chrX 509000 510000 4421 SHOX 63 135.5 chr2 132736000 132737000 -4488 ANKRD30BL 89.5 207 chr13 29933000 29934000 4581 HMGB1 3 36.5 chr2 72993000 72994000 -4612 EMX1 127 268.5 chr1 154670000 154671000 -4692 C1orf61 167 393 chr6 50793000 50795000 4784 TFAP2D 176 312 chr18 18008000 18009000 5086 GATA6 130.5 251.5 chr13 27455000 27456000 5274 PRHOXNB 156 330 chr17 44149000 44150000 5381 PRAC 132.5 304.5 chr7 8444000 8447000 5390 NXPH1 411.5 773 chr8 55536000 55541000 5452 SOX17 750 1497 chr1 28941000 28942000 5461 YTHDF2 3.5 34 chr2 174902000 174903000 -5567 SP9 151.5 341.5 chr6 111905000 111906000 5607 REV3L 95.5 187.5 chr1 198268000 198270000 5607 NR5A2 127.5 252.5 chr3 159299000 159303000 5646 SHOX2 575.5 990 chr12 105506000 105508000 5837 RFX4 374 661.5 chr12 101880000 101883000 5918 ASCL1 476 883 chr10 15948000 15949000 -5975 FAM188A 20 48 chr5 72773000 72775000 6108 FOXD1 463 927.5 chr15 70314000 70320000 -6262 PKM2 166.5 392 chr9 21992000 21994000 6312 CDKN2B 174.5 345 chrX 24937000 24938000 6486 ARX 272 412 183 chr10 92677000 92678000 -6488 ANKRD1 240 462 chr14 36188000 36192000 -6524 PAX9 647.5 1299.5 chr6 108600000 108601000 6592 NR2E1 218 432 chr2 85651000 85652000 -6625 VAMP8 104 206.5 chr3 148616000 148617000 6629 ZIC1 77 140 chr9 125806000 125808000 -6710 LHX2 301.5 588 chr16 71632000 71634000 6775 ZFHX3 223 546.5 chr2 177792000 177793000 6832 HNRNPA3 5 49.5 chr13 96679000 96680000 6925 MBNL2 98.5 200.5 chr17 24400000 24402000 6956 PIPOX 5.5 46 chr10 135350000 135352000 7127 233 530.5 chr9 119209000 119211000 7138 ASTN2 1.5 24 chr3 62341000 62342000 -7270 FEZF2 220 421.5 chr17 43955000 43957000 7271 HOXB1 170.5 355.5 chr14 36066000 36067000 -7319 NKX2-1 69 107 chr8 23611000 23614000 7367 NKX2-6 456.5 877.5 chr5 138810000 138811000 -7387 DNAJC18 2.5 11 chr17 44168000 44169000 -7390 HOXB13 140 261.5 chr1 23558000 23562000 7466 ZNF436 643.5 1279.75 chr7 68708000 68711000 7658 AUTS2 464.5 894.5 chr10 118908000 118911000 7775 MIR3663 353 619 chr2 176648000 176650000 7936 EVX2 80 200.5 chr1 146816000 146817000 -8312 NBPF15 65.5 151 chr2 210336000 210337000 -8462 UNC80 44 95.5 chr12 52722000 52723000 8499 MIR615 143.5 337 chr2 176752000 176754000 -8553 HOXD1 357.5 619 chr2 100093000 100102000 -9023 AFF3 742 1599.5 chr13 78065000 78068000 9196 POU4F1 332 633 chr3 75805000 75806000 9323 FRG2C 46.5 113 chr6 100151000 100153000 -9371 PRDM13 196 318.5 chr1 143717000 143719000 -9532 PDE4DIP 29.5 69.5 chr21 39036000 39037000 9551 NCRNA00114 5.5 56 chr11 127906000 127908000 -9585 ETS1 73.5 159.5 chr4 174697000 174699000 9816 NBLA00301 244.5 490.5 LPP5K27 over-enriched loci (>=50%) mapping to predicted enhancer regions (Ernst et al., 2011) chr1 143717066 143719066 chr2 44856300 44857300 chr2 101985150 101987150 chr2 104621050 104622050 chr2 113285850 113286850 chr2 222844800 222845800 chr3 46595300 46596300 184 chr6 413800 414800 chr6 90355750 90356750 chr7 121756181 121757181 chr7 156946233 156948233 chr10 102537083 102538083 chr12 2699100 2700100 chr14 53108100 53109100 chr15 84240200 84242200 chr17 24399550 24401550 chr17 37099300 37100300 chr18 43024100 43025100 chr19 35531550 35533550 chr20 21018475 21019475 chr21 33406683 33407683 chr21 39036300 39037300 chr21 46225000 46226000 185 Appendix B: H3K27me3 Quantitative Changes in HP HP P2K27 over-enriched loci (>=50%) compared to HP P5K27, within 10,000bp around TSS. Chromosome start, end, distance to TSS, gene name, quantile normalized tag count in HPP2 and HPP5 shown Chr Start End Dist. To TSS Gene Name #HPP2 #HPP5 chr18 17074000 17078000 -201 GREB1L 264 85 chr19 53100000 53107000 753 SNAR-A13 152.5 19 chr19 55290000 55298000 -985 SNAR-A11 165 27.5 chrX 119944000 119948000 1319 CT47A1 78.5 4 chrX 119900000 119914000 1414 CT47A8 293.5 5 chr19 55308000 55311000 -1943 SNAR-A3 79.5 8 chr4 8934000 8980000 2028 USP17 1908.5 59.5 chr8 86742000 86764000 2157 REXO1L2P 2079.5 189 chrX 119895000 119897000 -2193 CT47B1 52 0 chrX 119930000 119931000 2239 CT47A8 24 0 chrX 119934000 119936000 -2261 CT47A1 52.5 0 chrX 119925000 119926000 2379 CT47A5 30.5 0 chr19 55325000 55327000 -2409 SNAR-A5 58.5 2 chrX 49125000 49127000 3348 GAGE2B 51 5 chr8 86851000 86910000 4861 REXO1L2P 6259 545.5 chrX 151755000 151757000 5833 NSDHL 41.5 11 chr11 3631000 3633000 9063 ART1 342.5 10 HP P2K27 over-enriched loci (>=50%) mapping to predicted enhancers chr15 86171833 86179833 chr18 17219650 17224650 chr19 53100350 53107350 chr19 55290520 55298520 chr19 55325500 55327500 chrX 406500 409500 HP P5K27 over-enriched loci (>=50%) compared to HP P2K27, within 10,000 bp around TSS. Chromosome start, end, distance to TSS, gene name, quantile normalized tag count in HPP2 and HPP5 shown Chr Start End Dist To TSS Gene Name #HPP2 #HPP5 chr1 224992000 224995000 -1 ITPKB 51 150.5 chr4 2390000 2391000 1 LOC402160 13 51.5 186 chr7 93357000 93359000 1 TFPI2 15 77.5 chr7 140419000 140422000 -1 LOC100131199 32 104 chr17 45633000 45635000 -1 COL1A1 18 76.5 chr10 116153000 116156000 5 AFAP1L2 63 172.5 chr10 90629000 90631000 -6 STAMBPL1 18 76 chr5 131733000 131734000 7 LOC553103 17 53.5 chr9 21983000 21986000 -10 CDKN2A 180.5 324.5 chr14 41143000 41150000 -14 LRFN5 191.5 478 chr16 87047000 87048000 -15 ZFPM1 26.5 63.5 chr17 3813000 3816000 -15 ATP2A3 27 105 chr15 43209000 43210000 16 DUOX1 3.5 25 chr2 219633000 219634000 -18 IHH 14 39 chr2 152663000 152664000 -19 CACNB4 32 70 chr4 55685000 55688000 19 KDR 33.5 118.5 chr20 17984000 17989000 21 OVOL2 81 298 chr16 12901000 12905000 22 SHISA9 36.5 128 chr2 180434000 180435000 -23 ZNF385B 19 49 chr7 100330000 100333000 -23 ACHE 56 148 chr10 42892000 42893000 -23 RET 2 14.5 chr11 68363000 68369000 -25 CPT1A 150 514.5 chr4 78037000 78039000 26 ANKRD56 64.5 163.5 chr2 219881000 219883000 -27 PTPRN 17.5 58.5 chr10 129423000 129428000 -28 FOXI2 84.5 321 chr3 4509000 4511000 -32 ITPR1 9.5 37.5 chr3 193609000 193610000 32 FGF12 4.5 20 chr13 19704000 19705000 34 GJB6 20.5 61 chr1 4613000 4617000 35 AJAP1 84 191.5 chr4 89298000 89300000 35 ABCG2 13 58.5 chr17 6557000 6558000 -36 SLC13A5 19 58 chr15 38922000 38925000 -38 SPINT1 10.5 116.5 chr8 95720000 95725000 -40 ESRP1 24.5 212.5 chr10 106004000 106005000 42 GSTO1 2 12.5 chr15 30513000 30516000 42 FAM7A2 33 123.5 chr5 1498000 1499000 43 SLC6A3 1 18 chr1 2450000 2453000 44 HES5 64 155 chr10 7492000 7495000 -46 SFMBT2 108.5 235 chr2 94885000 94888000 47 ANKRD20A8P 32 112 chr6 31476000 31477000 48 MICA 12 32.5 chr3 131546000 131548000 -49 COL6A5 46.5 131 chr5 577000 578000 49 SLC9A3 11 39 chr7 122313000 122315000 49 CADPS2 21 65 chr22 36153000 36154000 -49 ELFN2 13.5 43 chr10 44199000 44202000 51 CXCL12 27 108 chr11 65815000 65817000 51 TMEM151A 38 88 chr22 29833000 29834000 51 SELM 5.5 21 chr5 180563000 180565000 -54 TRIM7 69 177.5 chr3 185461000 185463000 -55 CAMK2N2 36.5 92.5 187 chr16 19329000 19330000 -58 TMC5 11.5 40 chr16 65516000 65518000 -60 RRAD 32.5 89 chr18 19523000 19524000 -60 LAMA3 20.5 79.5 chr3 199291000 199293000 -61 LOC348840 67.5 135 chr11 63824000 63825000 61 C11orf20 11.5 38 chr17 45401000 45402000 -61 DLX4 23.5 89 chr8 41872000 41875000 -63 ANK1 19 83 chr3 12304000 12307000 64 PPARG 68 161 chr13 90848000 90850000 64 GPC5 33.5 94.5 chr1 6401000 6404000 66 HES2 23.5 88.5 chr1 108308000 108310000 68 VAV3 9.5 55.5 chr8 10625000 10626000 -68 SOX7 48 98 chr6 31246000 31247000 -70 POU5F1 1 18 chr3 53503000 53505000 -71 CACNA1D 43.5 117 chr13 110010000 110014000 72 RAB20 29.5 165 chr17 34561000 34562000 -72 PLXDC1 8 31.5 chr11 65423000 65426000 73 FOSL1 63.5 215.5 chr2 17923000 17924000 74 KCNS3 3.5 16.5 chr2 238432000 238434000 74 RAMP1 20 66.5 chr8 53640000 53641000 74 FAM150A 12 45.5 chr17 40654000 40656000 -75 FMNL1 11.5 43.5 chr17 37144000 37145000 -76 HAP1 10.5 33.5 chr6 125516000 125517000 -78 TPD52L1 14.5 34 chr20 60484000 60485000 -79 GATA5 15 64 chr13 31318000 31320000 80 EEF1DP3 35.5 80 chr3 124649000 124651000 82 ADCY5 28.5 103 chr7 115952000 115955000 -83 CAV1 33.5 123.5 chr1 233879000 233881000 -84 GNG4 5.5 31 chr1 208473000 208475000 89 C1orf133 23.5 81.5 chr22 49050000 49052000 -94 MAPK11 14 75.5 chr10 47974000 47976000 -95 ZNF488 7.5 60 chr6 29798000 29800000 -96 HLA-F 20 72 chr10 133849000 133852000 96 DPYSL4 38.5 129.5 chr11 74629000 74630000 -98 LOC441617 6.5 23 chr15 38332000 38333000 -98 C15orf56 7.5 51 chr3 156280000 156281000 101 MME 6.5 35 chr20 22512000 22514000 101 FOXA2 56.5 147 chr1 208045000 208047000 102 IRF6 11 49.5 chr14 91372000 91373000 102 TC2N 1 13.5 chr19 38484000 38487000 -103 LOC80054 12.5 39 chr19 44681000 44682000 103 DLL3 45.5 117 chr1 234294000 234296000 104 NID1 20 80 chr2 74594000 74596000 -104 TLX2 36.5 110.5 chr8 23596000 23597000 -105 NKX3-1 11 52.5 chr11 65309000 65313000 -105 OVOL1 56 217 chr2 73282000 73284000 106 NOTO 75 174 chr9 72219000 72220000 -107 KLF9 26.5 49.5 188 chr16 1322000 1325000 -107 BAIAP3 35.5 168.5 chr19 539000 543000 107 HCN2 22.5 103 chr3 129024000 129025000 -109 MGLL 14 32 chr3 172659000 172663000 -109 TNIK 84.5 243.5 chr17 61728000 61731000 112 PRKCA 11 50 chr2 70634000 70635000 113 TGFA 2 2 chr4 42094000 42095000 -113 SHISA3 15 29.5 chr20 3166000 3170000 -113 SLC4A11 45 197.5 chr1 85235000 85236000 -116 MCOLN2 48 119 chr8 71144000 71148000 116 PRDM14 30 149.5 chr2 238060000 238061000 -117 MLPH 25 59.5 chr18 19848000 19849000 118 TTC39C 2 5 chr1 61316000 61324000 -122 NFIA 195 503.5 chr4 149581000 149585000 122 NR3C2 80 260 chr1 59021000 59024000 -127 JUN 24 133 chr14 64086000 64087000 127 C14orf50 13.5 47 chr12 113330000 113331000 130 TBX5 78.5 149 chr16 82959000 82960000 -134 ATP2C2 11.5 55 chr4 11038000 11041000 135 HS3ST1 70 153 chr3 47595000 47596000 -137 CSPG5 12 48.5 chr9 84867000 84869000 -137 RASEF 47.5 119 chr10 94822000 94825000 -137 CYP26A1 29.5 124.5 chr16 4926000 4928000 137 PPL 4 32 chr1 38032000 38033000 139 MANEAL 5.5 14.5 chr16 22733000 22734000 139 HS3ST2 25.5 68 chr7 100156000 100157000 141 EPO 12 37 chr5 114543000 114545000 142 TRIM36 44.5 107.5 chr17 1904000 1906000 -143 HIC1 23.5 74.5 chr22 19120000 19124000 146 SCARF2 39.5 165 chr4 55218000 55220000 148 KIT 21.5 71.5 chr6 20319000 20322000 149 MBOAT1 20 106.5 chr12 43730000 43732000 149 DBX2 41.5 104.5 chr6 31479000 31480000 150 MICA 4.5 34.5 chr3 31998000 31999000 -154 OSBPL10 6.5 29.5 chr8 85258000 85261000 -155 RALYL 71 156.5 chr22 36244000 36246000 156 CARD10 13.5 78.5 chr5 7447000 7452000 157 ADCY2 46 188.5 chr9 23810000 23814000 -157 ELAVL2 96 228.5 chr6 32918000 32922000 159 LOC100507463 85.5 203 chr6 78229000 78231000 -161 HTR1B 57.5 146.5 chr5 175989000 175991000 163 SNCB 26 92.5 chr22 44924000 44926000 -163 PPARA 22.5 64.5 chr9 90339000 90341000 164 NXNL2 9.5 47.5 chr20 21441000 21444000 164 NKX2-2 131 240.5 chr12 128952000 128956000 165 TMEM132D 60 218.5 chr19 2979000 2981000 165 TLE2 12 49 chr11 60475000 60477000 -167 SLC15A3 8.5 40 189 chr19 34847000 34849000 -167 PLEKHF1 4 49.5 chr7 24290000 24291000 168 NPY 4.5 31.5 chr20 61107000 61111000 -168 BHLHE23 32 128.5 chr16 27982000 27983000 -169 GSG1L 12 44 chr19 34709000 34710000 169 VSTM2B 37.5 88.5 chr1 13712000 13714000 -171 LRRC38 35.5 96 chr10 104143000 104146000 171 NFKB2 43 119 chr15 81567000 81568000 172 TM6SF1 16 46.5 chr18 55716000 55720000 -172 PMAIP1 33 130.5 chr1 200945000 200947000 174 SYT2 45.5 136.5 chr9 132874000 132875000 175 LAMC3 5.5 23 chr22 16886000 16889000 -175 MICAL3 59 137.5 chr12 129211000 129213000 176 FLJ31485 26 100.5 chr19 43502000 43503000 176 KCNK6 9.5 34.5 chr2 241155000 241158000 -177 RNPEPL1 64.5 136 chr3 40403000 40404000 -177 ENTPD3 21 75.5 chr18 43916000 43919000 178 ZBTB7C 54.5 169 chr1 184915000 184917000 182 PTGS2 17 73 chr16 3035000 3038000 -183 MMP25 25.5 98 chr21 27260000 27263000 -190 ADAMTS5 49.5 127 chr4 15694000 15695000 192 PROM1 10.5 28.5 chr8 22463000 22467000 -196 SORBS3 71.5 219 chr9 21548000 21551000 197 LOC554202 39 115.5 chr4 142776000 142778000 -199 IL15 30.5 105 chr8 21937000 21940000 200 NPM2 77 197 chr11 10428000 10430000 200 AMPD3 25.5 92.5 chr17 70401000 70402000 -200 FADS6 12.5 46.5 chr12 70952000 70954000 204 TRHDE 73.5 172.5 chr17 35036000 35037000 -205 PPP1R1B 5.5 32.5 chr2 95054000 95056000 -206 MAL 37.5 114.5 chr15 98698000 98701000 206 ADAMTS17 23.5 77 chr6 73388000 73389000 208 KCNQ5 18.5 38.5 chr2 47449000 47451000 209 EPCAM 12 52 chr2 219825000 219827000 -209 TUBA4B 32.5 150.5 chr12 16652000 16653000 -209 LMO3 9 22 chr12 109858000 109860000 211 LOC100131138 4.5 48 chr1 151917000 151919000 212 NPR1 42.5 152.5 chr2 17584000 17587000 212 VSNL1 16.5 45 chr6 39389000 39391000 214 KCNK17 53 132 chr10 62882000 62884000 214 TMEM26 55.5 134.5 chr17 71891000 71894000 215 SPHK1 64.5 201 chr19 44266000 44268000 215 PAPL 23.5 78 chr17 46066000 46068000 -217 ABCC3 53.5 127 chr3 127170000 127171000 -218 ROPN1B 6.5 24 chr11 10279000 10287000 -218 ADM 173 611 chr12 50912000 50914000 -221 KRT7 16 70 chr11 19692000 19693000 222 LOC100126784 25 44.5 190 chr2 10361000 10362000 223 HPCAL1 24.5 78.5 chr2 233060000 233062000 -224 ECEL1 12.5 50 chr14 67051000 67053000 -226 TMEM229B 20 100.5 chr22 42618000 42620000 226 PNPLA5 16.5 77 chr4 6523000 6527000 227 PPP2R2C 31 158 chr1 205694000 205695000 232 CR2 18.5 40.5 chr3 54130000 54133000 -233 CACNA2D3 38 105.5 chr21 37300000 37301000 -233 DSCR6 3 29.5 chr1 203804000 203805000 -235 MFSD4 6.5 24 chr3 51680000 51681000 238 TEX264 1 18 chr10 81881000 81883000 -238 PLAC9 22 74.5 chr18 12648000 12649000 -238 PSMG2 7.5 25.5 chr17 72093000 72094000 240 ST6GALNAC2 23.5 51 chr1 85286000 85288000 -243 MCOLN3 27.5 89 chr7 143164000 143165000 243 LOC154761 1 23 chr9 96441000 96442000 244 FBP1 21.5 66 chr14 73295000 73299000 -246 C14orf43 47.5 122 chr9 91408000 91411000 -247 GADD45G 24.5 113.5 chr2 121758000 121760000 248 TFCP2L1 20.5 76.5 chr11 129802000 129805000 249 ADAMTS8 50.5 114 chr17 71385000 71387000 251 TRIM47 11 52.5 chr4 110442000 110445000 -252 COL25A1 57.5 115.5 chr18 41906000 41907000 -252 PSTPIP2 10 26.5 chr5 131374000 131376000 254 ACSL6 34.5 115 chr1 170076000 170079000 256 DNM3 31.5 97.5 chr9 126253000 126254000 256 GPR144 7 37 chr11 27677000 27682000 256 BDNF 121 263.5 chr20 60743000 60746000 258 SLCO4A1 49.5 149 chr19 6414000 6416000 -260 CRB3 4 83 chr14 99695000 99696000 265 DEGS2 21.5 53.5 chr2 128148000 128151000 -266 LIMS2 35 121 chr14 74813000 74818000 266 FOS 38.5 202.5 chr22 40652000 40653000 267 TNFRSF13C 2.5 17.5 chr11 57038000 57041000 268 SLC43A1 42 196.5 chr3 24510000 24512000 270 THRB 15 43 chr12 42516000 42517000 271 TMEM117 18 51 chrX 8659000 8662000 -273 KAL1 55 130.5 chr11 2906000 2909000 -274 PHLDA2 49 162 chr14 66777000 66778000 -274 MPP5 1 14.5 chr14 69020000 69023000 276 PLEKHD1 45.5 170 chr12 12935000 12936000 277 GPRC5A 13.5 61 chr6 5030000 5032000 281 PPP1R3G 41 131.5 chr15 81113000 81114000 283 CPEB1 8.5 26 chr18 13208000 13209000 -286 C18orf1 8.5 41.5 chr16 65754000 65755000 -289 HSF4 69 131 chr7 8268000 8270000 -290 ICA1 14.5 57 chr10 62162000 62164000 290 ANK3 13.5 68.5 191 chr18 68684000 68687000 290 NETO1 74.5 174 chr20 38749000 38753000 290 MAFB 146 325 chr1 199634000 199636000 292 LAD1 20.5 82.5 chr6 84619000 84621000 296 RIPPLY2 55 150.5 chr22 17516000 17519000 296 GSC2 68 169.5 chr2 232499000 232500000 -297 NPPC 38 74.5 chr8 99374000 99377000 297 NIPAL2 25 124.5 chr22 34266000 34268000 -298 RASD2 12 80.5 chr20 22977000 22979000 301 THBD 29.5 99.5 chr19 11452000 11453000 303 ELAVL3 15 35.5 chr7 72675000 72678000 306 MLXIPL 34.5 142.5 chr3 39825000 39827000 -307 MYRIP 39 96.5 chr1 26245000 26246000 -309 SLC30A2 27.5 69.5 chr16 84342000 84343000 -310 C16orf74 5.5 33 chr17 64108000 64110000 -310 FAM20A 19.5 73 chr19 3752000 3753000 310 MATK 5.5 27.5 chr7 959000 962000 315 ADAP1 16.5 85 chr13 31899000 31901000 315 N4BP2L1 7.5 40 chr1 15352000 15353000 -316 TMEM51 4 9.5 chr8 73612000 73613000 320 KCNB2 14 37 chr15 39008000 39009000 -323 DLL4 32 79.5 chr6 1257000 1259000 325 FOXQ1 38.5 83 chr17 9488000 9492000 325 USP43 25 128 chr9 139067000 139069000 326 ENTPD2 10.5 57 chr17 38976000 38981000 331 ETV4 67.5 245.5 chr13 27392000 27393000 332 PDX1 98 158 chr7 87095000 87097000 335 RUNDC3B 38 117 chr1 150232000 150234000 338 S100A10 11.5 62.5 chr19 48865000 48867000 338 PLAUR 16 89.5 chr12 54760000 54761000 341 ERBB3 2 26.5 chr5 150706000 150708000 344 SLC36A2 31.5 94 chr2 115636000 115637000 346 DPP10 11 48 chr5 150583000 150584000 347 CCDC69 5.5 34.5 chr20 30103000 30105000 348 HCK 23.5 90.5 chr19 41039000 41041000 350 KIRREL2 16 54.5 chr10 6059000 6062000 -352 IL15RA 58.5 164 chr11 69298000 69300000 352 FGF4 7 54.5 chr19 8314000 8315000 -354 KANK3 6 32.5 chr7 71440000 71441000 -356 CALN1 12 34.5 chr17 78630000 78631000 -356 METRNL 0 1 chr1 25128000 25130000 357 RUNX3 86 160.5 chr7 21548000 21550000 -358 DNAH11 37.5 110.5 chr22 17891000 17894000 360 CLDN5 46.5 125.5 chr6 166640000 166643000 361 PRR18 55 141.5 chr21 41800000 41803000 362 TMPRSS2 17 119.5 chr6 33697000 33698000 366 ITPR3 6.5 41 chr20 60810000 60812000 366 NTSR1 40.5 89.5 192 chr9 115151000 115153000 367 BSPRY 6 58 chr16 6009000 6010000 367 RBFOX1 26.5 69 chr21 36428000 36431000 367 CBR3 85.5 267 chr17 47590000 47593000 -369 CA10 38 113.5 chr3 53055000 53056000 -371 SFMBT1 12.5 45 chr1 117949000 117952000 373 FAM46C 46 125.5 chr17 44662000 44665000 -373 PHOSPHO1 31 98 chr9 128716000 128717000 -374 RALGPS1 0 11.5 chr11 61104000 61105000 374 SYT7 3.5 25.5 chr6 158322000 158323000 -376 SYNJ2 1 13.5 chr2 132731000 132732000 -377 MIR663B 6 48.5 chr8 68036000 68038000 379 TCF24 67.5 153.5 chr3 13896000 13898000 -381 WNT7A 46.5 110.5 chr12 87496000 87500000 381 KITLG 30 126 chr17 44042000 44044000 382 HOXB7 168.5 338.5 chr3 161425000 161428000 383 LOC401097 130.5 279 chr14 76676000 76679000 387 ZDHHC22 24 86.5 chr9 78823000 78825000 -391 FOXB2 47 101 chr9 34366000 34367000 394 KIAA1161 2 16.5 chr3 139636000 139637000 395 ESYT3 6 24.5 chr16 31135000 31136000 396 PYDC1 9 23 chr11 406000 408000 397 SIGIRR 13 50 chr10 134900000 134902000 -398 VENTX 21 104.5 chr12 68418000 68419000 -398 RAB3IP 0 8.5 chr2 200879000 200881000 399 SPATS2L 6.5 42 chr2 24436000 24437000 401 ITSN2 0 7 chr16 31121000 31123000 -402 PYCARD 17.5 76 chr21 41460000 41464000 402 BACE2 53.5 135 chr6 29824000 29829000 404 IFITM4P 90.5 250.5 chr1 39729000 39730000 -405 BMP8A 2 25 chr5 148631000 148633000 406 AFAP1L1 32 94 chr16 65760000 65763000 -406 NOL3 43.5 101.5 chr1 243384000 243385000 -410 KIF26B 40 57.5 chr20 538000 539000 410 TCF15 1 3 chr10 128883000 128885000 412 FAM196A 13 59.5 chr16 55217000 55218000 414 MT1E 37 85 chr6 160688000 160690000 -415 SLC22A3 29.5 90 chr5 136862000 136863000 417 SPOCK1 11.5 36 chr7 124192000 124193000 417 GPR37 4.5 30 chr8 123861000 123866000 418 ZHX2 45 166.5 chr4 44145000 44147000 -419 KCTD8 11.5 40 chr5 137829000 137830000 420 EGR1 8 47.5 chr11 129823000 129826000 421 ADAMTS15 98.5 228 chr21 42512000 42515000 423 ABCG1 55.5 141 chr2 31214000 31217000 -425 GALNT14 65 160.5 chr14 98805000 98811000 -425 BCL11B 29 225 chr10 118990000 118992000 426 SLC18A2 37.5 88.5 193 chr12 88268000 88272000 427 DUSP6 35 138.5 chr9 76301000 76304000 428 RORB 52.5 123 chr16 70217000 70219000 429 MARVELD3 7.5 54.5 chr1 202387000 202388000 430 ETNK2 2.5 19.5 chr5 132175000 132178000 -432 ANKRD43 70.5 174 chr8 22492000 22495000 -432 PDLIM2 33 96.5 chr12 61614000 61615000 432 PPM1H 2.5 12.5 chr22 27798000 27801000 434 KREMEN1 28.5 119.5 chr1 6406000 6408000 -435 ESPN 45.5 115.5 chr5 10616000 10618000 -435 ANKRD33B 27.5 90 chr18 98000 101000 435 ROCK1P1 922.75 1518.75 chr1 179148000 179149000 -436 KIAA1614 0 8.5 chr3 129690000 129691000 -437 GATA2 32 78 chr2 119632000 119633000 441 C1QL2 21 42.5 chr21 34963000 34965000 442 CLIC6 35.5 141 chr1 178465000 178468000 444 LHX4 148 271.5 chr17 3545000 3547000 447 P2RX5 20.5 77 chr12 55167000 55169000 448 GLS2 30 94.5 chr11 65945000 65946000 449 NPAS4 36 87.5 chr13 24152000 24154000 451 ATP12A 25 71.5 chr6 2848000 2850000 -456 SERPINB9 13.5 43 chr1 224137000 224138000 -457 TMEM63A 4.5 45 chr7 119700000 119701000 -458 KCND2 3.5 19.5 chr8 70907000 70908000 458 SLCO5A1 6 28 chr4 8210000 8212000 459 ABLIM2 16.5 90.5 chr20 57585000 57586000 -459 PHACTR3 16.5 53 chr2 111593000 111596000 -462 BCL2L11 86 193 chr5 140326000 140328000 464 PCDHAC2 23.5 79.5 chr22 17303000 17304000 464 PRODH 9 34.5 chr5 94646000 94647000 -465 MCTP1 20.5 35 chr17 70431000 70432000 -465 OTOP2 36 83 chr19 40336000 40338000 -465 FXYD5 9 53.5 chr17 6887000 6888000 466 SLC16A11 5.5 31.5 chr13 35903000 35904000 -467 CCNA1 22.5 74.5 chr6 31432000 31433000 468 HLA-B 4 33.5 chr19 60383000 60385000 -468 SYT5 42 103 chr10 71869000 71873000 471 NODAL 52.5 289 chr9 92603000 92606000 472 SYK 56 147 chr17 4589000 4590000 472 CXCL16 29 78.5 chr16 155000 156000 -473 HBM 5 27.5 chr22 36927000 36930000 -473 MAFF 12.5 77.5 chr1 85702000 85704000 477 DDAH1 29.5 97 chr16 2009000 2011000 478 NPW 68.5 151.5 chr18 32020000 32022000 -478 MOCOS 25.5 108.5 chr2 29997000 29998000 481 ALK 21 52 chr8 35212000 35214000 483 UNC5D 15.5 44 chr9 95148000 95150000 -483 C9orf129 12 80.5 194 chr5 140284000 140288000 -486 PCDHAC1 42 128 chr15 41213000 41214000 486 TMEM62 24.5 63 chr18 5532000 5535000 486 EPB41L3 22.5 70 chr1 27158000 27160000 488 C1orf172 10 73.5 chr17 7105000 7108000 488 CLDN7 5 145.5 chr8 22044000 22046000 -490 HR 27.5 69 chr10 125640000 125642000 490 CPXM2 12.5 67 chr2 11211000 11214000 -491 PQLC3 12.5 74 chr10 115989000 115990000 492 VWA2 9 48.5 chr6 152169000 152173000 493 ESR1 104 255 chr1 224478000 224479000 494 MIXL1 15 43 chr13 101366000 101367000 496 FGF14 21.5 56 chr1 35019000 35021000 -498 GJB3 30.5 106 chr1 94779000 94782000 -499 F3 31 97 chr9 90981000 90985000 502 SHC3 117 260.5 chr18 33400000 33401000 -502 CELF4 12 52 chr7 138443000 138446000 505 ZC3HAV1 21.5 100.5 chr14 103653000 103655000 505 MIR203 7.5 38.5 chr11 30561000 30567000 506 MPPED2 75.5 221.5 chr1 29321000 29324000 508 TMEM200B 47.5 217.5 chr14 63263000 63265000 509 SGPP1 22.5 68 chr2 10138000 10139000 -511 CYS1 11 33 chr3 185430000 185431000 -511 VWA5B2 23.5 78.5 chr2 160362000 160363000 512 CD302 13 43 chr13 19997000 19998000 512 CRYL1 14.5 40 chr20 17456000 17463000 513 BFSP1 71.5 261 chr10 102780000 102783000 514 SFXN3 32.5 128.5 chr17 28227000 28228000 515 MYO1D 4.5 23 chr1 85131000 85133000 -516 LPAR3 17.5 65.5 chr19 52216000 52217000 517 NPAS1 32.5 64 chr15 32834000 32835000 -519 GJD2 22.5 49 chr10 111960000 111961000 521 MXI1 3.5 21 chr11 133707000 133708000 522 GLB1L2 6 28 chr15 58477000 58479000 -523 ANXA2 7.5 36.5 chr11 123029000 123031000 525 SCN3B 31.5 91 chr1 53300000 53302000 527 PODN 34 79.5 chr16 14695000 14696000 527 PLA2G10 5 52 chr6 54818000 54820000 -528 FAM83B 12.5 64 chr4 84249000 84250000 536 PLAC8 12.5 40.5 chr14 77035000 77036000 -537 ISM2 38.5 106.5 chr16 66021000 66023000 -537 HSD11B2 22.5 91.5 chr1 201363000 201365000 541 ADORA1 11 63 chr7 29569000 29572000 548 PRR15 40.5 158 chr2 166357000 166360000 549 GALNT3 20 156.5 chr9 135140000 135142000 -549 ABO 39.5 115 chr2 935000 937000 -554 SNTG2 8.5 31 chr3 125163000 125164000 -555 CCDC14 16 48 195 chr17 41329000 41330000 555 LOC100130148 132 284 chr11 69598000 69605000 -556 ANO1 130 283 chr5 5193000 5195000 557 ADAMTS16 21 76 chr11 110916000 110918000 557 LAYN 48.5 128 chr5 79901000 79902000 560 ANKRD34B 26.5 85 chr9 121170000 121172000 560 DBC1 25 98 chr2 25244000 25245000 563 POMC 9 64.5 chr17 40864000 40867000 565 ARHGAP27 33 119 chr4 142273000 142274000 566 RNF150 5.5 13.5 chr1 173427000 173429000 572 KIAA0040 11.5 66 chr2 241587000 241588000 572 SNED1 12.5 46.5 chr9 4480000 4482000 573 SLC1A1 11 68.5 chr10 118490000 118493000 575 HSPA12A 26.5 114.5 chr15 38359000 38364000 579 ANKRD63 127.5 317.5 chr19 17767000 17768000 581 B3GNT3 6.5 21.5 chr3 198240000 198241000 583 MFI2 3 26 chr10 79066000 79068000 583 KCNMA1 27.5 87.5 chr15 39573000 39575000 586 ITPKA 102 216 chr18 18003000 18005000 586 GATA6 77 156 chr1 19154000 19158000 -587 IFFO2 35.5 119.5 chr17 1560000 1562000 -588 TLCD2 12 38.5 chr22 44818000 44822000 -588 LOC100271722 27 87.5 chr10 105027000 105028000 590 INA 15 59.5 chr18 6405000 6406000 -590 L3MBTL4 8 39 chr17 10041000 10043000 593 GAS7 18.5 63 chr2 20730000 20731000 595 GDF7 10.5 25 chr5 135294000 135295000 595 FBXL21 37 83 chr3 4994000 4997000 -597 BHLHE40 22 90.5 chr4 128922000 128925000 597 HSPA4L 47 146 chr4 148872000 148875000 597 ARHGAP10 39 90.5 chr3 198179000 198180000 601 PIGZ 27.5 72 chr4 94967000 94970000 -601 ATOH1 67 177.5 chr22 38720000 38723000 601 FAM83F 22 119 chr7 130442000 130445000 602 FLJ43663 30 109.5 chr5 156818000 156820000 -605 NIPAL4 31.5 101 chr17 56843000 56846000 606 C17orf82 84 236 chr20 57309000 57310000 606 EDN3 14.5 63 chr5 170669000 170670000 607 TLX3 80.5 158.5 chr3 42518000 42519000 -608 VIPR1 6.5 43 chr5 175724000 175725000 -608 ARL10 5.5 36.5 chr11 129535000 129536000 608 ST14 26.5 75.5 chr7 98304000 98306000 609 TMEM130 30 113.5 chr4 155882000 155886000 -613 LRAT 71 250.5 chr4 37568000 37569000 -615 TBC1D1 20.5 52 chr21 34907000 34909000 615 RCAN1 22 82 chr12 50703000 50704000 617 NR4A1 6 27.5 chr17 29932000 29933000 619 TMEM132E 20 59 196 chr1 9634000 9636000 623 PIK3CD 26 86 chr1 109808000 109812000 -623 SYPL2 79 251 chr1 77521000 77522000 625 AK5 22.5 68.5 chr3 148593000 148594000 -626 ZIC4 8 39 chr1 40193000 40195000 629 MFSD2A 18.5 79.5 chr4 72271000 72274000 633 SLC4A4 41.5 123 chr12 116020000 116022000 634 TESC 14 53 chr1 150275000 150276000 635 S100A11 10 49 chr16 66826000 66828000 637 ESRP2 10 68 chr8 19841000 19842000 638 LPL 21.5 78 chr7 102873000 102876000 -640 SLC26A5 31 89 chr18 12244000 12246000 640 CIDEA 22 62.5 chr3 100838000 100841000 -644 COL8A1 46 133 chr4 9392000 9394000 644 DRD5 26.5 89.5 chr14 73319000 73327000 649 C14orf43 215.5 439.5 chr22 15946000 15947000 649 IL17RA 11 55.5 chr3 46709000 46710000 650 ALS2CL 9 47 chr6 136911000 136915000 650 MAP7 28.5 128.5 chr16 632000 633000 650 FAM195A 3 42.5 chr5 101659000 101660000 652 SLCO4C1 7 31.5 chr6 144513000 144515000 653 STX11 29 90 chr10 101079000 101080000 654 CNNM1 19.5 59 chr15 28471000 28474000 656 CHRFAM7A 28 110 chr22 28446000 28448000 656 CABP7 14 59.5 chr1 15958000 15959000 658 FBLIM1 4.5 19 chr17 45488000 45490000 661 ITGA3 13 56.5 chr17 24917000 24918000 668 ABHD15 24 69.5 chr4 89731000 89733000 -670 HERC3 29 73.5 chr14 50631000 50632000 672 TRIM9 5.5 27 chr2 38157000 38158000 -673 CYP1B1 8.5 18.5 chr6 41712000 41715000 -673 MDFI 27.5 114.5 chr9 138234000 138237000 -675 LHX3 82 204.5 chr5 122399000 122403000 -676 PPIC 60 229.5 chr3 37469000 37470000 683 ITGA9 15.5 64.5 chr15 70275000 70278000 690 GRAMD2 42.5 129.5 chr15 97463000 97464000 691 SYNM 36.5 74 chr16 972000 973000 691 SOX8 11 55.5 chr20 6051000 6052000 691 FERMT1 4.5 21.5 chr19 10058000 10059000 694 C19orf66 4.5 16.5 chr14 104401000 104403000 -695 KIAA0284 7.5 45.5 chr7 49784000 49785000 697 VWC2 39 84.5 chr1 181422000 181423000 703 LAMC2 3.5 24.5 chr11 2423000 2424000 703 KCNQ1 42 96 chr11 64266000 64269000 706 RASGRP2 57.5 175.5 chr1 152644000 152646000 707 IL6R 27.5 88.5 chr22 29421000 29422000 707 OSBP2 35 80 chr1 86393000 86395000 709 COL24A1 64 148.5 197 chr12 56406000 56408000 710 LOC100130776 76.5 184.5 chr4 6252000 6253000 719 JAKMIP1 0 6 chr2 101133000 101137000 -722 TBC1D8 19 87.5 chr3 69871000 69873000 724 MITF 21 69.5 chr6 32927000 32931000 726 TAP1 134 284 chr12 26240000 26241000 727 SSPN 19 40.5 chr1 53080000 53083000 729 ZYG11A 31 116.5 chr5 180008000 180009000 730 FLT4 2.5 11.5 chr16 73365000 73366000 730 FA2H 14 49 chr5 174804000 174805000 -731 DRD1 5 39 chr16 65765000 65767000 734 NOL3 46.5 111 chr20 54637000 54640000 735 TFAP2C 32 118 chr18 65219000 65221000 736 DOK6 65 161.5 chr21 44484000 44488000 -738 ICOSLG 22.5 100 chr14 104514000 104516000 739 AHNAK2 16.5 82 chr18 75254000 75261000 740 NFATC1 96 301.5 chr10 26542000 26547000 -742 GAD2 171 371.5 chr6 3695000 3698000 745 C6orf145 39 104.5 chrX 53366000 53367000 747 IQSEC2 6.5 28.5 chr10 103523000 103530000 -751 FGF8 94 354.5 chr14 94304000 94307000 752 GSC 126.5 253.5 chr12 6179000 6182000 757 CD9 17.5 83 chr16 372000 374000 758 LOC100134368 9.5 61.5 chr1 892000 893000 760 PLEKHN1 11 18 chr18 72972000 72974000 762 MBP 10 60 chr13 27965000 27968000 765 FLT1 27.5 141 chr6 56928000 56929000 768 BEND6 3.5 23 chr9 138702000 138703000 -768 AGPAT2 2 9 chr3 130807000 130808000 772 PLXND1 12 61 chr15 28183000 28185000 773 FAM7A3 22 85.5 chr22 18124000 18126000 774 TBX1 65 138.5 chr10 105244000 105245000 775 NEURL 17 43.5 chr11 122571000 122573000 -783 CLMP 10 64.5 chr9 94610000 94615000 786 ANKRD19P 128 343 chr20 60559000 60561000 -787 C20orf200 12 37.5 chr10 25505000 25507000 -789 LOC100128811 53 121 chr17 8033000 8034000 789 C17orf59 6.5 24 chr5 160908000 160909000 -792 GABRB2 18 49.5 chr8 17702000 17705000 -794 MTUS1 16 88.5 chr8 25098000 25100000 796 DOCK5 17 64.5 chr1 218987000 218988000 -799 MOSC2 16.5 53 chr6 16869000 16872000 -800 ATXN1 23.5 74.5 chr15 79214000 79215000 801 C15orf26 2 14.5 chr5 141682000 141686000 804 SPRY4 24.5 130.5 chr16 67329000 67330000 804 CDH1 5 33.5 chr12 48026000 48027000 -808 DNAJC22 36 90.5 chr17 75422000 75432000 808 CBX4 513.5 1184 198 chr20 32754000 32756000 -809 TP53INP2 16.5 56.5 chr22 18382000 18385000 809 ARVCF 55 141 chr19 49942000 49944000 -818 BCL3 16.5 49.5 chr21 42056000 42063000 818 RIPK4 163.5 400 chr12 121254000 121256000 819 B3GNT4 29 81 chr16 970000 971000 819 LMF1 5.5 19 chr6 168585000 168586000 820 SMOC2 26.5 100.5 chr7 115926000 115929000 820 CAV2 38 133 chr1 78284000 78286000 823 GIPC2 45.5 139 chr18 53618000 53623000 825 ATP8B1 136.5 325 chr2 219575000 219576000 -826 MIR375 34 117.5 chr8 143691000 143693000 835 ARC 11 69.5 chr13 98536000 98539000 -839 DOCK9 13 61.5 chr6 75970000 75973000 843 COL12A1 184.5 344.5 chr17 58908000 58910000 846 ACE 24 85 chr19 37858000 37860000 847 RGS9BP 5 46.5 chr16 67235000 67238000 848 CDH3 20 92 chr3 159307000 159308000 -854 SHOX2 51.5 126 chr12 47678000 47679000 855 DDN 30.5 68.5 chr20 56522000 56523000 855 APCDD1L 11 50 chr12 61831000 61833000 857 AVPR1A 46 117.5 chr9 97822000 97824000 858 NCRNA00092 48.5 116 chr16 19087000 19089000 861 SYT17 22 69 chr17 37827000 37829000 864 PTRF 23.5 98 chr14 22376000 22377000 867 MMP14 2 48.5 chr9 109290000 109292000 868 KLF4 41.5 111.5 chr10 64245000 64249000 -868 EGR2 104.5 251 chr2 160626000 160627000 872 PLA2R1 6 21 chr8 11599000 11601000 874 GATA4 198.5 408 chr1 224803000 224805000 876 C1orf95 62.5 141 chr14 54101000 54106000 -887 SAMD4A 49 210 chr19 17922000 17926000 889 KCNN1 50.5 237.5 chr20 42871000 42872000 893 RIMS4 9 37.5 chr2 233501000 233503000 -895 NGEF 28.5 103 chr12 4789000 4790000 897 KCNA6 29.5 84 chr7 31059000 31060000 899 ADCYAP1R1 14.5 68 chr5 76151000 76152000 911 F2RL1 14.5 43.5 chr1 164403000 164404000 -918 FAM78B 11.5 36 chr3 45161000 45163000 918 CDCP1 30 131.5 chr4 85723000 85725000 919 CDS1 12 62 chr3 46593000 46597000 920 TDGF1 34.5 235 chr19 18146000 18147000 921 IFI30 6 31.5 chr1 199884000 199886000 927 NAV1 18.5 71.5 chr19 50694000 50695000 929 PPM1N 4.5 23 chr21 43670000 43671000 930 SIK1 3.5 14.5 chr1 150078000 150079000 931 LOC100132111 3.5 21 chr3 115148000 115149000 -938 ZDHHC23 2 15.5 199 chr9 114953000 114955000 941 SLC31A2 29 97 chr11 68208000 68211000 941 GAL 23.5 124.5 chr17 39437000 39440000 942 NAGS 85 216 chr18 8598000 8599000 -943 RAB12 8.5 37 chr2 171380000 171381000 -946 GAD1 24.5 47 chr15 28652000 28654000 950 FAM7A2 24 77 chr11 1551000 1552000 953 LOC338651 46 115.5 chr17 45965000 45967000 953 EPN3 17 65 chr2 172657000 172658000 -954 DLX1 47.5 139 chr3 124269000 124270000 954 PDIA5 10.5 30.5 chr2 46378000 46380000 955 EPAS1 143 289 chr8 16903000 16907000 -955 FGF20 50.5 143 chr5 131658000 131660000 956 SLC22A4 42 115.5 chr13 40930000 40931000 958 C13orf15 48 89.5 chr10 124211000 124213000 969 HTRA1 19 68.5 chr15 81742000 81745000 972 BNC1 103 196.5 chr9 139317000 139318000 -976 NRARP 6.5 27 chr11 133648000 133653000 -985 GLB1L3 31 318 chr20 5243000 5245000 -985 PROKR2 50.5 136.5 chr1 3557000 3559000 -989 TP73 59.5 180.5 chr7 139408000 139410000 990 PARP12 30 91.5 chr14 58175000 58176000 990 DACT1 27.5 73.5 chr6 137406000 137408000 991 IL20RA 34 110 chr2 95375000 95376000 -995 KCNIP3 10.5 31.5 chr4 113286000 113288000 998 C4orf32 47.5 117.5 chr11 100503000 100505000 1004 PGR 123 256 chr5 38591000 38592000 1005 LIFR 6.5 33 chr7 51350000 51352000 1009 COBL 19.5 84 chr17 73739000 73741000 1014 TMEM235 14 57.5 chr6 131497000 131498000 -1019 AKAP7 4 29.5 chr1 148787000 148788000 -1022 ADAMTSL4 10 48.5 chr6 111516000 111517000 1026 SLC16A10 12 31.5 chr1 159843000 159844000 1027 HSPA7 13.5 47.5 chr8 86345000 86347000 1032 CA13 16 71.5 chr17 42284000 42286000 1033 WNT9B 48 104 chr6 36755000 36756000 1035 CDKN1A 22.5 70.5 chr10 126125000 126130000 1040 NKX1-2 48.5 311 chr14 72774000 72776000 1042 PAPLN 48.5 86.5 chr21 44108000 44109000 -1044 AGPAT3 2 21 chr2 100801000 100803000 -1045 NPAS2 30.5 91.5 chr11 82121000 82122000 1054 FAM181B 12 51.5 chr1 65385000 65390000 1062 AK4 45.5 159.5 chr17 34633000 34636000 1066 STAC2 66.5 192 chr1 37272000 37275000 -1069 GRIK3 67.5 133 chr14 73776000 73778000 1072 VSX2 198.5 350 chr4 93443000 93444000 -1073 GRID2 8.5 21.5 chr5 179713000 179715000 -1079 GFPT2 20 95 200 chr10 125842000 125844000 -1080 CHST15 10 45 chr22 36287000 36288000 1083 CDC42EP1 47.5 126 chr17 60206000 60208000 1084 LOC146880 38.5 131 chr2 34000 37000 1088 FAM110C 37.5 133.5 chr6 137283000 137285000 -1095 SLC35D3 38.5 129.5 chr13 102250000 102251000 -1095 KDELC1 5.5 30.5 chr2 213110000 213111000 1097 ERBB4 25.5 68.5 chr2 30308000 30310000 1099 LBH 16 67 chr7 28185000 28186000 -1101 LOC100128081 2 21 chr6 45498000 45500000 1108 RUNX2 101.5 181 chr3 135230000 135231000 1110 SLCO2A1 17 68.5 chr4 994000 995000 -1110 FGFRL1 24.5 68.5 chr4 4438000 4442000 1116 D4S234E 63 178 chr20 24985000 24988000 1116 ACSS1 54.5 155.5 chr14 23872000 23874000 1117 ADCY4 64.5 181 chr6 166500000 166502000 1121 T 74 173 chr2 219557000 219558000 1123 FEV 77.5 163.5 chr3 57089000 57090000 -1124 ARHGEF3 9.5 30.5 chr5 76046000 76047000 -1124 F2R 23 62 chr1 231817000 231818000 1127 KCNK1 17 61 chr21 35182000 35186000 -1143 RUNX1 100.5 246.5 chr10 112391000 112395000 -1145 RBM20 114 240 chr10 92606000 92607000 1151 HTR7 25.5 70 chr15 67379000 67380000 1152 PAQR5 15.5 52 chr8 102573000 102577000 1156 GRHL2 20 188 chr4 1154000 1157000 1157 SPON2 62.5 199.5 chr17 63610000 63611000 1167 LOC100499466 6 25 chr2 191722000 191724000 1170 STAT4 21.5 75.5 chr19 46550000 46551000 1171 TGFB1 7.5 50 chr3 140146000 140149000 1172 FOXL2 92 248.5 chr7 120757000 120758000 1174 WNT16 14.5 53 chr17 37586000 37590000 -1178 KCNH4 72.5 199.5 chr17 50698000 50699000 1180 HLF 20.5 31.5 chr1 63555000 63570000 1182 FOXD3 298 1195.5 chr8 31617000 31619000 1190 NRG1 47.5 116 chr19 12763000 12766000 1190 JUNB 44 158.5 chr9 71128000 71133000 1192 FAM189A2 52 180.5 chr12 48028000 48029000 1192 DNAJC22 10 32.5 chr15 76699000 76700000 1192 CHRNA3 17 61 chr7 149668000 149669000 1196 RARRES2 3 26.5 chrX 152747000 152749000 1197 PDZD4 3.5 60 chr1 222870000 222874000 1198 CNIH3 204.5 429.5 chr19 51096000 51097000 1202 MYPOP 3 28.5 chr17 35752000 35754000 1203 RARA 43.5 88 chr17 21221000 21222000 1208 KCNJ12 18.5 75 chr4 89597000 89600000 1209 HERC5 63.5 170 chr4 185981000 185985000 1209 ACSL1 88.5 213 201 chr15 83323000 83327000 -1209 PDE8A 40.5 146.5 chr1 27193000 27195000 1218 TRNP1 11 81 chr12 26170000 26171000 -1230 BHLHE41 9 45.5 chr6 56926000 56927000 -1232 BEND6 26.5 74 chr10 134894000 134896000 1232 UTF1 9 52.5 chr3 50625000 50626000 -1234 CISH 17.5 58 chr16 19033000 19035000 1245 ITPRIPL2 10 37 chr20 60896000 60898000 -1250 C20orf20 29.5 111 chr11 2246000 2249000 1258 ASCL2 17 90 chr1 158090000 158091000 1261 C1orf204 6.5 38 chr22 17274000 17276000 1264 DGCR6 52 114.5 chr19 56212000 56215000 1266 KLK10 84.5 181 chr10 97039000 97040000 1271 PDLIM1 5.5 33.5 chr18 9698000 9701000 1272 RAB31 36 108 chr18 18970000 18972000 1275 CABLES1 19.5 61 chr6 137856000 137861000 -1276 OLIG3 260.5 465 chr8 48811000 48813000 1279 CEBPD 27.5 89 chr11 69225000 69229000 1287 FGF19 19 168.5 chr6 3399000 3402000 1292 SLC22A23 28 131.5 chr14 54437000 54439000 1292 GCH1 35 114 chr10 91282000 91286000 1293 SLC16A12 33 160 chr12 56416000 56418000 1296 AGAP2 141 268.5 chr1 183208000 183210000 1305 FAM129A 30 94.5 chr2 96567000 96568000 1309 ARID5A 31 84.5 chr9 832000 834000 1310 DMRT1 77.5 158 chr22 28206000 28209000 1319 NEFH 37.5 139 chr17 77781000 77782000 1322 SLC16A3 10 38 chr4 115118000 115120000 1327 ARSJ 23 68 chr2 88534000 88535000 -1332 FOXI3 12 48 chr19 35556000 35557000 1332 ZNF536 7.5 30.5 chr9 134028000 134029000 1345 NTNG2 14 30.5 chr10 6283000 6284000 -1346 PFKFB3 30.5 68 chr4 172971000 172974000 1350 GALNTL6 56 156 chr14 89596000 89597000 -1361 KCNK13 4.5 45.5 chr21 36992000 36993000 -1361 SIM2 40 111 chr2 60633000 60638000 -1363 BCL11A 22 110.5 chr9 203000 204000 -1365 DOCK8 13 55 chr5 83714000 83716000 1367 EDIL3 17 63 chr19 2428000 2429000 1377 GADD45B 4.5 24.5 chr10 128068000 128069000 -1383 ADAM12 8 24 chr4 37570000 37571000 1385 TBC1D1 7.5 19.5 chr10 112248000 112250000 1385 DUSP5 29.5 122.5 chr15 37998000 38000000 1385 GPR176 16 85 chr17 32366000 32369000 -1385 LHX1 106.5 324.5 chr8 127638000 127639000 1393 FAM84B 4.5 31.5 chr5 95093000 95095000 1394 RHOBTB3 34 98.5 chr8 80686000 80688000 1396 STMN2 35.5 101 202 chrX 118777000 118779000 1396 ANKRD58 11 46 chr1 2974000 2980000 1398 PRDM16 153 409 chr17 34112000 34116000 -1399 MLLT6 73 277.5 chr20 22515000 22516000 -1399 FOXA2 68 119 chr5 88002000 88005000 1402 LOC645323 95.5 245.5 chr8 116748000 116750000 1402 TRPS1 6.5 34.5 chr22 37969000 37970000 1403 PDGFB 37.5 90 chr7 101244000 101245000 -1404 CUX1 14 44 chr3 114731000 114734000 -1408 SIDT1 113 222.5 chr6 126108000 126114000 -1425 HEY2 57 289 chr2 151050000 151052000 1426 RND3 13.5 84 chr7 100594000 100600000 -1428 VGF 105.5 383 chr17 17338000 17340000 1434 RASD1 67 143 chr22 18092000 18093000 1434 GP1BB 14 27.5 chr2 200028000 200037000 -1436 SATB2 249 613.5 chr4 115739000 115742000 1440 UGT8 20.5 103.5 chr8 56953000 56954000 -1440 LYN 10.5 44 chr7 526000 530000 1446 FLJ44511 89.5 329.5 chr17 45991000 45993000 -1448 CACNA1G 15 75 chr1 44644000 44646000 1453 RNF220 60.5 134.5 chr4 107036000 107039000 1454 NPNT 92 209 chr16 3026000 3028000 -1457 CCDC64B 8 76 chr8 22355000 22357000 1459 PPP3CC 21.5 78.5 chr2 113751000 113752000 1468 PAX8 73.5 176.5 chr15 99238000 99240000 1468 ALDH1A3 52 133.5 chr4 15390000 15391000 1471 CD38 16 49.5 chr16 31390000 31397000 1473 TGFB1I1 70 319 chr8 140781000 140785000 1481 KCNK9 124.5 228 chr8 132123000 132124000 -1483 ADCY8 29 61 chr12 116282000 116283000 1490 NOS1 17 54 chr3 32124000 32125000 1493 GPD1L 4.5 25 chr4 158218000 158219000 1495 GLRB 7 18 chr2 163404000 163406000 -1497 KCNH7 36 91.5 chr17 76065000 76068000 -1501 NPTX1 112.5 298.5 chr8 67851000 67852000 1530 SGK3 20.5 44.5 chr12 50072000 50074000 -1533 GALNT6 12 49 chr17 62391000 62395000 1558 CACNG4 49.5 131 chr16 22294000 22296000 -1561 CDR2 22 59.5 chr17 32922000 32923000 -1564 DUSP14 10 42 chr6 107067000 107069000 1577 AIM1 49 133 chr1 243386000 243387000 1590 KIF26B 2.5 7 chr3 187306000 187310000 1595 ETV5 70 175 chr11 13256000 13259000 1599 ARNTL 103 264.5 chr16 55231000 55236000 -1600 MT1DP 96 236 chr19 47519000 47521000 -1601 MEGF8 25.5 90 chr2 159533000 159537000 1608 TANC1 82 194.5 chr8 9798000 9802000 -1608 MIR124-1 63 157 203 chr16 65776000 65778000 -1616 KIAA0895L 28.5 86 chr7 92305000 92306000 -1623 CDK6 11.5 45 chr7 26159000 26161000 1628 NFE2L3 10 52 chr11 69341000 69342000 1629 FGF3 49.5 132 chr8 93182000 93184000 1630 RUNX1T1 15 74.5 chr19 7759000 7761000 1630 CLEC4GP1 15.5 67 chr22 18386000 18388000 -1631 C22orf25 28 89 chr1 156417000 156419000 1639 CD1D 48.5 121.5 chr15 74140000 74142000 1646 C15orf27 12.5 38 chr14 75519000 75520000 -1655 TGFB3 15 40 chr19 868000 872000 1658 KISS1R 132.5 313 chr4 2388000 2389000 1668 ZFYVE28 27.5 55.5 chr14 61655000 61656000 1672 FLJ43390 12.5 41.5 chr10 98932000 98936000 1673 SLIT1 58 149 chr1 235273000 235275000 1675 RYR2 39 116.5 chr16 159000 171000 -1679 HBA1 201.5 591 chr4 5943000 5945000 1686 CRMP1 116.5 220.5 chr11 117171000 117172000 1686 DSCAML1 41 86 chr8 25959000 25961000 -1691 EBF2 126 257 chr2 279000 281000 -1692 FAM150B 74.5 184.5 chr5 133887000 133889000 -1697 PHF15 5.5 56 chr7 17306000 17307000 1699 AHR 4.5 25 chr17 35014000 35018000 1701 NEUROD2 175 407 chr13 46026000 46028000 1703 LRCH1 20 74 chr3 181234000 181237000 1711 PEX5L 37 116.5 chr7 115639000 115640000 1717 TES 8.5 41 chr15 73282000 73284000 1726 C15orf39 9 38.5 chr5 2806000 2808000 1738 C5orf38 13 34 chr20 36868000 36871000 1738 PPP1R16B 23.5 117.5 chr3 113062000 113064000 1742 PHLDB2 21 59 chr10 124901000 124903000 -1750 BUB3 57.5 156.5 chr17 63421000 63423000 -1773 C17orf58 24 92.5 chr7 43119000 43122000 1777 HECW1 27.5 98 chr11 19694000 19695000 -1778 LOC100126784 5.5 11 chr11 75595000 75599000 -1778 WNT11 222 406 chr10 28071000 28075000 1784 MKX 181.5 428.5 chr19 38805000 38808000 1787 CHST8 70.5 185.5 chr3 181238000 181240000 -1789 PEX5L 21 64.5 chr3 58196000 58197000 -1799 ABHD6 8.5 24.5 chr2 275000 278000 1808 FAM150B 40 141.5 chr14 103385000 103386000 -1820 PPP1R13B 2 20 chr6 31570000 31574000 -1834 MICB 35.5 109.5 chr17 44929000 44930000 1846 NGFR 58 127.5 chr19 16297000 16300000 1849 KLF2 108.5 229 chr5 72453000 72455000 1856 TMEM171 36.5 101.5 chr16 85103000 85104000 1866 FOXF1 101 183 chr6 31757000 31759000 -1871 LY6G5C 14 63 204 chr1 154606000 154609000 1873 RHBG 16 47 chr5 175903000 175905000 1882 CDHR2 7 52.5 chr11 122032000 122035000 1892 UBASH3B 12.5 63 chrX 23260000 23262000 -1906 PTCHD1 29.5 94 chr17 7280000 7283000 -1913 FGF11 34 90 chr17 42251000 42255000 -1919 WNT3 102 287.5 chr3 73021000 73023000 1925 GXYLT2 21 63.5 chr11 124135000 124136000 1933 ESAM 32.5 87 chr3 174596000 174598000 -1938 NLGN1 23 75 chr10 124887000 124888000 1943 HMX3 125 215 chr19 53947000 53950000 1959 FUT1 13 73.5 chr19 7888000 7890000 1968 LOC645781 22.5 68.5 chr19 36530000 36538000 -1970 TSHZ3 85.5 270 chr1 20753000 20754000 1981 FAM43B 28.5 68.5 chr2 10971000 10972000 1986 KCNF1 7 29.5 chrX 153683000 153687000 1996 MPP1 60 186.5 chr14 35073000 35077000 2001 INSM2 103.5 258 chr11 35118000 35120000 2007 CD44 28 62 chrX 24945000 24947000 -2014 ARX 63.5 139.5 chr19 12856000 12858000 2017 KLF1 12 57 chr9 126307000 126308000 2020 NR5A1 11.5 47 chr16 55201000 55203000 2021 MT2A 22.5 66.5 chr5 76286000 76287000 2064 CRHBP 17 56.5 chr8 10627000 10628000 -2068 SOX7 63.5 167 chr4 44143000 44144000 2081 KCTD8 32.5 74 chr1 164399000 164402000 2082 FAM78B 83 190 chr5 88006000 88008000 -2098 LOC645323 63 202 chr5 132188000 132196000 -2099 SHROOM1 118 314 chr5 1934000 1942000 -2120 IRX4 305.5 718 chr2 132728000 132730000 2123 MIR663B 203 575.75 chr9 2004000 2011000 2158 SMARCA2 73 281 chr19 50864000 50867000 2158 GIPR 29.5 131.5 chr11 127960000 127961000 2163 ETS1 6 20 chr12 4247000 4255000 -2163 CCND2 164.5 376 chr19 48659000 48660000 2171 LYPD3 1 10 chr12 106237000 106240000 2173 BTBD11 119.5 263.5 chr10 102976000 102977000 2207 LBX1 86.5 162 chr6 73390000 73391000 2208 KCNQ5 11 36 chr8 22608000 22610000 -2240 EGR3 38.5 72 chr17 72091000 72092000 2240 ST6GALNAC2 57.5 81 chr15 72830000 72831000 2263 CYP1A2 3 11 chr20 20292000 20297000 -2265 INSM1 247 439 chr15 58084000 58088000 2287 FOXB1 41.5 126 chr14 73149000 73153000 -2301 ACOT6 41.5 142.5 chr9 132526000 132529000 -2302 PRDM12 347 670.5 chr2 120822000 120823000 2311 INHBB 17 46 chr1 146822000 146823000 -2312 NBPF15 10 55.5 205 chr14 105013000 105016000 2324 CRIP2 42.5 119.5 chr10 94440000 94444000 2339 HHEX 90 283.5 chr19 18578000 18584000 -2340 CRLF1 213.5 491.5 chr21 35180000 35181000 2357 RUNX1 21.5 64 chr2 113975000 113976000 2369 FOXD4L1 75 137 chr15 39589000 39593000 2377 LTK 183 372.5 chr17 56841000 56842000 -2394 C17orf82 85 141.5 chr4 148870000 148871000 -2403 ARHGAP10 9 19 chr3 199289000 199290000 2439 LOC348840 26.5 72 chr2 119630000 119631000 2441 C1QL2 10 54.5 chr3 57170000 57174000 2443 IL17RD 47 154 chr11 819000 822000 -2452 CD151 37 115.5 chr14 28307000 28310000 2462 FOXG1 259.5 428 chr18 28604000 28605000 2472 KLHL14 13 24.5 chr10 134450000 134454000 -2473 NKX6-2 68 225.5 chr10 95353000 95354000 -2517 RBP4 10.5 37.5 chr8 140786000 140788000 -2519 KCNK9 47.5 132 chr14 53483000 53494000 2520 BMP4 140.5 447 chr15 28270000 28276000 -2531 DKFZP434L187 127 387.5 chr6 3174000 3177000 -2533 TUBB2B 55.5 151.5 chr6 85526000 85530000 2618 TBX18 216 406.5 chr4 55221000 55222000 2648 KIT 14.5 54.5 chr19 18575000 18577000 2660 CRLF1 17.5 76 chr2 233095000 233098000 -2666 CHRND 106.5 226 chr10 51158000 51160000 -2667 AGAP7 54.5 140.5 chr6 72184000 72185000 2669 C6orf155 3.5 18 chr1 199350000 199352000 -2683 CACNA1S 28 80.5 chr2 120817000 120818000 -2689 INHBB 53.5 110 chr10 13432000 13434000 -2696 SEPHS1 19 83.5 chr13 40135000 40137000 2734 FOXO1 5 59 chr10 47124000 47127000 -2740 ANTXRL 35.5 139 chr12 55917000 55919000 2742 NDUFA4L2 51.5 139.5 chr5 176762000 176764000 -2757 PFN3 71 209 chr15 70311000 70316000 -2762 PKM2 90 219.5 chr7 35262000 35264000 -2764 TBX20 110.5 231.5 chr2 176669000 176671000 -2776 HOXD12 67 203.5 chr5 50717000 50718000 2785 ISL1 52.5 139 chr20 769000 772000 -2785 FAM110A 34 129 chr15 82977000 82980000 2805 SCAND2 36.5 156.5 chr13 27438000 27439000 2817 CDX2 20 81 chr7 1235000 1249000 2820 UNCX 752.5 1463.5 chr15 86948000 86951000 -2842 MIR1179 89.5 265.5 chr18 58344000 58345000 2862 ZCCHC2 2 18 chr13 19666000 19670000 -2886 GJB2 79.5 173.5 chr9 72216000 72217000 2893 KLF9 26 55.5 chr19 50872000 50874000 -2894 MIR642B 17 76 chr21 44949000 44957000 2923 TSPEAR 120 570 206 chr2 72225000 72226000 2971 CYP26B1 13 61 chr11 306000 308000 3009 IFITM1 7.5 62.5 chr22 23150000 23151000 -3030 ADORA2A 12 52 chr15 88092000 88093000 3044 MESP1 31.5 64.5 chr2 176764000 176766000 -3068 LOC401022 44.5 127.5 chr1 198266000 198267000 3107 NR5A2 16 45 chr5 170671000 170673000 3107 TLX3 213 366.5 chr19 18758000 18762000 3114 COMP 147 306.5 chr7 100395000 100398000 -3124 MUC12 24 89.5 chr14 22361000 22363000 -3146 SLC7A7 10 44.5 chr1 2970000 2972000 3149 FLJ42875 112 192.5 chr1 154652000 154668000 -3155 MIR9-1 288.5 992 chr6 1552000 1553000 -3180 FOXC1 12 53 chr7 100040000 100042000 3182 PCOLCE 13.5 91 chr7 5426000 5427000 3203 TNRC18 44 93.5 chr5 50711000 50712000 -3215 ISL1 40.5 119.5 chr4 158364000 158365000 3230 GRIA2 7 21 chr8 144579000 144582000 3245 MAFA 101 284.5 chrX 8663000 8664000 -3273 KAL1 23.5 41 chr3 27733000 27738000 3289 EOMES 199 484 chr8 100021000 100024000 -3307 OSR2 173.5 390.5 chr6 1260000 1262000 3325 FOXQ1 36 95.5 chr5 43057000 43059000 -3330 LOC648987 23 76 chr2 159529000 159531000 -3392 TANC1 20 53.5 chr10 79070000 79072000 -3417 KCNMA1 43 79.5 chr1 178469000 178470000 3444 LHX4 113.5 170.5 chr5 43080000 43083000 3507 LOC153684 33.5 122.5 chr17 30502000 30503000 3551 UNC45B 22.5 54.5 chr1 154452000 154454000 3597 PMF1-BGLAP 34.5 127 chr17 43980000 43982000 -3608 HOXB2 96.5 204.5 chr17 26905000 26910000 -3628 MIR193A 60.5 198.5 chr6 75975000 75977000 -3657 COL12A1 12.5 58 chr12 129095000 129098000 -3660 LOC100190940 77.5 172 chr5 1058000 1059000 -3668 NKD2 14 40 chr20 32759000 32760000 3691 TP53INP2 3.5 25 chr20 20299000 20302000 3735 INSM1 83.5 238.5 chr8 145526000 145528000 3751 SCRT1 48.5 119 chr2 74578000 74581000 -3752 LOC151534 40.5 119 chr17 73861000 73867000 3753 SOCS3 71 215.5 chr20 54633000 54635000 -3765 TFAP2C 84 195 chr12 92491000 92492000 3771 SOCS2 25.5 79 chr19 47193000 47195000 -3778 ATP1A3 23 69.5 chr6 1559000 1560000 3820 FOXC1 28.5 102 chr19 10263000 10268000 3845 ICAM5 91.5 235 chr17 44144000 44158000 3881 PRAC 492.5 1275.5 chr17 43972000 43975000 3892 HOXB2 188.5 410.5 chr7 129203000 129209000 -3910 MIR183 120 419.5 207 chr9 103000 106000 3917 FOXD4 252 589.5 chr9 76305000 76307000 3928 RORB 53 150 chr1 155160000 155162000 3952 LRRC71 45.5 122 chr17 71644000 71646000 3975 FOXJ1 67 151 chr14 49539000 49541000 3988 C14orf182 24.5 91 chr22 48628000 48631000 -4001 ZBED4 83.5 199.5 chr16 65746000 65747000 -4097 B3GNT9 21.5 68.5 chr9 78828000 78829000 4109 FOXB2 4.5 21 chr19 3306000 3307000 -4116 NFIC 15 33.5 chr13 52320000 52330000 -4224 PCDH8 133.5 353.5 chr16 65768000 65771000 4234 NOL3 28.5 81.5 chr14 94301000 94303000 4252 GSC 104.5 212 chr3 170859000 170861000 4257 MECOM 43 110.5 chr20 62139000 62142000 4359 NCRNA00176 17 80.5 chr5 158454000 158456000 4366 EBF1 20 60.5 chr2 220126000 220127000 -4387 MIR3132 10 40.5 chr19 56030000 56032000 -4409 KLK15 15.5 54 chr11 62449000 62451000 -4412 CHRM1 24.5 120.5 chr9 95751000 95755000 4429 BARX1 139 306.5 chr9 14303000 14314000 -4455 NFIB 127 423 chr17 56836000 56837000 4461 TBX2 57 200 chr2 132733000 132740000 -4488 ANKRD30BL 352.5 722 chr1 53976000 53978000 -4535 GLIS1 48 112.5 chr11 69160000 69161000 -4554 CCND1 7.5 32.5 chr7 44151000 44153000 -4559 MYL7 9 59 chr9 95760000 95764000 -4571 BARX1 167 356.5 chr10 131647000 131648000 4581 EBF3 25.5 65.5 chr2 236745000 236747000 -4609 GBX2 124.5 275 chr12 55903000 55907000 -4623 SHMT2 38 132 chr14 91104000 91109000 4636 C14orf184 72 265 chr14 32471000 32476000 -4710 NPAS3 189 409.5 chr6 33156000 33157000 4762 HLA-DPB1 4.5 24.5 chr2 88599000 88602000 -4784 C2orf51 39 109 chr16 53527000 53528000 4888 IRX5 24.5 58 chr4 123015000 123017000 -4898 BBS7 16 46 chr4 191180000 191181000 4906 LOC100288255 48.5 103 chr11 31798000 31804000 -4915 PAX6 249 537.5 chr2 174912000 174914000 4933 SP9 104 187 chr15 58472000 58473000 4977 ANXA2 2.5 21 chr16 31406000 31408000 5060 SLC5A2 15 75 chr18 75819000 75822000 -5070 HSBP1L1 37.5 93 chr19 1698000 1701000 -5162 ONECUT3 110.5 332 chr9 21989000 21991000 5210 CDKN2B-AS1 61.5 125.5 chr6 137851000 137853000 5224 OLIG3 60.5 178.5 chr6 33193000 33194000 5229 HLA-DPB2 7.5 29.5 chr3 170869000 170870000 -5243 MECOM 5.5 45 chr3 13672000 13673000 5278 LOC285375 19 40.5 208 chr2 191592000 191593000 -5279 STAT1 3 14.5 chr1 154860000 154862000 5290 HAPLN2 55 178.5 chr11 46369000 46371000 -5317 CHRM4 6 37.5 chr12 47672000 47676000 5355 DDN 60.5 137.5 chr1 178471000 178472000 5444 LHX4 87.5 142.5 chr10 102816000 102817000 5511 KAZALD1 21 53.5 chr2 176755000 176757000 -5553 HOXD1 50 112.5 chr4 154077000 154079000 -5585 FHDC1 12 67.5 chr12 129205000 129208000 5676 FLJ31485 82.5 183 chr6 1549000 1551000 -5680 FOXC1 141 290 chr15 58074000 58082000 -5713 FOXB1 131 306.5 chr15 67164000 67168000 5756 NCRNA00277 34 106.5 chr2 70985000 70989000 5772 VAX2 119 249.5 chr7 5434000 5437000 -5797 TNRC18 92 215.5 chr11 124121000 124122000 5819 VSIG2 15.5 53.5 chr9 138228000 138230000 5825 LHX3 69.5 148.5 chr16 683000 697000 5826 FBXL16 123 418 chr19 17813000 17815000 5841 JAK3 8 42 chr5 1927000 1933000 5880 IRX4 305 644.5 chr14 60178000 60182000 5908 SIX1 342.5 638.25 chr4 85632000 85633000 5911 NKX6-1 29.5 64 chr9 130165000 130170000 -5919 URM1 81.5 252 chr19 13068000 13070000 5974 LYL1 65.5 123 chr4 54654000 54656000 -6005 GSX2 71 172 chr6 10513000 10516000 -6037 LOC100130275 102 227 chr1 37278000 37279000 -6069 GRIK3 4.5 11 chr3 13889000 13892000 6119 WNT7A 21 100 chr17 44656000 44658000 6127 PHOSPHO1 60 170 chr19 49947000 49953000 6182 BCL3 108 274 chr7 5423000 5424000 6203 TNRC18 20.5 58 chr7 155289000 155294000 6228 SHH 216 478 chr17 43969000 43970000 -6229 HOXB1 4.5 23.5 chr7 35253000 35255000 6236 TBX20 55 138.5 chr14 102464000 102466000 6254 AMN 31.5 105.5 chr2 119327000 119330000 -6271 EN1 189.5 396 chr17 43949000 43965000 6271 HOXB1 322.5 933.5 chr16 71631000 71636000 6275 ZFHX3 178 335 chr2 176716000 176718000 -6277 MIR10B 81 161 chr5 134391000 134392000 6363 PITX1 74 167 chr8 11604000 11607000 6374 GATA4 86.5 244 chr1 147552000 147553000 6400 LOC388692 3 19 chr18 53245000 53250000 -6415 ONECUT2 212 430 chr17 75378000 75380000 6510 CBX8 235 449.5 chr17 77100000 77102000 -6513 ACTG1 6 67 chr9 107455000 107461000 -6559 TAL2 107 261.5 chr6 1327000 1330000 -6568 FOXF2 184 466.5 chr10 131645000 131646000 6581 EBF3 52 125.5 209 chr3 27745000 27746000 -6711 EOMES 77 125 chr19 15428000 15429000 -6738 WIZ 4.5 47.5 chr18 74834000 74835000 -6763 SALL3 22.5 73 chr9 21992000 21993000 6812 CDKN2B 39 79 chr1 210798000 210799000 -6820 ATF3 1 19 chr15 42808000 42810000 -6852 TRIM69 12.5 64.5 chr17 56894000 56897000 6911 TBX4 62.5 158.5 chr9 101000 102000 6917 FOXD4 165 249 chr22 19641000 19644000 -6918 AIFM3 13 72 chr2 172651000 172652000 -6954 DLX1 28 77 chr21 46217000 46221000 -7091 COL6A1 22.5 158 chr11 32420000 32422000 7139 WT1-AS 34.5 87.5 chr10 102986000 102987000 7159 FLJ41350 29.5 81 chr10 98926000 98931000 7173 SLIT1 30 95 chr14 94312000 94315000 -7248 GSC 46.5 151.5 chr22 40644000 40647000 7267 TNFRSF13C 19 119.5 chr7 100602000 100604000 7277 C7orf52 97.5 213.5 chr19 45806000 45807000 7383 LTBP4 8 28.5 chr10 131659000 131660000 -7419 EBF3 25 55.5 chrX 512000 513000 7421 SHOX 36 75.5 chr2 174915000 174916000 7433 SP9 137 238.5 chr3 57166000 57168000 7443 IL17RD 13 70.5 chr1 46781000 46783000 7474 KNCN 66 143.5 chr9 37016000 37018000 7476 PAX5 45.5 146 chr17 2605000 2607000 -7774 MIR1253 9 42 chr11 2839000 2841000 -7839 KCNQ1DN 13 53.5 chr2 236729000 236738000 7891 GBX2 304 823.5 chr1 178473000 178475000 7944 LHX4 37.5 139 chr1 44651000 44652000 7953 RNF220 20.5 46.5 chr15 97376000 97377000 -7953 PGPEP1L 6.5 25.5 chr8 37818000 37819000 8089 BRF2 1 23 chr5 151270000 151283000 8090 GLRA1 128 458.5 chr19 52435000 52437000 -8137 BBC3 21.5 72.5 chr19 8304000 8308000 8146 KANK3 72 228 chr14 36044000 36045000 8241 SFTA3 24 64 chr1 221374000 221376000 8247 TLR5 14.5 44 chr22 39922000 39924000 -8259 L3MBTL2 28 91 chr5 139465000 139466000 -8392 PURA 46.5 83 chr15 26026000 26027000 -8447 OCA2 8 24.5 chrX 153678000 153679000 8496 MPP1 7.5 28.5 chr14 20170000 20172000 8690 OR6S1 11 52.5 chr15 70319000 70320000 -8762 PKM2 26.5 63 chr2 37406000 37407000 -8774 PRKD3 8 23 chr2 88595000 88598000 -8784 C2orf51 29.5 101 chr4 185962000 185970000 -8806 SLED1 68 278 chr7 2693000 2696000 8811 AMZ1 44.5 159.5 chr9 21972000 21976000 -8868 CDKN2A 112 221.5 210 chr6 30203000 30204000 -8989 TRIM40 31 67 chr1 202613000 202616000 -9030 LOC127841 61.5 194 chr10 102988000 102989000 9159 FLJ41350 54.5 87 chr16 56576000 56578000 9160 TEPP 30 89 chr4 88658000 88663000 9179 SPARCL1 47 155 chr21 36449000 36450000 -9209 DOPEY2 30.5 85 chr15 74425000 74426000 9298 ISL2 23.5 94.5 chr6 1564000 1566000 9320 FOXC1 48.5 145.5 chr3 75804000 75807000 9323 FRG2C 64 148 chr9 138224000 138227000 9325 LHX3 65 166.5 chr12 113595000 113599000 9352 TBX3 167 401 chr5 134388000 134389000 9363 PITX1 3.5 23 chr7 156505000 156506000 -9392 MNX1 141 229 chr7 154952000 154954000 9415 EN2 110.5 210 chr8 11242000 11246000 9444 TDH 45.5 130 chr7 20802000 20803000 -9467 SP8 3.5 27.5 chr5 87987000 87991000 9513 MIR9-2 64.5 176.5 chr2 236748000 236754000 -9609 GBX2 165.5 426.5 chr22 18133000 18135000 9774 TBX1 38 109 chr14 28321000 28322000 9839 C14orf23 40 93 chr1 44653000 44654000 9953 RNF220 20.5 46.5 HPP5 over-enriched loci mapping to predicted enhancers chr16 372100 374100 chr3 191003483 191009483 chr10 130226950 130228950 chr3 193713750 193715750 chr11 306100 308100 chr3 198721150 198722150 chr11 406550 408550 chr4 4745700 4748700 chr11 2906800 2909800 chr4 44145300 44147300 chr11 10280750 10288750 chr4 55685500 55688500 chr11 13255150 13258150 chr4 85723850 85725850 chr11 27678050 27683050 chr4 88658883 88663883 chr11 60475450 60477450 chr4 89298350 89300350 chr11 65423100 65426100 chr4 93442650 93443650 chr11 82203650 82204650 chr4 107036700 107039700 chr11 82230250 82240250 chr4 113285400 113287400 chr11 127891100 127921100 chr4 115117650 115119650 chr11 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chr7 30989533 30992533 chr19 12855675 12857675 chr7 31047650 31049650 chr19 17813000 17815000 chr7 43119400 43122400 chr19 36529300 36537300 chr7 44151750 44153750 chr19 48865450 48867450 chr7 51350550 51352550 chr19 56211100 56214100 chr7 68623000 68624000 chr20 768250 771250 chr7 72674733 72677733 chr20 11177150 11179150 chr7 73839800 73842800 chr20 13125550 13129550 chr7 75734333 75735333 chr20 17457416 17464416 chr7 100039933 100041933 chr20 17984950 17989950 chr7 100394700 100397700 chr20 20299250 20302250 chr7 100728866 100730866 chr20 44684625 44685625 chr7 102873400 102876400 212 chr20 56238000 56239000 chr7 115638800 115639800 chr21 43670450 43671450 chr7 130442550 130445550 chr21 44947650 44955650 chr7 138442866 138445866 chr21 46217700 46221700 chr7 149375800 149378800 chr22 16885250 16888250 chr7 149667650 149668650 chr22 17275350 17277350 chr8 5509666 5516666 chr22 17303250 17304250 chr8 10855500 10857500 chr22 19641100 19644100 chr8 22493100 22496100 chr22 36051575 36053575 chr8 25098250 25100250 chr22 42617966 42619966 chr8 25601100 25605100 chr22 44817100 44821100 chr8 35211400 35213400 chr22 44843650 44848650 chr8 56953183 56954183 chrX 8659400 8662400 chr8 86345050 86347050 chrX 16504600 16506600 chr8 95719133 95724133 chrX 152746950 152748950 chr8 99373600 99376600 chr1 19153950 19157950 chr8 101545412 101547412 chr1 22405866 22408866 chr8 116748066 116750066 chr1 22409400 22413400 chr8 124241075 124242075 chr1 35019450 35021450 chr8 127637700 127638700 chr1 39056250 39058250 chr8 144232450 144234450 chr1 44655033 44658033 chr9 4480600 4482600 chr1 44658700 44661700 chr9 7768850 7772850 chr1 44739283 44752283 chr9 14305268 14316268 chr1 44762888 44764888 chr9 71127966 71132966 chr1 44777095 44786095 chr9 76300200 76303200 chr1 44802450 44804450 chr9 90338750 90340750 chr1 65386100 65391100 chr9 91407800 91410800 chr1 77521400 77522400 chr9 91480610 91484610 chr1 108308150 108310150 chr9 95148200 95150200 chr1 146201475 146205475 chr12 42516200 42517200 chr1 146242300 146243300 chr12 50072100 50074100 chr1 150232600 150234600 chr12 64190183 64191183 chr1 4671000 4672000 chr12 87495833 87499833 chr1 164403150 164404150 chr12 113577725 113580725 chr1 170075400 170078400 chr12 124236366 124239366 chr1 170595555 170597555 chr12 128921360 128922360 chr1 173426650 173428650 chr13 31899450 31901450 chr1 178473250 178475250 chr13 42345244 42348244 chr1 181422200 181423200 chr13 46026200 46028200 chr1 183207700 183209700 chr13 52320050 52330050 chr1 199633450 199635450 chr13 98536166 98539166 chr1 202387200 202388200 chr13 110010025 110014025 chr1 208044866 208046866 chr13 111108450 111111450 chr1 221374125 221376125 chr14 22361350 22363350 chr1 233879050 233881050 chr14 41140950 41147950 chr2 33816 36816 chr9 99021100 99024100 chr2 273900 276900 chr9 115151066 115153066 213 chr2 3246500 3253500 chr9 118119100 118124100 chr2 11210966 11213966 chr9 124149275 124151275 chr2 30308500 30310500 chr9 125666175 125668175 chr2 47449166 47451166 chr9 138701883 138702883 chr2 60633100 60638100 chr10 29266600 29270600 chr2 101133575 101137575 chr10 47974550 47976550 chr2 111593200 111596200 chr10 62882550 62884550 chr2 118945175 118956175 chr10 71870000 71874000 chr2 119629700 119630700 chr10 81730550 81732550 chr2 152599300 152604300 chr10 81880725 81882725 chr2 166356200 166359200 chr10 91281575 91285575 chr2 191592200 191593200 chr10 97038500 97039500 chr2 219824450 219826450 chr10 103026750 103038750 chr2 219880600 219882600 chr10 104143500 104146500 chr2 222844300 222845300 chr10 112390050 112394050 chr2 223324500 223328500 chr10 116517250 116519250 chr2 233095900 233098900 chr14 54102450 54107450 chr3 14617450 14619450 chr14 63262350 63264350 chr3 18263850 18265850 chr14 64085700 64086700 chr3 31998325 31999325 chr14 73169650 73170650 chr3 46593550 46597550 chr14 73294616 73298616 chr3 57166900 57168900 chr14 74814166 74819166 chr3 57169150 57173150 chr14 78625600 78629600 chr3 62368650 62381650 chr14 98782000 98786000 chr3 69871350 69873350 chr14 104401300 104403300 chr3 100837150 100840150 chr15 42808300 42810300 chr3 115148050 115149050 chr15 58084866 58088866 chr3 140063083 140065083 chr15 58472400 58473400 chr3 151234800 151238800 chr15 67164292 67168292 chr3 156279800 156280800 chr15 72829800 72830800 chr3 181233666 181236666 chr15 73282350 73284350 chr3 181237650 181239650 chr2 118846525 118849525 chr3 187306850 187310850 chr7 29447750 29451750 214 Appendix C: H3K27me3 Quantitative Changes in H9 H9 P2K27 over-enriched loci (>=50%) compared to H9 P5K27, within 10,000bp around TSS. Chromosome start, end, distance to TSS, gene name, quantile normalized tag count in H9P2 and H9P5 shown Chr Start End Dist to TSS Gene Name #H9P2 #H9P5 chr1 33107000 33111000 1 FNDC5 441 180 chr20 29920000 29923000 -38 DUSP15 442 182 chr13 37341000 37343000 -61 TRPC4 92.5 31 chr11 75104000 75109000 -82 MOGAT2 314 113.5 chr11 62070000 62072000 -92 AHNAK 446.5 194.5 chr1 68470000 68472000 -128 WLS 330 175 chr14 56345000 56354000 -154 OTX2OS1 1509 686 chr17 69149000 69155000 -178 SDK2 651.5 175 chr13 25522000 25524000 198 SHISA2 328 138.5 chr18 17073000 17079000 -201 GREB1L 1709 656.5 chr4 106287000 106288000 209 TET2 127.5 47 chr10 94820000 94826000 -222 CYP26A1 1047.5 437.5 chr6 17390000 17391000 226 RBM24 80.5 38 chr2 183438000 183441000 243 FRZB 267.5 92 chr11 117528000 117529000 340 SCN4B 66.5 35.5 chr9 119216000 119219000 -362 ASTN2 522 262 chr4 109307000 109310000 370 LOC641518 794.5 421.5 chr6 49626000 49627000 428 C6orf141 28 2.5 chr3 114414000 114415000 435 BOC 105 36.5 chr11 117251000 117254000 456 FXYD6- FXYD2 280.5 79 chr15 53821000 53823000 469 PRTG 671.5 277 chr6 89884000 89886000 -481 SRSF12 233.5 95 chr11 65358000 65359000 514 SNX32 47.5 11.5 chr14 99139000 99143000 -520 CCDC85C 230.5 96.5 chr2 26249000 26251000 536 FAM59B 49.5 13.5 chr17 7683000 7686000 540 KDM6B 378 98.5 chr7 107883000 107884000 577 NRCAM 102.5 37 chr11 2127000 2129000 -591 IGF2 114 29.5 chr12 1572000 1574000 592 FBXL14 400 198 chr17 7696000 7701000 -609 TMEM88 1227 322.5 chr13 94161000 94165000 -610 SOX21 305 80.5 chr11 75055000 75058000 627 MAP6 774.5 374 chr5 60664000 60665000 643 ZSWIM6 53 18.5 chr4 1763000 1768000 663 FGFR3 391 130.5 chr17 27617000 27619000 692 RHBDL3 80 27 chr22 41835000 41837000 -701 BIK 177 79.5 215 chr18 2642000 2643000 731 LOC645158 73.5 20 chr2 222143000 222146000 754 EPHA4 478 83 chr1 221967000 221968000 758 CAPN2 95.5 28.5 chr8 29262000 29264000 -759 DUSP4 537.5 300 chr12 112383000 112404000 760 LHX5 10393 5158 chr7 127836000 127837000 772 IMPDH1 224 97 chr12 6799000 6805000 776 GPR162 338.5 119 chr3 161957000 161958000 810 PPM1L 285.5 128 chr1 845000 846000 -820 FLJ39609 315.5 143 chr16 705000 707000 826 METRN 137 38.5 chr1 109594000 109596000 836 CELSR2 151.5 52.5 chr1 6243000 6246000 -878 GPR153 137.5 45 chr19 59158000 59160000 898 CACNG8 295 96 chr8 23156000 23160000 905 CHMP7 494.5 187 chr10 95508000 95509000 944 LGI1 30 11.5 chr19 53831000 53832000 951 DBP 47 9 chr16 49740000 49742000 1009 SALL1 69 17 chr15 45798000 45800000 1022 SEMA6D 218 82 chr1 38282000 38286000 1037 POU3F1 579 254 chr7 107429000 107431000 1040 LAMB1 335 117 chr17 5343000 5345000 1043 LOC728392 212 82 chr1 154940000 154942000 1083 CRABP2 152 40.5 chr19 59619000 59620000 1083 TTYH1 85.5 24 chr1 33586000 33588000 1086 PHC2 347.5 145 chr14 50366000 50367000 1089 NIN 43.5 12.5 chr19 58187000 58188000 1096 ZNF702P 260 115.5 chr20 55272000 55276000 1114 BMP7 872 447.5 chr8 65656000 65657000 1151 BHLHE22 144.5 52.5 chr17 72217000 72218000 1151 MXRA7 182.5 83.5 chr8 38506000 38507000 -1163 C8orf86 21.5 9 chr1 229222000 229225000 -1207 MIR1182 140.5 52 chr17 34011000 34018000 1209 SRCIN1 551.5 148.5 chr11 124540000 124542000 1231 PKNOX2 457 194 chr1 179325000 179326000 1239 IER5 73 22 chr11 40270000 40272000 1240 LRRC4C 124.5 62.5 chr16 60626000 60628000 1240 CDH8 241 89 chr2 42573000 42574000 1241 KCNG3 90 19.5 chr10 70882000 70883000 1268 TSPAN15 117 45 chr17 14145000 14148000 1269 HS3ST3B1 899 441 chr7 149102000 149103000 -1272 ZNF467 218.5 93 chr10 104254000 104256000 1291 SUFU 149.5 49 chr1 58486000 58489000 1299 DAB1 327 129 chr18 32132000 32134000 1300 FHOD3 166 69.5 chr7 86112000 86113000 1334 GRM3 63 26 chr13 22904000 22905000 1341 SACS 27.5 3 chr12 40915000 40919000 1356 YAF2 801 435 chr2 220088000 220089000 1364 ACCN4 43 14.5 216 chr5 127902000 127904000 -1366 FBN2 78.5 29.5 chr19 55764000 55765000 -1386 LRRC4B 40.5 13 chr19 42875000 42878000 -1444 ZNF781 252 96.5 chr5 31230000 31232000 1447 CDH6 495 202.5 chr3 16898000 16902000 -1456 PLCL2 195.5 70.5 chr19 60556000 60557000 1494 COX6B2 144 57 chr16 2459000 2461000 -1501 NTN3 228.5 81.5 chr16 76381000 76382000 1516 VAT1L 37.5 11 chr6 10524000 10526000 -1544 TFAP2A 465 237 chr2 8733000 8743000 -1564 ID2 507 107.5 chr22 49461000 49462000 1564 SHANK3 69 29.5 chr3 195889000 195890000 1589 FAM43A 137.5 74 chr19 52612000 52614000 1597 MEIS3 638.5 336 chr1 11463000 11464000 1618 PTCHD2 50.5 14.5 chr16 78189000 78192000 1623 MAF 143 38.5 chr19 10486000 10489000 1626 S1PR5 276 134 chr11 94139000 94140000 -1656 AMOTL1 122.5 54 chr12 119292000 119294000 -1659 MSI1 134.5 47 chr11 628000 630000 1695 DRD4 169 54 chr7 121733000 121734000 -1699 FEZF1 37.5 10.5 chr6 114283000 114284000 -1720 MARCKS 252 107.5 chr20 6698000 6699000 1755 BMP2 53 15.5 chr9 36126000 36131000 1758 GLIPR2 1026 457.5 chr10 104182000 104187000 -1759 MIR146B 1011.5 479.5 chr15 64782000 64785000 1772 SMAD6 469 220.5 chr16 51140000 51142000 -1785 TOX3 111.5 29.5 chr19 59105000 59107000 -1803 CACNG7 219 64 chr17 27615000 27616000 -1808 RHBDL3 85 44 chr1 177528000 177535000 1860 SOAT1 889.5 179 chr1 5975000 5979000 -1880 NPHP4 172 47 chr4 42095000 42098000 1887 SHISA3 376.5 141.5 chr3 50513000 50515000 1896 CACNA2D2 210 97 chr4 41057000 41062000 1939 LIMCH1 991 461.5 chr7 121726000 121732000 -1948 LOC154860 514 167 chr22 21854000 21855000 1948 BCR 42.5 13 chr19 60580000 60584000 1984 TMEM190 174 66 chr5 107032000 107033000 1995 EFNA5 39.5 15.5 chr11 75159000 75160000 2074 DGAT2 71 38 chr19 55759000 55763000 2114 LRRC4B 855.5 347 chr10 71482000 71487000 2137 H2AFY2 671.5 288.5 chr16 30585000 30586000 2221 FBRS 55 27.5 chr3 99936000 99937000 2238 ST3GAL6 33 11 chr18 74838000 74840000 -2263 SALL3 250 90 chr12 47493000 47500000 -2279 CACNB3 1266.5 556.5 chr16 49744000 49746000 -2316 SALL1 130.5 49.5 chr6 35290000 35295000 2332 SCUBE3 418.5 183.5 chr5 176172000 176173000 2334 UNC5A 42 23.5 217 chr19 37860000 37861000 2347 RGS9BP 65 17 chr5 60660000 60663000 -2357 ZSWIM6 585.5 268 chr4 30332000 30335000 2365 PCDH7 359.5 62 chr1 36704000 36706000 -2373 MRPS15 170 66 chr1 29437000 29439000 2385 PTPRU 363 177 chr1 23752000 23761000 2409 ID3 524 155 chr12 5409000 5419000 2459 NTF3 2430 1138.5 chr10 94812000 94815000 2489 CYP26C1 1150 565 chr19 55912000 55917000 -2493 SHANK1 250 114 chr17 17521000 17525000 -2512 RAI1 1141 620.5 chr2 26924000 26930000 2527 DPYSL5 1508.5 778 chr22 19652000 19656000 2553 AIFM3 347 152 chr13 99419000 99420000 2679 ZIC5 56.5 6 chr7 97342000 97343000 -2710 ASNS 69.5 22 chr16 30579000 30582000 -2779 FBRS 195 94 chr10 71478000 71481000 -2863 H2AFY2 247.5 115.5 chr9 126569000 126572000 2897 NR6A1 254 51.5 chr2 144991000 144992000 2928 ZEB2 95.5 49 chrX 67964000 67973000 2935 EFNB1 1369 636 chr7 107432000 107436000 -2960 LAMB1 629.5 333.5 chr13 60674000 60676000 -2985 MIR3169 231.5 85.5 chr8 23153000 23155000 -3095 CHMP7 69.5 24.5 chr17 76983000 76987000 -3135 BAHCC1 464 189.5 chr17 39991000 39996000 3162 FZD2 583 137.5 chr6 34078000 34080000 -3194 MIR1275 140.5 86 chr1 155100000 155101000 3205 NTRK1 81 25 chr6 47864000 47868000 3237 OPN5 332 131.5 chr19 1403000 1406000 3352 APC2 151 57.5 chr17 45408000 45409000 3371 DLX4 59 19.5 chr1 229622000 229626000 3413 EGLN1 581 250.5 chr9 88746000 88751000 3424 GAS1 398 158 chr6 144374000 144376000 -3440 HYMAI 121 40.5 chr6 154867000 154873000 3445 CNKSR3 583 212 chr17 45994000 46000000 3552 CACNA1G 1144.5 560 chr12 6753000 6758000 3569 LAG3 317 141 chr7 112509000 112511000 3629 GPR85 106.5 41.5 chr18 21181000 21184000 3712 ZNF521 414.5 140 chr19 58191000 58194000 -3904 ZNF702P 163 65.5 chr4 1837000 1841000 -3921 WHSC1 526 282.5 chr16 30613000 30615000 -3963 SRCAP 314 145.5 chr13 99433000 99439000 3973 ZIC2 203 62 chr1 229181000 229190000 4054 ARV1 1804.5 875 chrX 67961000 67962000 -4065 EFNB1 60 21 chr11 75161000 75162000 4074 DGAT2 28 12 chr4 108960000 108962000 -4170 SGMS2 176 73.5 chr10 115301000 115313000 4232 HABP2 3263 1478 chr4 106691000 106704000 4274 ARHGEF38 2042 825 218 chr11 29999000 30000000 -4347 KCNA4 466 261 chr19 52618000 52620000 -4403 MEIS3 355.5 138 chr1 855000 856000 4516 SAMD11 172.5 66 chr2 45016000 45020000 -4541 SIX3 1292.5 641.5 chr1 37868000 37869000 4578 RSPO1 164 28.5 chr14 85070000 85072000 4759 FLRT2 305.5 133 chr8 41281000 41301000 -4853 SFRP1 1400.5 366 chr1 112847000 112849000 -4893 WNT2B 489 216.5 chrX 39916000 39917000 5026 BCOR 47.5 10.5 chr15 88090000 88091000 5044 MESP1 323 137.5 chr9 116719000 116736000 5091 TNFSF8 2707.5 1215 chr12 40920000 40928000 -5579 YAF2 1305.5 588.5 chr12 48729000 48735000 -5754 ACCN2 1013 496.5 chr16 66080000 66082000 -5783 AGRP 346 176 chr8 65441000 65444000 -5829 LOC100130155 248 47.5 chr20 177000 184000 -5877 DEFB132 1252 630 chr6 170446000 170449000 -5878 DLL1 201 104.5 chr17 17628000 17631000 -5932 SMCR5 345.5 175.5 chr1 6230000 6236000 6161 HES3 691 252 chr10 21828000 21837000 -6281 C10orf114 418.5 149.5 chr12 52006000 52010000 -6321 AAAS 399 180.5 chr6 49628000 49637000 6428 C6orf141 646.5 263.5 chr11 66251000 66253000 -6554 SPTBN2 70 14.5 chr7 79594000 79597000 -6576 GNAI1 169 65 chr8 124483000 124486000 -6614 ATAD2 93.5 34.5 chr9 34572000 34574000 6722 CNTFR 128 59 chr19 7838000 7839000 6826 FLJ22184 40.5 10.5 chr17 45411000 45413000 6871 DLX4 95.5 52.5 chr20 2627000 2630000 6976 EBF4 257 122 chr11 121515000 121516000 7011 MIRLET7A2 102 35.5 chr11 75164000 75165000 7074 DGAT2 20 5.5 chrX 128609000 128610000 7114 APLN 101.5 35.5 chr1 155122000 155124000 -7147 PEAR1 75.5 33 chr1 150097000 150103000 -7203 THEM5 695 255 chr3 27731000 27732000 7289 EOMES 363.5 153 chr19 1407000 1410000 7352 APC2 90.5 34.5 chr2 144986000 144988000 7428 ZEB2 877 504 chr14 50359000 50361000 7589 NIN 89.5 25.5 chr6 34083000 34084000 -7694 MIR1275 227.5 114 chr3 46906000 46907000 7757 PTH1R 75.5 35 chr17 45418000 45421000 8087 DLX3 303 131 chr5 135556000 135574000 -8250 LOC389332 2756 1264.5 chr6 4729000 4731000 8321 CDYL 101.5 31 chr1 203692000 203693000 -8351 MIR135B 7 2 chr8 61740000 61751000 -8393 CHD7 520 179.5 chr1 2459000 2461000 -8456 HES5 146 52 chr1 226326000 226331000 -8484 ARF1 549 221 219 chr17 17529000 17539000 8488 RAI1 1260 571.5 chr1 23767000 23768000 -8591 ID3 63 26 chr22 36463000 36464000 -8687 TRIOBP 93 40 chr10 71472000 71475000 -8863 H2AFY2 203.5 96 chr17 23766000 23767000 -9145 SLC46A1 71.5 20.5 chr1 834000 837000 9180 FLJ39609 53 15 chr20 2445000 2449000 -9222 ZNF343 273.5 119.5 chr20 29911000 29913000 9462 DUSP15 72.5 18.5 chr10 92679000 92682000 -9488 ANKRD1 450.5 192 chr8 54946000 54947000 -9495 RGS20 40.5 17 chr1 37862000 37865000 9578 RSPO1 95.5 30 chr3 185770000 185774000 9719 EPHB3 670 295.5 chr3 46908000 46909000 9757 PTH1R 151.5 83.5 chr5 137457000 137458000 9827 WNT8A 190 80 chr9 123090000 123094000 -9900 GSN 250 131 H9P2 over-enriched loci mapping to predicted enhancer regions chr1 23751866 23760866 chr9 123090050 123094050 chr1 23766800 23767800 chr9 123454050 123456050 chr1 33108000 33112000 chr9 126568550 126571550 chr1 56672250 56676250 chr9 131260475 131262475 chr1 67749900 67760900 chr9 133830400 133831400 chr1 109594550 109596550 chr10 21827850 21836850 chr1 174155666 174160666 chr10 21842450 21844450 chr1 229178900 229187900 chr10 71471000 71474000 chr1 229221933 229224933 chr10 95508350 95509350 chr1 229411350 229415350 chr10 104254350 104256350 chr1 229622850 229626850 chr11 2127450 2129450 chr1 242175500 242178500 chr11 65358250 65359250 chr2 5968775 5973775 chr11 66251150 66253150 chr2 8733075 8743075 chr11 75163700 75164700 chr2 26248650 26250650 chr11 98801050 98802050 chr2 44886300 44889300 chr11 117251750 117254750 chr2 139841700 139844700 chr11 124539650 124541650 chr2 155981441 155984441 chr12 1572450 1574450 chr2 172754250 172755250 chr12 6798400 6804400 chr2 183438850 183441850 chr12 13789216 13792216 chr2 222039800 222042800 chr12 13793500 13795500 chr2 233473016 233474016 chr12 50523825 50527825 chr2 239230400 239232400 chr12 112405300 112407300 chr1 5974150 5978150 chr12 112434900 112440900 chr3 4248800 4250800 chr13 22903950 22904950 chr3 16898750 16902750 chr13 52813100 52818100 chr3 129875650 129879650 chr13 57923050 57926050 220 chr3 185774900 185775900 chr13 60673750 60675750 chr3 185783325 185801325 chr13 99432850 99438850 chr5 32535200 32538200 chr14 56380100 56387100 chr5 36452366 36455366 chr14 101478950 101483950 chr5 60663600 60664600 chr15 34976600 34977600 chr5 85223000 85230000 chr16 51139750 51141750 chr5 107031700 107032700 chr16 78188925 78191925 chr5 127902150 127904150 chr16 85398100 85399100 chr6 27832787 27840787 chr17 29100550 29102550 chr6 49626600 49635600 chr17 39969300 39978300 chr6 89814400 89817400 chr17 69960150 69963150 chr6 170446950 170449950 chr17 76982400 76986400 chr7 23727450 23731450 chr18 10464725 10470725 chr7 34360600 34366600 chr18 17215400 17227400 chr7 97341500 97342500 chr19 5443450 5445450 chr8 23153100 23155100 chr19 55742100 55754100 chr8 23155250 23159250 chr19 55911966 55916966 chr8 26362450 26364450 chr19 59157500 59159500 chr8 38505883 38506883 chr19 60579150 60583150 chr8 41283366 41303366 chr20 2632450 2637450 chr8 41318800 41320800 chr20 42677360 42681360 chr8 56154466 56156466 chr22 19653050 19657050 chr8 61743100 61754100 chrX 28250900 28253900 chr8 61994450 61999450 chrX 128911700 128918700 chr8 124483400 124486400 chrX 128940666 128948666 chr9 36127350 36132350 chr12 89900966 89903966 H9 P5K27 over-enriched loci (>=50%) compared to H9 P2K27, within 10,000bp around TSS. Chromosome start, end, distance to TSS, gene name, quantile normalized tag count in H9P2 and H9P5 shown Chr Start End Dist. To TSS Gene Name #H9P2 #H9P5 chr2 166358000 166360000 49 GALNT3 37 93 chrX 146167000 146170000 54 MIR514-1 36 99.5 chr11 57038000 57040000 -66 SLC43A1 105.5 290 chr1 143052000 143054000 112 LOC728875 22.5 140 chr1 143233000 143234000 -174 LOC728875 33.5 120.5 chr1 16201000 16206000 182 C1orf64 126 406.5 chr3 181235000 181239000 211 PEX5L 204.5 429 chr14 60818000 60819000 -217 TMEM30B 11.5 45 chrX 109649000 109652000 304 TDGF3 47.5 138 chr4 191226000 191227000 355 DUX2 0.5 28 chr20 31737000 31738000 371 E2F1 40.5 166 chr3 46592000 46597000 420 TDGF1 108 704.5 221 chr11 133648000 133654000 -485 GLB1L3 111 478.5 chr1 27158000 27160000 488 C1orf172 38.5 120.5 chr17 7105000 7108000 488 CLDN7 78.5 276.5 chr10 71871000 71873000 -529 NODAL 189 515 chr8 95720000 95724000 -540 ESRP1 89 349.5 chr22 38721000 38722000 601 FAM83F 53.5 187 chr1 208045000 208046000 602 IRF6 9.5 45.5 chr19 53138000 53154000 635 SNAR-C3 76.5 415 chr1 63556000 63568000 682 FOXD3 1555 3656 chr5 80292000 80294000 686 RASGRF2 157.5 367 chr1 168382000 168383000 688 METTL11B 40 96 chrX 152746000 152751000 697 PDZD4 140 469 chr16 372000 374000 758 LOC100134368 41.5 123.5 chr1 150232000 150233000 838 S100A10 54.5 125 chr10 135329000 135330000 -858 DUX4 0 32 chr4 155885000 155886000 887 LRAT 41.5 72 chr1 142536000 142538000 -890 LOC100286793 69.5 177 chr1 146399000 146400000 -896 FLJ39739 18.5 137.5 chr11 406000 407000 897 SIGIRR 27.5 84.5 chr11 127896000 127897000 915 ETS1 46.5 72 chr13 110012000 110014000 -928 RAB20 67.5 172.5 chrX 107221000 107224000 945 ATG4A 22 78 chr4 8966000 8980000 -953 USP17 40.5 585.5 chr11 68364000 68366000 975 CPT1A 156.5 381 chr1 12046000 12048000 979 TNFRSF8 138.5 328.5 chr20 17456000 17462000 1013 BFSP1 368.5 968.5 chrX 15591000 15593000 1075 TMEM27 17.5 44.5 chr19 55294000 55296000 1080 SNAR-A7 8 52.5 chrX 131399000 131406000 -1101 MBNL3 75 182.5 chr3 172661000 172663000 -1109 TNIK 189 455.5 chr11 67106000 67107000 -1142 GSTP1 22 65 chr1 27193000 27195000 1218 TRNP1 19.5 71.5 chr19 53100000 53108000 1253 SNAR-A13 26.5 255 chr12 6180000 6182000 1257 CD9 31.5 122 chrX 146172000 146173000 -1260 MIR514-3 7 32 chr11 69226000 69228000 1287 FGF19 87 277.5 chr5 141683000 141684000 1304 SPRY4 68.5 190 chr17 38976000 38979000 1331 ETV4 74.5 243.5 chr17 26912000 26913000 1372 MIR193A 50 157.5 chr5 88003000 88004000 1402 LOC645323 59.5 183 chr14 98808000 98810000 -1425 BCL11B 60 221.5 chrX 15594000 15595000 -1425 TMEM27 9.5 19 chr16 3026000 3028000 -1457 CCDC64B 108 354 chr1 20488000 20489000 -1499 VWA5B1 62.5 167 chr11 127898000 127900000 -1585 ETS1 66.5 197 chr20 16656000 16658000 -1609 SNRPB2 41.5 112.5 chr8 102574000 102577000 1656 GRHL2 38 131 222 chr1 16353000 16354000 1669 EPHA2 59 177 chr4 8934000 8963000 -1727 USP17 52.5 1228.5 chr14 54101000 54104000 -1887 SAMD4A 141 322 chr3 109425000 109427000 -1893 IFT57 34.5 124.5 chr6 126109000 126112000 -1925 HEY2 115.5 280 chr9 95149000 95152000 -1983 C9orf129 36.5 205.5 chr11 68367000 68369000 -2025 CPT1A 251.5 542.5 chr9 107460000 107465000 -2059 TAL2 246.5 591.5 chr4 155881000 155884000 -2113 LRAT 117 260.5 chr19 53119000 53123000 2152 SNAR-A7 14 112 chr8 86742000 86764000 2157 REXO1L2P 248 2288.5 chr12 95114000 95115000 2162 ELK3 19 44 chr11 117625000 117627000 2221 MPZL3 56.5 123.5 chr19 55304000 55305000 -2297 SNAR-A11 0 28 chr2 275000 277000 2308 FAM150B 192.5 390 chr19 55289000 55291000 -2322 SNAR-A7 12 62 chr19 55325000 55327000 -2409 SNAR-A5 6 61 chr19 55309000 55311000 -2443 SNAR-A3 8.5 84 chr16 31393000 31396000 2473 TGFB1I1 230.5 439 chrX 100430000 100431000 2481 TAF7L 3.5 19 chrX 153683000 153686000 2496 MPP1 247.5 523 chr1 246638000 246639000 2581 OR2T1 6 56.5 chr19 41491000 41492000 -2585 LOC100134317 4 44.5 chr22 30218000 30219000 -2626 EIF4ENIF1 29 64.5 chrX 104949000 104952000 -2692 NRK 149.5 342 chr11 117629000 117633000 -2779 MPZL3 86 203 chr9 125207000 125208000 -2797 MIR601 6.5 35.5 chr2 159529000 159532000 -2892 TANC1 201 507 chr9 14302000 14312000 -2955 NFIB 304 842.5 chr1 208027000 208028000 -2987 C1orf74 18 60.5 chr2 132728000 132742000 -2988 ANKRD30BL 1424.5 2711 chrX 140421000 140422000 2991 CXorf18 7 31.5 chr2 101137000 101138000 -3222 TBC1D8 12.5 26 chr1 46036000 46041000 -3372 MAST2 87 223.5 chrX 110256000 110257000 3539 PAK3 2 18 chr19 63742000 63746000 -3648 TRIM28 1050 2197.5 chrX 106242000 106248000 3713 RBM41 59.5 174 chr20 20300000 20301000 3735 INSM1 35.5 135 chr10 135337000 135338000 3768 DUX2 0 45.5 chr3 113203000 113206000 3803 TAGLN3 217.5 588 chr16 32067000 32068000 3875 HERC2P4 9 37 chr8 11099000 11102000 -4215 XKR6 135 300 chrX 509000 510000 4421 SHOX 593.5 879.5 chr1 1003000 1005000 -4450 RNF223 98 243.5 chr20 31728000 31733000 -4575 NECAB3 370.5 1322 chr19 42454000 42459000 -4748 LOC284412 32.5 186 chrX 131091000 131098000 -4769 FRMD7 59 181 223 chr15 63160000 63162000 4793 KBTBD13 106.5 259 chr8 86851000 86910000 4861 REXO1L2P 641 6255.5 chr15 58472000 58473000 4977 ANXA2 16 47 chrX 16802000 16805000 -5045 RBBP7 32.5 93 chr9 21987000 21993000 5210 CDKN2B-AS1 482.5 1020.5 chr10 104138000 104140000 -5219 NFKB2 40 117 chr13 52324000 52328000 -5224 PCDH8 102.5 342 chr14 91102000 91109000 5636 C14orf184 730.5 1609 chr14 73317000 73319000 5649 C14orf43 215 424 chr10 15948000 15949000 -5975 FAM188A 47.5 109 chr17 26902000 26908000 -6128 MIR193A 573.5 1594 chrX 137769000 137773000 6383 FGF13 40 106.5 chrX 15586000 15587000 6575 TMEM27 2.5 18 chr21 46218000 46221000 -6591 COL6A1 68.5 266 chr3 13888000 13892000 6619 WNT7A 359.5 853 chr15 70315000 70320000 -6762 PKM2 607.5 1149 chrX 130977000 130979000 -6926 MST4 16 62.5 chrX 131080000 131085000 7231 FRMD7 71.5 147 chrX 135390000 135391000 -7291 BRS3 10.5 38 chr5 151271000 151283000 7590 GLRA1 660.5 2040 chr3 57165000 57168000 7943 IL17RD 125.5 377.5 chrX 131408000 131412000 -8601 MBNL3 33 97.5 chr2 132282000 132287000 -8796 C2orf27B 103 354.5 chr12 113597000 113598000 8852 TBX3 155 425.5 chr5 169582000 169585000 -9028 C5orf58 73.5 190.5 chr11 3631000 3633000 9063 ART1 16 169 chr19 41483000 41487000 -9085 LOC100134317 0 156 chr7 31046000 31053000 -9101 ADCYAP1R1 210 790.5 chrX 16788000 16789000 9399 RBBP7 3.5 13 chr4 88656000 88664000 9679 SPARCL1 319 1089 HPP5 overenriched loci mapping to predicted enhancer regions chr1 44741580 44752580 chr9 95148450 95151450 chr1 44777383 44781383 chr9 107448583 107456583 chr1 46037225 46042225 chr9 134290250 134294250 chr1 59220300 59221300 chr10 71871500 71873500 chr1 161796350 161798350 chr10 90987100 90995100 chr1 183765383 183769383 chr11 26912300 26921300 chr1 203237025 203241025 chr11 30429725 30430725 chr1 208044700 208045700 chr11 30726250 30729250 chr2 274150 276150 chr11 67106050 67107050 chr2 3246250 3254250 chr11 82202900 82204900 chr2 7246950 7249950 chr11 82232000 82237000 chr2 65636783 65643783 chr11 127897350 127912350 chr2 106782450 106784450 chr11 132602283 132606283 224 chr2 118844366 118852366 chr11 133649225 133655225 chr2 118947050 118955050 chr12 74493550 74495550 chr2 223316812 223328812 chr12 95113900 95114900 chr2 225755450 225757450 chr12 128919860 128924860 chr3 18248950 18253950 chr13 39817033 39822033 chr1 16352800 16353800 chr13 42344416 42346416 chr1 22408011 22412011 chr13 52323550 52327550 chr3 46592800 46597800 chr13 110011900 110013900 chr3 57166150 57169150 chr13 111109200 111111200 chr3 62369900 62380900 chr14 78624850 78627850 chr3 72721000 72724000 chr14 86688800 86707800 chr3 113202600 113205600 chr15 58472400 58473400 chr3 120524525 120526525 chr15 63160650 63162650 chr3 140062083 140066083 chr15 90756625 90759625 chr3 142274650 142278650 chr16 372100 374100 chr3 151234050 151239050 chr16 8540875 8545875 chr3 160350700 160351700 chr16 31393700 31396700 chr3 181235133 181239133 chr16 49200000 49205000 chr3 191003733 191010733 chr17 17709811 17714811 chr4 4746450 4748450 chr18 5263800 5270800 chr4 4833344 4839344 chr18 6010612 6015612 chr4 88657216 88665216 chr18 33318300 33319300 chr4 93754600 93756600 chr19 11662675 11668675 chr4 124647000 124648000 chr19 41482750 41486750 chr4 139536900 139541900 chr19 41490900 41491900 chr4 139801350 139805350 chr19 53101166 53109166 chr4 179908716 179914716 chr19 53118750 53122750 chr4 185324300 185326300 chr19 53139934 53155934 chr4 185961466 185970466 chr19 55293900 55295900 chr5 17581366 17583366 chr19 55325500 55327500 chr5 118394944 118396944 chr20 11175400 11180400 chr5 151271400 151283400 chr20 17456850 17462850 chr5 163655100 163657100 chr20 20300250 20301250 chr5 166934600 166936600 chr20 44680875 44686875 chr6 19418600 19422600 chr20 46799900 46803900 chr6 26837400 26840400 chr21 17959450 17965450 chr6 26878600 26880600 chr21 22211400 22218400 chr6 112315355 112319355 chr21 46218450 46221450 chr6 126107900 126110900 chr22 30217725 30218725 chr6 155482070 155486070 chrX 15645400 15647400 chr6 156537825 156540825 chrX 104949650 104952650 chr7 30989737 30994737 chrX 106243250 106249250 chr7 31045900 31052900 chrX 109171750 109178750 chr7 68620925 68622925 chrX 109648950 109651950 chr7 70882000 70885000 chrX 113739800 113742800 chr7 102020000 102023000 chrX 114988750 114989750 chr7 102115450 102117450 chrX 130435750 130440750 225 chr8 5504666 5520666 chrX 130943100 130944100 chr8 25598100 25606100 chrX 131078933 131083933 chr8 95719466 95723466 chrX 131091200 131098200 chr8 101543412 101549412 chrX 131408850 131412850 chr9 7767350 7773350 chrX 132293150 132300150 chr9 14304358 14314358 chrX 137243200 137246200 chr9 27737061 27742061 chrX 138249866 138254866 chr9 90354625 90356625 chrX 152746650 152751650 226 Appendix D: H3K4me3 Quantitative Changes in LP LP P2K4 over-enriched loci (>=50%) compared to LP P5K4, within 10,000bp around TSS. Chromosome start, end, distance to TSS, gene name, quantile normalized tag count in LPP2 and LPP5 shown Chr Start End Dist. To TSS Gene Name #LPP2 #LPP5 chr10 92680350 92681350 -9838 ANKRD1 33.5 9 chrX 102907950 102908950 -9645 PLP1 58.5 21 chr1 154674300 154676300 -9492 C1orf61 71.5 18.5 chr19 60654266 60656266 -9224 SHISA7 141 50.5 chr1 226837300 226838300 -9217 RHOU 141 50.5 chr19 13836833 13837833 -9180 MIR181C 87.5 18 chr4 986050 987050 -9060 FGFRL1 31 5 chr3 50239260 50240260 -8891 GNAI2 50 50 chr1 202613650 202614650 -8680 LOC127841 63 21.5 chr1 203665866 203666866 -8529 LEMD1 18 1 chr13 111760966 111761966 -8448 SOX1 13 6.5 chr15 26025250 26026250 -7697 OCA2 90 36 chr15 50876537 50877537 -7536 ONECUT1 7 2.5 chr9 95154700 95156700 -7183 C9orf129 43.5 7.5 chr9 129544100 129545100 -7175 TOR2A 28.5 5 chr19 18204300 18205300 -6552 PDE4C 90.5 21 chr12 101868650 101869650 -6432 ASCL1 60 16.5 chr18 74834900 74835900 -5863 SALL3 79 28 chr16 12250 13250 -5426 LOC100288778 19.5 4 chr17 45396000 45397000 -5061 DLX4 35 30 chr11 46369100 46370100 -4917 CHRM4 88.5 31 chr22 48628100 48629100 -4901 ZBED4 78 22.5 chr12 52629900 52630900 -4581 HOXC12 21.5 11.5 chr8 21962350 21963350 -4177 EPB49 105.5 35.5 chr4 32800 46800 -3427 ZNF595 1505 670.5 chr17 17625960 17627960 -3392 SMCR5 203 74 chr12 129095450 129096450 -3110 LOC100190940 26.5 13 chr9 97313233 97314233 -3081 PTCH1 84 107.5 chr17 21245000 21247000 -3041 KCNJ18 104 30 chr7 100598100 100599100 -3028 VGF 21 15 chr11 1976650 1980650 -3009 H19 152 43.5 chr17 26907900 26908900 -2728 MIR193A 17.5 1.5 chr7 100395900 100397900 -2724 MUC12 190 69.5 chr22 19663433 19664433 -2625 LZTR1 9 9 chr20 30635400 30637400 -2542 LOC149950 167.5 68.5 chr22 48606450 48607450 -2494 BRD1 45.5 11.5 chr11 43557000 43558000 -2020 MIR129-2 237.5 84.5 chr4 191223600 191224600 -1974 DUX4L6 34 9 227 chr6 3174263 3175263 -1796 TUBB2B 112 75 chr17 75429050 75430050 -1742 CBX4 29 20 chr3 180461533 180462533 -1660 KCNMB3 21 1 chr10 127567033 127580033 -1565 FANK1 2170 901 chr19 55912975 55913975 -1468 SHANK1 95.5 33.5 chr15 80126328 80127328 -1412 MEX3B 36.5 30 chrX 82648025 82649025 -1400 POU3F4 7.5 6 chr14 54102650 54103650 -1237 SAMD4A 83.5 69.5 chr5 178354900 178356900 -1170 GRM6 32 12.5 chr12 55920900 55922900 -1158 NDUFA4L2 117.5 50.5 chr8 141715466 141716466 -1138 EIF2C2 21 21.5 chr2 28467650 28468650 -1133 FOSL2 71.5 59 chr19 36532620 36533620 -1090 TSHZ3 114.5 70.5 chr14 99141066 99142066 -1086 CCDC85C 132.5 65.5 chr17 39631600 39632600 -1045 ATXN7L3 114.5 34 chr20 56859387 56861387 -1034 GNAS-AS1 24 6.5 chr18 68686300 68687300 -1010 NETO1 78 56.5 chr8 144399066 144400066 -918 ZFP41 24.5 7 chr14 41144600 41146600 -914 LRFN5 262.5 187.5 chr22 48949900 48950900 -887 PANX2 66.5 12 chr2 45731200 45732200 -847 PRKCE 99 54 chr18 13206950 13208950 -836 C18orf1 187.5 73 chr9 34579500 34581500 -778 CNTFR 171.5 72 chr16 2460225 2461225 -776 NTN3 32 14 chr8 140784250 140786250 -769 KCNK9 124 54 chr6 34313366 34314366 -680 HMGA1 231.5 128 chr16 88168500 88169500 -677 CPNE7 75 33 chrX 152747850 152751850 -653 PDZD4 147 49 chr2 191220950 191221950 -643 NAB1 107.5 40.5 chr10 134449666 134450666 -639 NKX6-2 24 16 chr1 147195900 147196900 -629 LOC645166 28.5 8 chr4 78959675 78960675 -607 CNOT6L 56.5 65 chr14 103652400 103653400 -595 MIR203 38 6.5 chr2 200038150 200039150 -574 SATB2 13 13 chr3 173648950 173649950 -510 GHSR 36.5 7.5 chr4 994600 995600 -510 FGFRL1 59 9 chrX 55531866 55533866 -510 USP51 93.5 8 chr2 227371750 227372750 -500 IRS1 168 70.5 chr7 121731780 121732780 -479 FEZF1 70.5 69.5 chr7 100331450 100332450 -473 ACHE 76.5 28 chr17 30783616 30784616 -460 SLFN12 25.5 11.5 chr12 48218250 48219250 -457 KCNH3 86.5 39.5 chr17 76064950 76065950 -451 NPTX1 36.5 18 chr12 119291275 119292275 -434 MSI1 102 94.5 chr12 48736850 48737850 -404 ACCN2 177.5 51 chr11 68537300 68538300 -374 MRGPRF 35 5.5 chr2 239987450 239988450 -370 HDAC4 28.5 50.5 228 chr22 45311100 45313100 -369 CELSR1 161 70 chr4 37568250 37569250 -365 TBC1D1 159 68.5 chrX 101861450 101862450 -348 BHLHB9 56.5 15.5 chr16 87534950 87535950 -341 CBFA2T3 56.5 6.5 chr19 7565950 7566950 -338 KIAA1543 92.5 31.5 chr21 32166666 32167666 -333 HUNK 123 50.5 chr19 11452633 11453633 -330 ELAVL3 18 9.5 chr4 3737266 3738266 -328 ADRA2C 46 18 chr9 132804100 132805100 -324 FIBCD1 102 38.5 chr1 41020950 41022950 -321 KCNQ4 104 38.5 chr1 48234960 48235960 -311 LOC388630 56.5 24 chr15 35178700 35179700 -311 MEIS2 70.5 129.5 chr19 18577966 18579966 -306 CRLF1 123.5 50.5 chr17 56831250 56832250 -289 TBX2 53 13 chrX 50230250 50231250 -289 DGKK 129.5 42 chr19 876250 877250 -287 ARID3A 214.5 95.5 chr9 111300200 111301200 -286 PTPN3 32.5 36 chr3 27738550 27739550 -261 EOMES 32.5 46 chr11 7651150 7652150 -253 CYB5R2 54.5 12.5 chr3 50167100 50168100 -252 SEMA3F 135 73 chr12 131415700 131416700 -222 GALNT9 32.5 2.5 chr16 29929600 29930600 -198 DOC2A 141 36.5 chr1 200429050 200430050 -191 LGR6 31 3.5 chr17 75623933 75624933 -191 TBC1D16 183.5 87 chr7 154425300 154426300 -185 PAXIP1 32 40 chr10 135042050 135043050 -181 PAOX 28.5 21 chr1 15608300 15609300 -178 EFHD2 129 51.5 chr11 128265850 128266850 -173 KCNJ5 119 37.5 chr4 74953300 74954300 -173 CXCL1 29 2 chr17 42250750 42251750 -169 WNT3 58 61.5 chr16 65195400 65196400 -168 CMTM3 81 63 chr22 42137300 42138300 -164 MPPED1 44 9 chr1 59022020 59023020 -147 JUN 70.5 69.5 chr10 62430850 62431850 -146 RHOBTB1 37.5 19 chr19 2979800 2980800 -135 TLE2 76 30 chr9 113401166 113402166 -134 PTGR1 16 2 chr19 61679925 61681925 -130 LOC100128252 115.5 7.5 chr20 41251600 41252600 -129 PTPRT 62 20 chr9 98421557 98422557 -124 CDC14B 52 89.5 chr19 9295250 9297250 -121 ZNF559- ZNF177 131 61 chrX 111809650 111810650 -119 LHFPL1 44.5 10 chr1 21868050 21869050 -107 RAP1GAP 77 44 chr18 59137200 59138200 -107 BCL2 123.5 61.5 chr11 65815350 65816350 -99 TMEM151A 105.5 22 chr19 38979000 38980000 -91 KCTD15 222 83.5 chr14 100103250 100105250 -90 BEGAIN 206.5 73 229 chr20 26135500 26138500 -86 MIR663 683 198.5 chr1 2312500 2313500 -74 RER1 90 26.5 chr4 17391800 17392800 -67 FAM184B 36.5 5 chr1 205560875 205561875 -65 CD55 68.5 65 chr17 76622980 76623980 -62 BAIAP2 74 22 chr3 129023950 129024950 -59 MGLL 200 59.5 chr1 15882860 15883860 -54 PLEKHM2 96.5 36 chr19 42553350 42554350 -49 ZNF527 78 28.5 chr19 63642950 63643950 -49 ZNF132 45 19 chr3 9570033 9571033 -47 LHFPL4 33 28 chr19 1236633 1237633 -35 EFNA2 127 49.5 chr17 958600 959600 -26 ABR 81 25.5 chr1 31855366 31856366 -22 HCRTR1 92.5 40.5 chr14 90352033 90353033 -19 TTC7B 23 10.5 chr8 144312350 144313350 -18 LY6H 78.5 30.5 chrX 107865280 107867280 -17 IRS4 45.5 8 chr19 41787050 41789050 -11 ZNF382 249 50.5 chr12 128953675 128954675 -10 TMEM132D 80.5 27 chr2 120939900 120940900 -5 LOC84931 29 2 chr7 100047550 100048550 0 MOSPD3 140 80 chr8 42028160 42029160 2 MYST3 47 35.5 chrX 151991000 151992000 5 PNMA6A 26.5 5.5 chr20 57308400 57309400 6 EDN3 14.5 4 chr10 134060200 134061200 8 PWWP2B 77.5 27 chr19 42098025 42100025 8 ZNF829 124 16 chr9 68470350 68471350 11 LOC440896 24 8 chr16 2185950 2186950 16 CASKIN1 117 32 chr17 4348400 4349400 22 SPNS2 24 17.5 chr15 68176780 68177780 30 TLE3 52 82 chr1 6162250 6163250 31 CHD5 52.5 7 chr10 79066550 79068550 33 KCNMA1 206 96.5 chrX 17787833 17789833 35 RAI2 180 57 chr10 75214650 75216650 39 KIAA0913 315 141.5 chr14 104515200 104516200 39 AHNAK2 24 19 chr22 18635275 18636275 41 RTN4R 27.5 24 chrX 128804680 128805680 62 ZDHHC9 209.5 100 chr22 48981100 48982100 65 SELO 117 51 chr19 40213000 40214000 68 SCN1B 164 63.5 chrX 151974500 151976500 78 PNMA3 50.5 2.5 chr3 75915866 75917866 79 ZNF717 210.5 76.5 chr16 51137720 51138720 87 TOX3 102.5 19 chr6 14225433 14226433 89 CD83 144 75.5 chr19 1055240 1056240 91 GPX4 85 48 chr1 94084575 94085575 99 MIR760 69 65.5 chr14 91372000 91373000 102 TC2N 132 52 chr9 138125850 138127850 102 NACC2 204 63.5 chr17 62391050 62392050 108 CACNG4 109 41.5 230 chr11 133331475 133332475 115 IGSF9B 144 77 chr13 97593050 97594050 115 FARP1 124.5 63.5 chr2 242322325 242323325 122 D2HGDH 187 88.5 chr21 44109166 44110166 122 AGPAT3 55 10.5 chr10 43081750 43082750 123 RASGEF1A 47 9 chr16 695200 696200 126 FBXL16 54.5 15 chr19 17216520 17217520 131 NR2F6 128.5 21 chr4 48186200 48188200 134 ZAR1 35.5 4.5 chr8 24869400 24870400 148 NEFL 198.5 95.5 chr1 242690950 242691950 154 C1orf101 15 0 chr19 42516575 42518575 155 HKR1 117.5 35 chr22 49050250 49051250 156 MAPK11 50 17 chr7 732840 733840 164 PRKAR1B 75 24 chr2 197883100 197884100 166 ANKRD44 121 47.5 chrX 56274766 56276766 171 KLF8 125.5 10 chr8 99508100 99509100 174 KCNS2 38 10 chr10 105242900 105244900 175 NEURL 183.5 59 chr5 6767400 6768400 182 PAPD7 103 90 chr7 30510300 30511300 182 GGCT 76 2 chr3 161425800 161426800 183 LOC401097 108 41.5 chr6 157722233 157723233 188 ZDHHC14 142 68.5 chr11 119615850 119616850 189 POU2F3 44.5 13 chr9 110968700 110969700 192 C9orf4 99 32 chr1 11461580 11462580 198 PTCHD2 119 59 chr17 77602750 77603750 198 GPS1 179.5 82.5 chr16 23754500 23755500 199 PRKCB 116.5 36.5 chr7 44331050 44332050 205 CAMK2B 91.5 21 chr1 234916100 234917100 207 ACTN2 92 25.5 chr12 124115600 124116600 222 AACS 136.5 53 chr8 126053950 126055950 230 ZNF572 59.5 15 chr12 116020900 116021900 234 TESC 46 30 chr19 61710260 61712260 236 ZNF471 50 11.5 chr10 11905140 11906140 237 C10orf47 125 75 chrX 3640933 3641933 242 PRKX 48.5 10.5 chrX 53366500 53367500 247 IQSEC2 85.5 26.5 chr3 45242066 45243066 252 TMEM158 50 30.5 chr19 63237000 63238000 254 ZSCAN1 73.5 7 chr13 18816450 18818450 261 LOC100287114 47 6.5 chr9 139127750 139129750 266 DPP7 37 3 chr11 43920450 43921450 268 C11orf96 87.5 45 chrX 100758875 100759875 272 ARMCX6 38.5 6.5 chr9 78710050 78711050 273 PRUNE2 126.5 54 chr11 65802050 65803050 278 CNIH2 87.5 27.5 chrX 152844125 152845125 283 ARHGAP4 28.5 5 chr20 2728500 2729500 292 CPXM1 132.5 70 chr6 90177920 90178920 294 RRAGD 165.5 58.5 chr22 17517000 17518000 296 GSC2 81.5 16 231 chr6 118029100 118031100 298 GOPC 302 161.5 chr15 54812280 54813280 299 ZNF280D 20 3.5 chr11 56984220 56986220 305 RTN4RL2 162 81.5 chr5 148631400 148632400 306 AFAP1L1 80.5 70 chr19 57647950 57649950 309 ZNF578 110.5 9.5 chr10 12431400 12432400 311 CAMK1D 222 101.5 chr16 82559550 82560550 312 NECAB2 48 28 chr8 48812466 48813466 313 CEBPD 89.5 28 chrX 55760150 55762150 315 RRAGB 59 10 chr10 128065800 128067800 317 ADAM12 208 80 chr16 5023120 5024120 323 NAGPA 169.5 70.5 chr1 41099780 41100780 325 CITED4 56 19 chr15 84138366 84139366 327 KLHL25 83 74 chr22 45037050 45038050 333 PKDREJ 12.5 1 chr18 9603766 9604766 334 PPP4R1 77 22 chr22 15897300 15898300 340 CECR7 34 5.5 chr22 36152100 36154100 351 ELFN2 87 31.5 chr2 231967933 231969933 354 B3GNT7 123.5 46.5 chr1 148388650 148389650 356 PLEKHO1 213.5 97 chr19 43411300 43412300 357 DPF1 108 40 chr22 43442950 43443950 359 PRR5 87.5 23 chr1 12045900 12046900 379 TNFRSF8 19 1 chr21 45785933 45786933 380 SLC19A1 109.5 37 chr11 63815250 63816250 381 KCNK4 59 14.5 chr21 42171760 42172760 391 PRDM15 62 15 chr12 67425600 67427600 397 SLC35E3 136.5 8.5 chr22 17337925 17338925 398 DGCR5 161 75.5 chr2 88250850 88251850 400 THNSL2 54 2.5 chr1 1464700 1465700 403 C1orf70 105 41.5 chr6 31889900 31890900 414 HSPA1L 44 10.5 chr19 3310033 3312033 417 NFIC 99 32.5 chr22 44750250 44752250 422 WNT7B 179 56.5 chr14 103058000 103059000 423 CKB 86.5 44.5 chr11 14868900 14870900 427 CYP2R1 62 10 chr16 53915900 53916900 428 IRX6 130 68.5 chr20 61599500 61601500 449 EEF1A2 107 36 chr2 46696800 46697800 455 PIGF 49 87.5 chr16 658100 659100 466 RHOT2 131 57 chr7 127788000 127789000 475 PRRT4 65 35.5 chr20 3713850 3715850 487 CENPB 126.5 50 chr10 134823950 134824950 489 KNDC1 118.5 45.5 chr11 1925075 1926075 497 MRPL23 172.5 86.5 chr10 115793800 115794800 504 ADRB1 43.5 12.5 chr16 4683700 4684700 505 NUDT16L1 113 48.5 chr12 110328150 110329150 515 SH2B3 114.5 46 chr1 227635450 227636450 516 ACTA1 43.5 17 chr20 62042325 62043325 516 MIR1914 32 2.5 232 chr6 26124000 26127000 519 HIST1H1A 115 14.5 chr12 2032700 2033700 523 CACNA1C 155.5 77.5 chr16 65591950 65592950 533 CES4A 132 29.5 chrX 72583850 72584850 535 CDX4 16 3.5 chr7 148787950 148788950 536 ZNF777 134.5 63.5 chr7 101246050 101247050 542 CUX1 45 21.5 chr19 660000 661000 547 PALM 71.5 25.5 chr8 22465250 22466250 554 SORBS3 27 13 chr12 47650850 47651850 558 WNT10B 137.5 49 chr7 5289150 5290150 563 SLC29A4 97.5 30.5 chr6 52637225 52638225 567 LOC730101 43 7.5 chr7 24289900 24291900 568 NPY 86 12 chr2 42649250 42650250 575 MTA3 172 144.5 chr12 109956300 109957300 589 CUX2 144.5 76 chr9 20611425 20612425 589 MLLT3 75 80 chr4 91267800 91268800 593 FAM190A 193.5 95.5 chr6 7053183 7054183 598 RREB1 124.5 111 chr5 1575975 1576975 601 LPCAT1 134 53.5 chr16 673325 674325 615 JMJD8 97.5 30 chr14 103251950 103252950 616 ZFYVE21 149 62 chr5 88015250 88016250 626 LOC645323 73.5 29.5 chr19 4015680 4018680 636 ZBTB7A 240.5 84.5 chrX 55494900 55496900 637 MAGEH1 77.5 10.5 chr9 139890700 139894700 638 CACNA1B 102 15 chr3 213800 214800 650 CHL1 144 56 chr17 35109950 35110950 670 ERBB2 300.5 168.5 chr19 6374650 6375650 672 KHSRP 203 119 chr6 16868525 16869525 675 ATXN1 100.5 61.5 chr12 56300150 56301150 690 SLC26A10 28 6.5 chr7 128365700 128366700 693 IRF5 27.5 2.5 chr12 56417100 56418100 696 AGAP2 88.5 27.5 chr1 1157700 1158700 708 B3GALT6 38.5 3 chr6 91062075 91063075 708 BACH2 137.5 40 chr7 37453866 37454866 714 ELMO1 71 62.5 chr1 92720700 92721700 744 GFI1 73.5 41 chr4 808700 809700 745 CPLX1 40 5.5 chr8 22604600 22605600 747 EGR3 147 51 chr9 99785566 99786566 756 ANP32B 191.5 89 chr7 73710225 73711225 759 GTF2I 108 83 chr7 71438883 71439883 761 CALN1 106 31.5 chr1 55278000 55279000 763 PCSK9 54.5 21 chr1 22910200 22911200 782 EPHB2 53 35 chr9 124029550 124030550 790 LHX6 128.5 57 chr9 99655650 99656650 792 FOXE1 56 15.5 chr22 20200475 20201475 805 PI4KAP2 99.5 39.5 chr19 541200 542200 807 HCN2 25.5 10 chr6 3100966 3102966 816 TUBB2A 229.5 126 233 chr17 78071200 78072200 817 FOXK2 146 91.5 chr19 3523766 3525766 823 HMG20B 169 76.5 chr16 3253633 3255633 864 FLJ39639 91.5 14 chr1 235272700 235273700 875 RYR2 192.5 83 chr11 117983900 117984900 884 PHLDB1 86 30.5 chr17 71384850 71385850 901 TRIM47 68.5 18.5 chr17 34014300 34015300 909 SRCIN1 64 22.5 chr22 37123450 37124450 927 LOC400927 212 108 chr21 38954100 38955100 974 ERG 103 35.5 chr6 2819760 2821760 983 MGC39372 45.5 5.5 chr20 61572950 61573950 987 KCNQ2 85.5 33 chr3 79898250 79899250 999 ROBO1 142.5 76 chr17 78631366 78632366 1010 METRNL 35.5 19 chr8 125808400 125809400 1011 MTSS1 83.5 63.5 chr11 112337733 112338733 1028 NCAM1 169.5 75 chr9 130491866 130492866 1036 SET 176.5 166 chr1 178466600 178467600 1044 LHX4 203.5 76 chr6 166500550 166501550 1071 T 23.5 33 chrX 68642375 68643375 1072 FAM155B 22 12 chr22 43782666 43783666 1079 PHF21B 197.5 102.5 chr19 1812960 1813960 1104 KLF16 74.5 79 chr20 57613650 57614650 1152 PHACTR3 28 5 chr6 31243775 31246775 1155 POU5F1 425.5 222 chrX 117842500 117843500 1185 ZCCHC12 23.5 12.5 chr3 32835266 32836266 1252 TRIM71 152.5 208.5 chr1 10777533 10778533 1261 CASZ1 111.5 62 chr7 49784600 49785600 1297 VWC2 95 47.5 chrX 117993550 117994550 1309 LONRF3 83 39.5 chr2 25416433 25417433 1345 DNMT3A 82 23 chr16 2227333 2228333 1364 DNASE1L2 4 1 chr12 129210250 129211250 1426 FLJ31485 52 31.5 chr7 30291400 30292400 1452 ZNRF2 149 191 chr5 63291340 63292340 1462 HTR1A 8 6 chr19 55293600 55295600 1480 SNAR-A7 24 3.5 chr14 105012700 105014700 1524 CRIP2 18 19 chr18 44320466 44321466 1541 CTIF 119.5 37.5 chr20 3011200 3012200 1670 AVP 12 6.5 chr14 64078150 64079150 1711 HSPA2 16.5 5 chr7 523750 524750 1757 PDGFA 121.5 61.5 chr10 23522750 23523750 1784 PTF1A 14.5 25 chr9 138860000 138861000 1812 C9orf172 8 2.5 chr6 5950300 5951300 1832 NRN1 37 24.5 chr20 44092700 44093700 1972 SLC12A5 61.5 24.5 chr19 1201050 1202050 1998 MIDN 180.5 75 chr18 68359550 68361550 2153 CBLN2 45 7.5 chr9 67380580 67382580 2429 FAM27B 365 106 chr19 35557300 35558300 2632 ZNF536 61 13.5 234 chr16 71646850 71647850 2685 ZFHX3 10.5 10.5 chr8 145527450 145528450 2801 SCRT1 29.5 12 chr9 44932033 44934033 2801 FAM27C 412 132 chr3 46595950 46597950 2870 TDGF1 203 81.5 chr6 101956100 101957100 3018 GRIK2 26 1 chr17 35013750 35014750 3451 NEUROD2 51 18 chr15 82947733 82949733 3480 ZSCAN2 47.5 6 chr1 155160100 155161100 3552 LRRC71 108 33 chr1 17633000 17635000 3644 RCC2 45.5 12 chr10 135286000 135287000 3789 FRG2B 14.5 2.5 chr1 2969600 2970600 4049 FLJ42875 80.5 23 chr9 138229900 138230900 4425 LHX3 50.5 13 chr12 122439166 122440166 5009 SETD8 96 31.5 chr5 2809933 2810933 5171 C5orf38 86 36.5 chr4 1150833 1151833 5324 SPON2 19 1.5 chr19 13068500 13069500 5974 LYL1 39 9 chr5 145704350 145705350 6070 POU4F3 74.5 46 chr21 36999550 37000550 6189 SIM2 20.5 23.5 chr11 840150 841150 6204 TSPAN4 53.5 35.5 chr8 65661100 65662100 6251 BHLHE22 66.5 48.5 chr2 233449000 233450000 6262 C2orf82 144.5 45.5 chr6 50900500 50901500 6602 TFAP2B 19 9 chr20 22500050 22501050 6730 NCRNA00261 18.5 9 chr4 191175100 191182100 6806 LOC100288255 1045.5 291 chr15 34958650 34959650 6876 LOC145845 105.5 36 chr3 75801200 75805200 7023 FRG2C 608 315.5 chr16 66244300 66245300 7419 ACD 69.5 29 chr10 101290050 101291050 7870 NKX2-3 29.5 9.5 chr3 75770250 75771250 8146 MIR1324 78.5 36 chr19 8305450 8306450 8196 KANK3 64.5 18 chr15 67168700 67169700 8956 NCRNA00277 24.5 9 chr17 39786800 39787800 9283 GRN 11.5 1 chr9 138224450 138225450 9875 LHX3 57 13 LPP2K4 overenriched loci mapping to predicted enhancer regions Chr Start Stop Chr Start Stop chr2 198169300 198170300 chr7 37452825 37453825 chr2 204845000 204846000 chr7 41293675 41294675 chrX 139729300 139730300 chr7 45569500 45570500 chr2 94903900 94906900 chr7 48152050 48153050 chr2 96468700 96471700 chr7 52575210 52578210 chr2 97280125 97281125 chr7 55862200 55863200 chr16 46274600 46275600 chr19 1237250 1238250 235 chr16 51138000 51139000 chr19 1814150 1815150 chr16 52154200 52155200 chr19 3523625 3525625 chr16 56062325 56063325 chr19 4015700 4018700 chr16 60562800 60563800 chr19 6375050 6376050 chr16 66243850 66244850 chr19 6528150 6529150 chr16 80301190 80302190 chr19 7566200 7567200 chr17 2703100 2704100 chr19 17217200 17218200 chr2 207379283 207381283 chr10 42826146 42828146 chr2 207695733 207697733 chr10 44048966 44049966 chr6 63380550 63382550 chr10 54907040 54909040 chr2 210998050 211000050 chr10 55174200 55175200 chr2 220079650 220080650 chr10 58014575 58018575 chr2 224604450 224605450 chr10 59327983 59328983 chr2 225004900 225005900 chr10 65814133 65816133 chr2 227222350 227223350 chr19 22257900 22259900 chr2 227372250 227373250 chr19 23134800 23135800 chr2 231968266 231970266 chr19 24049550 24051550 chr2 232153300 232154300 chr19 33270100 33271100 chr2 233538383 233539383 chr19 33617900 33618900 chr2 235214100 235215100 chr19 36532050 36533050 chr2 235972825 235973825 chr19 38979000 38980000 chr6 67060650 67062650 chr19 42517250 42519250 chr2 237431850 237432850 chr19 42554100 42555100 chr2 239986700 239987700 chr19 42740000 42742000 chr2 242321800 242322800 chr19 43116666 43117666 chr3 598100 599100 chr19 43410650 43411650 chr3 5725283 5727283 chr7 63973450 63976450 chr3 16702400 16705400 chr7 63980000 63981000 chr3 16809325 16810325 chr7 66446950 66447950 chr11 56837950 56838950 chr7 73709700 73710700 chr17 17625850 17627850 chr1 14489750 14491750 chr11 62976850 62977850 chr1 15883000 15884000 chr11 63026600 63027600 chr1 17632966 17634966 chr17 21254100 21255100 chr1 19494300 19495300 chr11 65001550 65002550 chr1 22909750 22910750 chr11 65801900 65802900 chr1 34304200 34305200 chr11 68146350 68147350 chr1 41100100 41101100 chr11 68672900 68673900 chr1 48235100 48236100 chr11 70933550 70935550 chr1 48607250 48608250 chr11 71809300 71810300 chr1 53671550 53673550 chr11 91542125 91543125 chr1 54565500 54567500 chr11 92445735 92446735 chr1 58617000 58618000 chr11 93492487 93494487 chr1 64068300 64070300 chr11 94021000 94022000 chr19 52817975 52818975 chr11 102287275 102288275 chr14 20242800 20243800 chr17 30744950 30745950 chr14 20358907 20359907 chr11 108766300 108767300 chr14 23740200 23741200 236 chr11 113909150 113910150 chr19 55293900 55295900 chr17 39632150 39633150 chr19 55913200 55914200 chr3 34538300 34539300 chr19 56655725 56656725 chr3 43499050 43501050 chr19 56925100 56927100 chr3 43608700 43609700 chr19 57648150 57650150 chr3 46595550 46597550 chr19 58851000 58854000 chr3 50238800 50239800 chr19 58977450 58979450 chr3 61895300 61896300 chr14 27139850 27141850 chr3 72154050 72156050 chr19 60654650 60656650 chr3 75765300 75766300 chr7 100048300 100049300 chr3 75770100 75771100 chr4 63653000 63656000 chr3 75836450 75838450 chr7 100395450 100397450 chr6 74113975 74117975 chr7 101246000 101247000 chr3 76793760 76794760 chr7 105065300 105066300 chr6 77331833 77333833 chr7 105139000 105140000 chr3 78454833 78455833 chr7 120317950 120318950 chrX 151975000 151977000 chr7 125337166 125338166 chrX 152270450 152274450 chr7 126258150 126259150 chrX 152308500 152310500 chr4 72940250 72947250 chrX 152590683 152592683 chr4 73256100 73258100 chrX 152661300 152662300 chr7 131522250 131523250 chrX 152731700 152734700 chr7 131523987 131524987 chrX 152747525 152751525 chr7 137765500 137767500 chrX 152782000 152783000 chr7 139986200 139987200 chrX 152796816 152798816 chr7 140922860 140923860 chrX 152932100 152933100 chr4 79645950 79646950 chrX 152934100 152935100 chr4 84349250 84350250 chrX 152980000 152981000 chr4 85478650 85480650 chrX 153152650 153153650 chr7 143538000 143539000 chrX 154339200 154340200 chr4 87600350 87602350 chr11 123149718 123151718 chr4 89003300 89004300 chr12 4010070 4011070 chr4 89261325 89263325 chr12 13639150 13640150 chr4 91970062 91971062 chr12 14302779 14303779 chr7 146933287 146934287 chr12 25934700 25935700 chr7 147658700 147659700 chr12 27949750 27951750 chr7 147714900 147715900 chr12 30827100 30830100 chr7 148787900 148788900 chr12 32065966 32066966 chr4 93572283 93574283 chr12 33564950 33565950 chr4 95238900 95239900 chr12 33693225 33696225 chr1 83989400 83990400 chr3 87857770 87861770 chr1 94083750 94084750 chr3 105746500 105748500 chr1 96764350 96766350 chr3 106024625 106027625 chr1 103110100 103111100 chr3 108129050 108130050 chr1 104428633 104429633 chr3 108535100 108536100 chr1 106101300 106102300 chr3 113516650 113517650 chr19 61709650 61711650 chr3 118050716 118051716 chr14 60449800 60450800 237 chr3 120321300 120322300 chr14 62317616 62318616 chr3 128002800 128003800 chr19 63237033 63238033 chr3 131353500 131355500 chr14 64077750 64078750 chr3 138267650 138268650 chr14 69982800 69983800 chr12 39766800 39767800 chr14 71309350 71311350 chr12 45349230 45350230 chr20 3306250 3307250 chr12 46638500 46640500 chr20 3714366 3716366 chr12 46823275 46824275 chr20 12511150 12512150 chr12 51896225 51897225 chr14 90352100 90353100 chr3 143491250 143492250 chr20 16843550 16845550 chr3 149616770 149617770 chr20 25775100 25776100 chr3 164922000 164923000 chr20 25999675 26000675 chr3 173337500 173339500 chr7 151399175 151400175 chr3 180461900 180462900 chr7 151709750 151710750 chr3 188096325 188097325 chr7 151738500 151741500 chr3 195013565 195015565 chr4 95528900 95529900 chr12 64274950 64275950 chr4 98281900 98282900 chr12 65201700 65202700 chr4 109031170 109032170 chr12 66199866 66200866 chr4 110592733 110594733 chr12 67426550 67428550 chr4 111903200 111904200 chr12 69187100 69188100 chr4 115632650 115634650 chr12 71495950 71496950 chr4 121138733 121140733 chr12 71688900 71689900 chr4 125093944 125094944 chr12 75436050 75438050 chr4 142909275 142910275 chr8 49588850 49589850 chr7 154425100 154426100 chr8 50167836 50170836 chr4 146839225 146840225 chr8 50873133 50875133 chr4 153958866 153960866 chr8 53621150 53622150 chr4 154167450 154169450 chr8 53674616 53676616 chr7 157636200 157637200 chr12 98640633 98641633 chr4 164555875 164557875 chr8 57094030 57095030 chr1 110174800 110175800 chr8 59528700 59529700 chr1 117205400 117206400 chr8 59723200 59724200 chr1 119685483 119686483 chr8 60153875 60155875 chr1 119691900 119692900 chr8 63478350 63480350 chr1 121047950 121048950 chr12 109955800 109956800 chr14 105152400 105154400 chr12 110327700 110328700 chr20 46054616 46055616 chr8 69299850 69300850 chr7 157722400 157724400 chr12 116715750 116716750 chr7 157733350 157735350 chr12 119290950 119291950 chr7 157758500 157767500 chr8 69922800 69923800 chr4 171097983 171098983 chr8 69943100 69944100 chr4 175538716 175539716 chr8 75109100 75110100 chr7 157811850 157812850 chr4 3451750 3452750 chr4 183427300 183428300 chr4 3570200 3571200 chr4 185775750 185776750 chr4 5257650 5258650 chr4 185784071 185785071 chr4 5496766 5497766 chr7 157844475 157846475 238 chr4 5638250 5641250 chr4 187051250 187052250 chr4 6157716 6158716 chr4 190729400 190730400 chr4 6387300 6388300 chr4 190737783 190738783 chr4 11660583 11662583 chr4 190753350 190759350 chr4 14055150 14056150 chr8 146300 148300 chr4 14784350 14785350 chr8 456100 457100 chr4 15452066 15453066 chr8 557900 558900 chr4 22495150 22496150 chr8 628000 629000 chr12 123195725 123196725 chr4 190779700 190782700 chr12 123291450 123292450 chr4 190818340 190819340 chr12 125496550 125497550 chr4 190823800 190824800 chr8 82822050 82823050 chr8 5009680 5011680 chr8 89039300 89040300 chr8 7001050 7002050 chr8 91763000 91764000 chr8 8021300 8022300 chr8 92802600 92805600 chr4 190986200 190987200 chr8 102751350 102753350 chr8 8022900 8023900 chr8 103272066 103273066 chr8 8025450 8026450 chr8 104082100 104084100 chr8 8061366 8063366 chr8 113276200 113278200 chr4 191048000 191053000 chr8 115368625 115370625 chr8 16185275 16186275 chr8 126054033 126056033 chr8 23472014 23473014 chr8 127304100 127306100 chr5 5568275 5569275 chr8 136004050 136005050 chr1 151362256 151363256 chr8 139047933 139048933 chr1 153932800 153933800 chr8 140509675 140510675 chr1 163320250 163322250 chr4 45126900 45127900 chr1 168020800 168021800 chr4 45250650 45252650 chr1 169454350 169455350 chr13 30310700 30311700 chr1 176519700 176520700 chr13 30315071 30316071 chr1 180154850 180155850 chr8 141714700 141715700 chr20 62041550 62042550 chr8 142387100 142388100 chr21 17413400 17415400 chr13 35786066 35787066 chr8 34883280 34884280 chr8 143976700 143977700 chr8 39008900 39009900 chr8 144277000 144278000 chr5 6766850 6767850 chr8 144399300 144400300 chr5 7283325 7284325 chr13 59062800 59063800 chr8 39703650 39704650 chr13 60556400 60558400 chr8 42027850 42028850 chr13 65240100 65242100 chr5 15604850 15606850 chr13 84125650 84127650 chr5 18636300 18637300 chr9 4520850 4521850 chr8 47050650 47052650 chr13 95402550 95406550 chr5 26522700 26523700 chr9 12945250 12946250 chr1 186983200 186987200 chr13 97592950 97593950 chr1 203666250 203667250 chr9 24281550 24283550 chr1 205560100 205561100 chr9 24499800 24500800 chr1 207369533 207372533 chr13 107025200 107026200 chr1 209655800 209656800 chr13 113082200 113083200 chr1 215613283 215614283 239 chr9 30107050 30108050 chr1 221264071 221265071 chr4 46188500 46191500 chr1 223098050 223100050 chr21 42171850 42172850 chr1 224144110 224145110 chr21 45786000 45787000 chr1 228802400 228803400 chr22 17337700 17338700 chr1 231675516 231677516 chr9 45300700 45301700 chr1 233115600 233117600 chr9 45302800 45304800 chr1 236609816 236610816 chr9 46279550 46281550 chr1 241960200 241961200 chr9 67787250 67793250 chr1 241962025 241963025 chr9 67945550 67947550 chr1 242691200 242692200 chr4 48908050 48909050 chr2 1026650 1028650 chr4 48909900 48911900 chr15 22486250 22487250 chr4 48930300 48934300 chr15 22569000 22570000 chr4 49284450 49288450 chr5 34581750 34583750 chr4 56235900 56236900 chr5 44513150 44514150 chr4 59163350 59164350 chr5 45240950 45242950 chr22 18635200 18636200 chr5 45883216 45884216 chr22 19662950 19663950 chr2 7106750 7107750 chr22 20125600 20126600 chr2 11571900 11572900 chr22 20201150 20202150 chr15 23323900 23324900 chr22 34236233 34237233 chr15 66085920 66087920 chr22 37124250 37125250 chr15 68178350 68179350 chr22 37709155 37712155 chr15 68587100 68588100 chr22 44845650 44846650 chr15 80126000 80127000 chr22 45351050 45352050 chr15 82947700 82949700 chr22 48606966 48607966 chr15 83310450 83312450 chr9 77261487 77262487 chr15 84139050 84140050 chr9 78376350 78377350 chr15 90854650 90855650 chr9 80200421 80202421 chr15 97191316 97193316 chr9 92303710 92304710 chr15 99430116 99431116 chr9 95155200 95157200 chr15 100335150 100338150 chr9 101471350 101472350 chr16 657700 658700 chr1 535975 536975 chr5 52599150 52600150 chr1 1158000 1159000 chr5 68254000 68257000 chr1 1465200 1466200 chr5 69421325 69423325 chr1 2615850 2623850 chr5 71908900 71909900 chr1 10675800 10677800 chr5 93707800 93708800 chr1 11534700 11535700 chr5 93718475 93722475 chr22 48981000 48982000 chr5 97267700 97268700 chrX 3641100 3642100 chrX 88642600 88643600 chrX 6237800 6238800 chrX 91355500 91357500 chrX 6886950 6887950 chrX 93846100 93849100 chrX 8649000 8650000 chrX 97714075 97719075 chrX 9880150 9882150 chrX 100759200 100760200 chrX 16287900 16288900 chr2 25166200 25167200 chrX 27737000 27738000 chr2 25417133 25418133 chr9 104442150 104443150 chr2 33686700 33687700 240 chr9 104684950 104685950 chrX 102870850 102871850 chrX 45947700 45948700 chrX 102908000 102909000 chrX 46230100 46231100 chr2 37737661 37738661 chr17 39786900 39787900 chr2 42648850 42649850 chrX 47352925 47353925 chr2 44213200 44214200 chrX 47680750 47681750 chr2 46695825 46696825 chr17 64240540 64242540 chr2 47778483 47780483 chr17 74690600 74691600 chr2 55917700 55918700 chr17 75581337 75582337 chr2 56637750 56638750 chr17 75623900 75624900 chr16 848000 849000 chrX 48442525 48443525 chr16 915850 916850 chrX 48930700 48931700 chr16 3253800 3255800 chr17 76622900 76623900 chr10 75214900 75216900 chr17 78070750 78071750 chr16 4684100 4685100 chr17 78630750 78631750 chr10 76003650 76004650 chr18 62566 64566 chr10 76005600 76006600 chrX 51488700 51489700 chr10 77578262 77580262 chrX 51652200 51653200 chr10 79538325 79539325 chr18 6603700 6604700 chr16 14488033 14489033 chrX 55494866 55496866 chr16 15964300 15965300 chrX 55532400 55534400 chr16 19723650 19724650 chrX 55760650 55762650 chr10 85185550 85187550 chrX 55950366 55952366 chr10 87626100 87627100 chrX 56275266 56277266 chr10 100686400 100687400 chr6 87687733 87688733 chr16 32228850 32230850 chr6 109160033 109161033 chr10 110792150 110793150 chr6 112160425 112162425 chr10 128966700 128969700 chr6 118029550 118031550 chr10 129130475 129131475 chr6 121988800 121989800 chr10 129889750 129890750 chr9 112628600 112629600 chr10 132082700 132084700 chr6 123950575 123951575 chr10 134060000 134061000 chr6 126033016 126034016 chr2 104738700 104739700 chr6 126064016 126065016 chr2 108379600 108382600 chr6 131665000 131666000 chr2 115295050 115296050 chr6 137671866 137672866 chr5 126620200 126621200 chr6 140999100 141000100 chr2 123400700 123401700 chr6 141751633 141752633 chr2 127416400 127418400 chr9 125290750 125291750 chr5 140729550 140731550 chr9 129543700 129544700 chr5 143370200 143371200 chr6 149033825 149034825 chr2 132197300 132198300 chr6 151407100 151409100 chr5 144975333 144977333 chr18 22440100 22441100 chr2 132306050 132307050 chr18 41591991 41593991 chr5 171761935 171762935 chr18 42709150 42710150 chr5 174963450 174965450 chr18 42806450 42810450 chrX 107866400 107868400 chr18 57818333 57820333 chrX 111809900 111810900 chr9 130490750 130491750 chrX 114965900 114966900 241 chr9 131184500 131185500 chrX 114997033 114998033 chr9 131371512 131372512 chrX 116344750 116346750 chr6 153804300 153807300 chrX 118635850 118636850 chr6 154608900 154609900 chrX 118700200 118701200 chr9 135718666 135720666 chr2 57206650 57208650 chr6 160571300 160572300 chrX 128005900 128006900 chr6 161639300 161641300 chrX 128805200 128806200 chr9 139329625 139330625 chrX 131971000 131972000 chr9 139717200 139718200 chrX 132320250 132321250 chr6 167478200 167479200 chr2 62408188 62410188 chr6 167563750 167566750 chr2 63652680 63654680 chr6 167579507 167580507 chr2 64338500 64340500 chr6 168773175 168774175 chr2 65129350 65130350 chr9 139963000 139964000 chr2 65693066 65694066 chr6 169148700 169149700 chr2 68776825 68777825 chr6 169252850 169253850 chr2 73540900 73541900 chr6 169659800 169660800 chr2 83363233 83367233 chr6 169967950 169968950 chr2 88251200 88252200 chr7 173700 174700 chr2 89387700 89388700 chr7 180600 181600 chr10 134484800 134490800 chr7 226000 227000 chr16 33264900 33265900 chr7 282450 283450 chr11 177750 182750 chr7 314150 315150 chr11 839700 840700 chr7 733200 734200 chr11 1082133 1084133 chr10 2232610 2234610 chr11 1976050 1980050 chr10 5315716 5316716 chr11 3877900 3878900 chr10 9928200 9929200 chr11 6778100 6779100 chr10 16268750 16269750 chr11 44855033 44856033 chr7 7357500 7359500 chr2 147598250 147600250 chr10 23389075 23390075 chr6 2799175 2800175 chr7 17445950 17446950 chr6 2820500 2822500 chr7 19990100 19991100 chr2 149563870 149565870 chrX 68016700 68017700 chr6 6156300 6157300 chrX 68272980 68273980 chr6 6244950 6245950 chrX 68415716 68416716 chr2 158882900 158884900 chrX 68455033 68456033 chr2 169226162 169227162 chrX 68643200 68644200 chr6 26124175 26127175 chrX 70806000 70807000 chr6 27642100 27643100 chr18 73842200 73843200 chr6 27901450 27903450 chr18 75873200 75874200 chr6 31890166 31891166 chr19 659750 660750 chr6 44152200 44153200 chr19 877350 878350 chr2 191221300 191222300 chr19 1054800 1055800 chr6 57318800 57319800 chr7 20082850 20083850 chr6 57588200 57589200 chr10 32005483 32007483 chr2 192473650 192475650 chr10 32056400 32058400 chr2 196316100 196318100 chr7 22674783 22675783 chrX 70239012 70240012 242 LP P5K4 over-enriched loci (>=50%) compared to LP P2K4, within 10,000bp around TSS. Chromosome start, end, distance to TSS, gene name, quantile normalized tag count in LPP2 and LPP5 shown Chr Start End Dist. To. TSS Gene Name #LPP2 #LPP5 chr1 143796000 143797000 -8957 PDE4DIP 5.5 13 chr10 111746000 111748000 -8716 ADD3 9 66 chr12 8068000 8069000 -8126 FOXJ2 1 10.5 chr1 143794000 143795000 -6957 PDE4DIP 8.5 11.5 chr8 122713000 122715000 -6767 HAS2-AS1 8 35 chr6 27555000 27556000 -6624 ZNF184 56.5 102 chr1 143914000 143916000 -5468 NOTCH2NL 4 37.5 chr1 143792000 143793000 -4957 PDE4DIP 1 10 chr8 122716000 122717000 -4267 HAS2-AS1 5 15 chr7 121734000 121738000 -4199 FEZF1 175.5 778.5 chr10 46499000 46500000 -4040 PPYR1 2 19 chr1 143790000 143791000 -2957 PDE4DIP 8 24.5 chr1 25449000 25450000 -2928 C1orf63 3 16 chr17 55270000 55271000 -2909 MIR21 106.5 270.5 chr14 56802000 56803000 -2859 MUDENG 3 18.5 chr15 64785000 64786000 -2484 SMAD6 13 37.5 chrX 133766000 133767000 -2371 FAM122C 5 34 chr12 88624000 88625000 -2363 LOC338758 5.5 36 chr8 122718000 122720000 -1767 HAS2-AS1 25 130 chr2 201703000 201706000 -1433 CFLAR 14.5 68 chr14 56343000 56344000 -1402 OTX2 38.5 202.5 chr15 70194000 70196000 -1246 SENP8 32.5 108 chr11 124176000 124177000 -991 C11orf61 4 18.5 chr10 18981000 18982000 -944 NSUN6 2 12.5 chr9 124734000 124735000 -900 ZBTB26 39.5 66.5 chr19 54885000 54886000 -677 CPT1C 57.5 166 chrX 131180000 131181000 -630 RAP2C 27 69 chr6 99980000 99981000 -572 SFRS18 37 81.5 chrX 135789000 135790000 -404 SNORD61 140 366 chr6 31618000 31619000 -269 DDX39B 37.5 58 chr5 159368000 159369000 -258 TTC1 87 297 chr12 112392000 112397000 -240 LHX5 152 444 chr12 54796000 54797000 -141 RPL41 86.5 266 chr1 78217000 78218000 -135 FUBP1 93 195 chr17 37967000 37968000 -118 COASY 61.5 154.5 chr8 74368000 74369000 -77 RPL7 15 70.5 chr15 91244000 91245000 -55 CHD2 51.5 169 chr1 148050000 148051000 52 HIST2H2BF 16.5 61.5 chr19 54691000 54692000 54 RPS11 184 386 chr17 34263000 34264000 79 RPL23 46 140.5 243 chr10 91393000 91394000 128 PANK1 95 216.5 chr11 62378000 62380000 131 SNORD27 423.5 946 chr3 151747000 151748000 236 EIF2A 186.5 396.5 chrX 71318000 71319000 249 PIN4 73 202.5 chr4 64957000 64958000 273 TECRL 0 20 chr4 109761000 109762000 302 RPL34 187 423 chr13 26723000 26725000 307 RPL21P28 242.5 550.5 chr4 46086000 46087000 313 GABRA2 13 48.5 chr1 148125000 148126000 351 HIST2H2AC 56 186.5 chr8 95634000 95635000 422 KIAA1429 87.5 286 chr1 159459000 159460000 542 APOA2 3 31.5 chr4 113786000 113790000 546 MIR367 422.5 944.5 chr12 64849000 64850000 574 TMBIM4 20.5 86.5 chr6 26129000 26132000 614 HIST1H4A 45.5 165.5 chr20 42418000 42419000 645 HNF4A 2 27.5 chr6 26232000 26234000 648 HIST1H2AC 317 737 chr12 7979000 7980000 659 SLC2A3 272.5 620.5 chr2 206731000 206732000 663 NDUFS1 186.5 392.5 chr19 63757000 63758000 667 CHMP2A 29 101 chr1 144150000 144151000 681 TXNIP 142.5 334.75 chrX 130984000 130988000 683 MST4 117.5 358 chr1 161438000 161439000 749 RGS5 7 34 chr18 27426000 27427000 772 TTR 1 21 chr1 78243000 78245000 776 DNAJB4 31.5 137 chr5 96503000 96504000 776 LIX1 10 68 chr14 100362000 100364000 802 MEG3 32.5 145.5 chr10 17210000 17212000 822 CUBN 10 73 chr6 27942000 27943000 838 HIST1H1B 38 139.5 chr13 96673000 96674000 925 MBNL2 17 74 chr6 36670000 36672000 932 SRSF3 384.5 857.25 chr7 26205000 26207000 938 HNRNPA2B1 272.5 576 chr15 91228000 91229000 970 LOC100507217 128.5 361 chr17 65583000 65585000 979 KCNJ16 8 64 chr3 112934000 112936000 983 PHLDB2 8 57.5 chr2 233348000 233349000 1019 KCNJ13 4 25.5 chr11 89508000 89509000 1034 NAALAD2 13.5 35 chr10 101677000 101679000 1044 DNMBP-AS1 18.5 89 chr9 74756000 74758000 1053 ALDH1A1 4 60.5 chr10 95230000 95232000 1064 MYOF 76 166.5 chr11 33138000 33139000 1113 CSTF3 134 410.5 chr3 187137000 187138000 1118 TRA2B 178.5 398.25 chr12 90028000 90029000 1173 LUM 3 20 chr17 6494000 6495000 1178 MED31 44 102 chr2 61987000 61988000 1193 COMMD1 10.5 17.5 chr2 64098000 64099000 1218 VPS54 23 90.5 chr2 216683000 216684000 1235 XRCC5 57.5 70 chr5 102482000 102483000 1241 GIN1 58.5 131 244 chr2 208840000 208841000 1264 PIKFYVE 11 34 chrX 105824000 105826000 1276 RNF128 4 51.5 chr3 57207000 57209000 1320 HESX1 45 187.5 chr4 84029000 84031000 1359 SEC31A 123 298 chr13 42462000 42464000 1377 EPSTI1 11.5 65 chr4 164636000 164637000 1377 C4orf43 9.5 55.5 chr5 33926000 33927000 1381 ADAMTS12 11.5 69.5 chr14 22353000 22354000 1412 SLC7A7 6.5 39.5 chr5 122210000 122211000 1441 SNX24 5 19 chr5 27072000 27074000 1446 CDH9 31 174.5 chr14 73619000 73620000 1449 ALDH6A1 61 97 chr2 188125000 188127000 1464 TFPI 7 51.5 chr7 86505000 86507000 1486 KIAA1324L 19.5 169.5 chr13 23043000 23045000 1491 TNFRSF19 10 81.5 chr9 14899000 14900000 1493 FREM1 14.5 67.5 chr4 74703000 74704000 1498 RASSF6 6 48.5 chr5 55324000 55326000 1578 IL6ST 104 212.5 chr1 145182000 145183000 1585 CHD1L 22.5 60.5 chr12 78851000 78852000 1609 PPP1R12A 132 290.5 chr14 61233000 61234000 1628 HIF1A 138 271 chr2 189548000 189550000 1656 COL3A1 5 47 chr20 42464000 42466000 1662 HNF4A 6 48 chrX 107571000 107572000 1690 COL4A5 43 105 chr2 183437000 183439000 1743 FRZB 15.5 88.5 chr7 20338000 20340000 1750 ITGB8 15.5 66 chr11 26973000 26975000 1796 FIBIN 3 43 chr8 38975000 38976000 1838 ADAM9 15.5 70.5 chr4 17423000 17424000 1877 NCAPG 20 57.5 chr4 83568000 83569000 1902 HNRPDL 116 407.75 chr1 167344000 167345000 1929 ATP1B1 8 30 chr11 6239000 6240000 1958 CCKBR 5 35 chr11 86428000 86429000 1966 TMEM135 14.5 50.5 chr5 75952000 75954000 1996 F2RL2 5 38 chr4 87736000 87737000 2008 PTPN13 17.5 50.5 chrX 21304000 21305000 2043 CNKSR2 6 38 chr12 32938000 32940000 2047 PKP2 46 155 chr17 64924000 64925000 2067 MAP2K6 15.5 47 chr5 108771000 108772000 2074 PJA2 16 68.5 chr4 134291000 134293000 2080 PCDH10 71.5 173 chr12 57596000 57599000 2094 LRIG3 39.5 187 chr2 224609000 224610000 2101 SERPINE2 8 52 chr17 38833000 38835000 2121 ARL4D 18.5 106.5 chr4 39085000 39089000 2132 KLB 8 119 chr2 201642000 201643000 2137 FAM126B 5 26 chr5 83049000 83052000 2152 HAPLN1 15 102.5 chr10 101757000 101758000 2166 DNMBP 8.5 56 chr4 159308000 159314000 2168 FAM198B 24 171.5 245 chr10 102214000 102216000 2198 WNT8B 4 30 chr13 25520000 25522000 2198 SHISA2 55 171.5 chr8 32527000 32528000 2230 NRG1 5 34.5 chr10 74524000 74525000 2238 P4HA1 21 85 chr1 115011000 115013000 2255 DENND2C 11.5 80 chr18 17078000 17079000 2299 GREB1L 7 29 chrX 107565000 107567000 2314 COL4A6 15.5 96 chr18 50141000 50142000 2331 C18orf54 1 18.5 chr6 55844000 55848000 2334 BMP5 16 185 chr12 113603000 113605000 2352 TBX3 51.5 169 chr20 13926000 13927000 2354 MACROD2 0 14.5 chr7 33913000 33915000 2363 BMPER 8 57.5 chrX 131177000 131178000 2370 RAP2C 23.5 69 chr4 87497000 87499000 2399 MAPK10 9 79.5 chr12 23990000 23993000 2404 SOX5 9 68.5 chr6 49628000 49629000 2428 C6orf141 12.5 54 chrX 105858000 105860000 2450 RNF128 3 44.5 chr10 53745000 53748000 2453 DKK1 4 68.5 chr1 46368000 46369000 2467 PIK3R3 33 84 chr8 58066000 58067000 2481 IMPAD1 9.5 50.5 chr6 139734000 139735000 2543 CITED2 38.5 149.5 chr8 49993000 49995000 2552 SNAI2 11 60 chr7 42240000 42241000 2643 GLI3 8 34 chr1 48232000 48233000 2649 LOC388630 4 22 chr1 67289000 67291000 2668 SLC35D1 13.5 80.5 chr2 28471000 28473000 2717 FOSL2 22 92.5 chr2 42131000 42132000 2835 PKDCC 21 62 chr5 43346000 43347000 2852 HMGCS1 21 64 chr6 19948000 19949000 2904 ID4 5 39.5 chr12 92298000 92300000 3123 NUDT4P1 26 103 chr16 78188000 78190000 3123 MAF 57 165 chrX 37588000 37589000 3333 DYNLT3 2 10.5 chr2 208338000 208340000 3388 FZD5 59.5 212.5 chr2 235066000 235068000 3432 ARL4C 96.5 214.5 chr5 31231000 31235000 3447 CDH6 18 100 chr5 38587000 38591000 3505 LIFR 22 173 chr3 77175000 77176000 3516 ROBO2 1 14 chr20 387000 388000 3687 TBC1D20 3 22 chr12 91059000 91061000 3804 BTG1 82.5 272 chr18 7102000 7106000 3813 LAMA1 40 153.5 chr20 14258000 14267000 3813 FLRT3 128.5 746 chr13 71335000 71336000 3831 DACH1 29.5 18 chr1 150229000 150230000 3838 S100A10 8 55 chr10 94827000 94828000 3863 CYP26A1 11 40 chr9 98837000 98838000 3913 CTSL2 4 28 chr2 216002000 216008000 4036 FN1 52 301.5 chr12 7975000 7977000 4159 SLC2A3 48 153 246 chr3 133864000 133868000 4170 UBA5 8 49 chr5 57787000 57788000 4170 PLK2 8.5 33.5 chr4 151725000 151729000 4473 MAB21L2 20.5 138 chr10 29010000 29012000 4570 BAMBI 20 89.5 chr11 65023000 65030000 4691 MALAT1 1126 3432.75 chr4 30334000 30338000 4865 PCDH7 64.5 283 chr18 24006000 24007000 4943 CDH2 1 23.5 chrX 40834000 40836000 5168 USP9X 60 150 chr6 127484000 127490000 5259 RSPO3 14 214 chr20 6700000 6705000 5755 BMP2 33.5 149.5 chr1 143699000 143702000 5879 PDE4DIP 20.5 111.5 chr14 64047000 64048000 6455 ZBTB1 5 20.5 chr4 39091000 39092000 6632 KLB 1 10 chrX 38312000 38313000 6825 TSPAN7 2 6.5 chr1 16804000 16807000 7069 NBPF1 19 59 chr1 143741000 143747000 7349 PDE4DIP 49.5 181.5 chr1 16851000 16854000 7844 MST1P2 17 69.5 chr1 143634000 143635000 8889 PDE4DIP 3 6 LPP5K4 overenriched loci mapping to predicted enhancer regions chr11 62377500 62379500 chr10 101677100 101679100 chr13 26723650 26725650 chr10 101756400 101757400 chr1 79937000 79939000 chr11 34671350 34672350 chr1 143791800 143792800 chr11 86428133 86429133 chr1 143902050 143904050 chr11 89508000 89509000 chr1 167344966 167345966 chr11 124176225 124177225 chr1 230829600 230830600 chr12 7670550 7672550 chr2 42131066 42132066 chr12 7979400 7980400 chr2 61986850 61987850 chr12 57596250 57599250 chr2 188125800 188127800 chr12 91059550 91061550 chr2 201702866 201705866 chr12 92297950 92299950 chr2 206730650 206731650 chr13 42528300 42531300 chr2 208840200 208841200 chr14 56802380 56803380 chr2 216003450 216009450 chr15 64785900 64786900 chr2 224609300 224610300 chr15 70194450 70196450 chr2 235065866 235067866 chr15 91243900 91244900 chr3 57207000 57209000 chr16 22113733 22114733 chr4 17423050 17424050 chr16 78188000 78190000 chr4 87497333 87499333 chr17 37966800 37967800 chr4 87736133 87737133 chr17 38832200 38834200 chr4 100714400 100716400 chr17 46477100 46478100 chr4 113786850 113790850 chr17 64924250 64925250 chr4 151724600 151728600 chr18 7101700 7105700 chr4 159309625 159315625 chr18 50140366 50141366 247 chr4 164636200 164637200 chr20 14258550 14267550 chr5 27072050 27074050 chrX 21304050 21305050 chr5 33925500 33926500 chrX 40834025 40836025 chr5 38586760 38590760 chrX 107570850 107571850 chr5 43345900 43346900 chrX 130985066 130989066 chr5 55324000 55326000 chrX 131177133 131178133 chr1 46367900 46368900 chrX 131180000 131181000 chr1 67289450 67291450 chrX 133765360 133766360 chr1 78243000 78245000 chr12 78849950 78850950 chr5 96503075 96504075 chr1 48231025 48232025 chr5 102481950 102482950 chr9 14897640 14898640 chr5 108771150 108772150 chr7 42238090 42239090 chr5 122209850 122210850 chr21 43225700 43226700 chr5 159367950 159368950 chr6 35640175 35641175 chr6 19948100 19949100 chr13 23041950 23043950 chr6 29018550 29019550 chr2 208337800 208339800 chr6 31618350 31619350 chr6 26129720 26132720 chr6 55845150 55849150 chr14 22353600 22354600 chr6 114436816 114438816 chr14 73619200 73620200 chr6 114766866 114768866 chr19 63756625 63757625 chr6 139733100 139734100 chr9 126397730 126398730 chr7 86504850 86506850 chr19 54691000 54692000 chr7 120934300 120937300 chr12 68009464 68011464 chr7 121733800 121737800 chr5 27507050 27509050 chr8 38975000 38976000 chr10 13787550 13788550 chr8 77774275 77776275 chr1 161438820 161439820 chr9 74756250 74758250 chr12 32936707 32938707 chr10 62187000 62188000 chr4 114432233 114434233 chr2 63695409 63697409 248 Appendix E: H3K4me3 Quantitative Changes in HP HP P2K4 over-enriched loci (>=50%) compared to HP P5K4, within 10,000bp around TSS. Chromosome start, end, distance to TSS, gene name, quantile normalized tag count in HPP2 and HPP5 shown Chr Start End Dist. To TSS Gene Name #HPP2 #HPP5 chr13 92667000 92668000 -9579 GPC6 36 5 chr19 59027000 59031000 -9540 NLRP12 335 46 chr2 38165000 38167000 -9173 CYP1B1 60 11.5 chr5 147228000 147234000 -7467 SCGB3A2 717 262 chr9 95150000 95156000 -4483 C9orf129 275.5 40 chr16 69210000 69213000 -3896 IL34 360 87.5 chr7 100447000 100448000 -2584 MUC17 24.5 5 chr6 31248000 31249000 -2070 POU5F1 42.5 19 chr14 41142000 41149000 -1014 LRFN5 470 228 chr11 133649000 133652000 -985 GLB1L3 386 155 chr7 100594000 100598000 -428 VGF 149.5 55 chr5 141683000 141687000 -196 SPRY4 537 249 chr7 115952000 115955000 -83 CAV1 260 108.5 chr17 44154000 44157000 -41 HOXB13- AS1 59.5 20 chr10 71870000 71873000 -29 NODAL 222 52.5 chr2 111968000 111970000 163 LOC541471 46.5 8 chr1 235272000 235273000 175 RYR2 97.5 34.5 chr5 80292000 80293000 186 RASGRF2 96 30.5 chr15 24568000 24570000 316 GABRB3 80.5 19 chr16 12903000 12904000 522 SHISA9 181.5 80 chr1 222871000 222872000 698 CNIH3 81.5 23.5 chr11 69227000 69228000 787 FGF19 84 32.5 chr10 112248000 112249000 885 DUSP5 81.5 24 chr1 224142000 224143000 969 LEFTY1 52.5 14 chr12 2939000 2941000 991 TEAD4 195 71.5 chr14 73885000 73887000 1081 VRTN 372.5 174 chr1 231496000 231498000 1082 PCNXL2 159 72.5 chrX 152747000 152749000 1197 PDZD4 131 43 chr9 14710000 14713000 1215 CER1 112 24.5 chr10 97039000 97040000 1271 PDLIM1 110 33.5 chr6 3400000 3401000 1292 SLC22A23 99 32 chr17 38977000 38978000 1331 ETV4 99.5 38 chr6 31243000 31247000 1430 POU5F1 608.5 261 chr11 68209000 68211000 1441 GAL 70 17.5 chr11 304000 307000 1509 IFITM1 329 127 chr11 127960000 127962000 1663 ETS1 106 28 chr12 7833000 7837000 1738 NANOG 521.5 189.5 249 chr3 171561000 171563000 1895 SKIL 109 39 chr3 46594000 46598000 1920 TDGF1 614.5 202 chr1 211192000 211193000 1990 VASH2 31.5 8.5 chr3 54645000 54648000 2424 ESRG 583 278.5 chr15 82947000 82949000 2747 ZSCAN2 32 3 chr17 44151000 44153000 2881 PRAC 55.5 14.5 chr3 110535000 110536000 3609 DPPA4 208.5 77 chr6 33193000 33195000 5729 HLA-DPB2 275 141 HPP2K4 overenriched loci mapping to predicted enhancers chr1 221264900 221269900 chr9 84689300 84691300 chr1 230321525 230330525 chr9 89216233 89222233 chr1 231495850 231497850 chr9 95150510 95156510 chr1 243046433 243048433 chr9 101945083 101949083 chr2 7932918 7939918 chr9 117276933 117280933 chr2 38164916 38166916 chr10 6882766 6883766 chr2 60497850 60500850 chr1 113153599 113156599 chr2 64334150 64339150 chr1 113165986 113168986 chr2 67410800 67414800 chr1 114376500 114378500 chr2 77170300 77175300 chr1 194082750 194083750 chr2 111967861 111969861 chr1 209640200 209641200 chr2 117754775 117756775 chr1 211149600 211154600 chr2 188135400 188138400 chr1 214386575 214389575 chr2 207380550 207385550 chr10 20698833 20700833 chr3 46594050 46598050 chr10 53462033 53468033 chr3 171561500 171563500 chr10 71870750 71873750 chr3 177520966 177525966 chr10 92044325 92046325 chr3 188377300 188380300 chr10 97038500 97039500 chr4 3981300 3982300 chr11 304166 307166 chr4 4097450 4100450 chr11 27606700 27611700 chr4 24111750 24113750 chr11 94015500 94026500 chr4 93409875 93417875 chr11 115820416 115821416 chr4 93580150 93584150 chr11 121017450 121020450 chr4 121135400 121142400 chr11 127959662 127961662 chr4 167873816 167875816 chr12 7832633 7836633 chr4 180322950 180326950 chr12 14749575 14754575 chr5 18706666 18709666 chr12 58002533 58007533 chr5 75689800 75690800 chr12 74046950 74049950 chr5 93720425 93723425 chr13 42339766 42343766 chr5 106343166 106349166 chr13 50642500 50646500 chr5 120060500 120065500 chr13 53601833 53604833 chr5 126205350 126208350 chr13 53740950 53744950 chr5 141684350 141688350 chr13 55047875 55054875 chr5 147226450 147232450 chr13 55128300 55131300 250 chr6 14408133 14411133 chr13 92666550 92667550 chr6 31248190 31249190 chr14 37730800 37734800 chr6 33193500 33195500 chr14 41140450 41147450 chr6 94596031 94601031 chr14 46644250 46646250 chr6 95323220 95326220 chr14 47804100 47807100 chr6 114849950 114854950 chr14 86773975 86777975 chr6 126066950 126069950 chr15 22484750 22487750 chr6 132265850 132270850 chr15 24568466 24570466 chr6 167560500 167565500 chr15 82946800 82948800 chr7 22709866 22711866 chr16 12902700 12903700 chr7 24402975 24409975 chr16 13356925 13360925 chr7 124687850 124691850 chr16 58671550 58672550 chr7 125351500 125354500 chr16 63819950 63824950 chr7 145571350 145575350 chr16 69209200 69212200 chr8 7001166 7004166 chr19 59027050 59031050 chr8 8023233 8026233 chr20 12273200 12274200 chr8 12436850 12438850 chr20 12681400 12686400 chr8 72750366 72758366 chr20 38331375 38337375 chr8 98282922 98284922 chrX 16110600 16112600 chr8 115364640 115372640 chrX 16504600 16506600 chr8 129694500 129698500 chrX 93844125 93846125 chr8 133164700 133166700 chrX 102907333 102909333 chr9 2266573 2271573 chrX 110675400 110677400 chr9 75890700 75895700 chrX 113744900 113746900 chrX 152746950 152748950 chrX 115832450 115835450 HP P5K4 over-enriched loci (>=50%) compared to HP P2K4, within 10,000bp around TSS. Chromosome start, end, distance to TSS, gene name, quantile normalized tag count in HPP2 and HPP5 shown Chr Start End Dist. To. TSS Gene Name #HPP2 #HPP5 chr8 65441000 65448000 -3829 LOC100130155 104 312 chr17 76984000 76985000 -3635 BAHCC1 79 144.5 chr10 21857000 21858000 -2883 C10orf140 16.5 70 chr2 45014000 45026000 -2541 SIX3 146.5 688 chr6 30957000 30958000 -2340 DDR1 13.5 68.5 chr17 76986000 76987000 -1635 BAHCC1 15.5 85 chr12 96381000 96382000 -1430 RMST 54.5 182.5 chr18 21187000 21188000 -1288 ZNF521 3 31 chr14 56343000 56353000 -1063 OTX2 302 1000 chr8 77757000 77761000 -935 LOC100192378 48 162.5 chr7 121729000 121736000 -699 FEZF1 447 924 chr2 222869000 222872000 -610 CCDC140 32.5 156 chr10 76827000 76837000 -481 ZNF503 193.5 596 251 chr2 63126000 63135000 -196 OTX1 140 567 chr15 35177000 35183000 -4 MEIS2 178 405.5 chr5 92941000 92949000 201 NR2F1 46 214 chr12 112386000 112402000 260 LHX5 205.5 1140.5 chr18 17074000 17079000 299 GREB1L 81.5 410.5 chr17 69151000 69152000 322 SDK2 77 167 chr9 130979000 130981000 361 IER5L 31.5 91.5 chr1 37795000 37796000 393 DNALI1 7 43.5 chr12 23991000 23996000 404 SOX5 56 264 chr11 113436000 113438000 502 ZBTB16 17.5 70 chr12 32546000 32548000 692 FGD4 20.5 113.5 chr9 125158000 125160000 731 CRB2 17 107.5 chr3 130889000 130890000 765 TMCC1 16 75 chr15 94669000 94673000 839 NR2F2 30 159 chr6 138934000 138935000 861 NHSL1 8 50 chr2 144992000 144995000 928 ZEB2 29 249.5 chr6 10522000 10523000 956 TFAP2A 21.5 48 chr2 66516000 66518000 964 MEIS1 16 88 chr14 100263000 100265000 1045 DLK1 8 71.5 chr12 46682000 46685000 1052 COL2A1 29 152.5 chr22 24896000 24897000 1060 SEZ6L 2 28 chr10 119291000 119296000 1069 EMX2OS 50.5 300.5 chr10 102212000 102216000 1198 WNT8B 14 146.5 chr6 99387000 99394000 1199 POU3F2 63 252 chr9 34578000 34579000 1222 CNTFR 11.5 77 chr3 62331000 62335000 1230 FEZF2 32 186.5 chr6 127480000 127486000 1259 RSPO3 101.5 374 chr5 96502000 96504000 1276 LIX1 15.5 144.5 chr14 60046000 60048000 1309 SIX6 9 51 chr7 129920000 129921000 1326 MEST 5 16 chr5 3650000 3651000 1332 IRX1 12.5 56.5 chr8 95516000 95518000 1356 RAD54B 112.5 270 chr4 87497000 87501000 1399 MAPK10 20 187.5 chr10 95508000 95510000 1444 LGI1 9 52.5 chr5 31230000 31232000 1447 CDH6 38 152.5 chr15 53820000 53822000 1469 PRTG 56 222 chr2 209998000 209999000 1484 MAP2 4 11.5 chr13 23042000 23046000 1491 TNFRSF19 23.5 230.5 chr5 102230000 102232000 1574 PAM 7 46.5 chr3 117645000 117646000 1575 LSAMP 38.5 57 chr1 163590000 163591000 1602 LMX1A 8 40.5 chr18 55089000 55091000 1605 RAX 18 130.5 chr8 65449000 65451000 1671 LOC100130155 50.5 143.5 chr13 25521000 25522000 1698 SHISA2 58 157.5 chr18 3445000 3446000 1728 TGIF1 11 69.5 chr8 32526000 32528000 1730 NRG1 10 75.5 chr20 14262000 14267000 1813 FLRT3 85.5 448.5 252 chr1 68468000 68470000 1817 WLS 56 168 chr6 55845000 55848000 1834 BMP5 21 81 chr11 94601000 94603000 1894 SESN3 135 297.5 chr16 63711000 63712000 1920 CDH11 31 97 chr15 94675000 94684000 1927 NR2F2 46 370 chrX 67967000 67968000 1935 EFNB1 12 42.5 chr10 53745000 53747000 1953 DKK1 11 62.5 chr1 46368000 46370000 1967 PIK3R3 133.5 298 chr6 139734000 139736000 2043 CITED2 135.5 328 chr12 1571000 1572000 2092 FBXL14 23 77 chr2 222142000 222144000 2254 EPHA4 94.5 290 chr3 57206000 57208000 2320 HESX1 27 119.5 chr2 42130000 42132000 2335 PKDCC 25.5 94 chr13 94159000 94161000 2390 SOX21 46 246.5 chr9 88749000 88750000 2424 GAS1 24.5 111.5 chr11 31782000 31792000 2455 PAX6 86 518 chr7 2526000 2531000 2495 LFNG 47.5 213 chr14 33487000 33488000 2535 EGLN3 38 105 chr4 78299000 78301000 2619 CCNG2 4 105 chr3 148612000 148613000 2629 ZIC1 0 18.5 chr6 83132000 83135000 2820 TPBG 13 135 chr15 91230000 91231000 2970 LOC100507217 6 34 chr5 107031000 107032000 2995 EFNA5 14.5 93.5 chr14 56338000 56340000 3098 OTX2 36.5 156.5 chr9 22439000 22441000 3160 DMRTA1 7 71 chr4 154926000 154927000 3178 SFRP2 28 74 chr1 144152000 144154000 3181 TXNIP 6 48.5 chr5 124104000 124107000 3204 ZNF608 98 262.5 chr9 81379000 81381000 3302 TLE4 12 77.5 chr8 28409000 28413000 3359 FZD3 19.5 107 chr13 105981000 105983000 3389 EFNB2 13.5 75 chr2 235066000 235068000 3432 ARL4C 65.5 250 chr7 42237000 42242000 3643 GLI3 87.5 307 chr17 39993000 39995000 3662 FZD2 20 95.5 chr4 151725000 151728000 3973 MAB21L2 17 136 chr9 73568000 73571000 4120 TMEM2 18 84.5 chr12 91058000 91061000 4304 BTG1 90.5 229 chr13 99436000 99438000 4973 ZIC2 67.5 214.5 chr1 82043000 82045000 5330 LPHN2 18.5 74 chr3 182917000 182919000 5594 SOX2 29 101 chr9 134454000 134455000 6686 BARHL1 3 13 chr11 65028000 65031000 7691 MALAT1 62 293 HPP5K4 overenriched loci mapping to prediced enhancer regions chr2 209998300 209999300 chr9 125157900 125159900 253 chr2 222141425 222143425 chr9 126396405 126399405 chr2 235065866 235067866 chr9 126460300 126463300 chr3 57205733 57207733 chr10 21837450 21838450 chr4 87497625 87501625 chr10 21839666 21844666 chr4 151724850 151727850 chr10 95508100 95510100 chr4 154925900 154926900 chr10 102211250 102215250 chr5 96501975 96503975 chr11 94600900 94602900 chr5 102230100 102232100 chr11 109506400 109509400 chr5 107031200 107032200 chr12 32545812 32547812 chr5 124104200 124107200 chr12 91058800 91061800 chr6 55845900 55848900 chr12 96381200 96382200 chr6 83131850 83134850 chr13 23041300 23045300 chr6 138933650 138934650 chr13 94158650 94160650 chr7 2525836 2530836 chr13 99108353 99110353 chr7 42236438 42241438 chr14 33487100 33488100 chr7 107754700 107755700 chr14 56422378 56424378 chr7 120933550 120937550 chr1 37795300 37796300 chr7 121730550 121737550 chr1 46367650 46369650 chr7 129920400 129921400 chr1 82042900 82044900 chr8 28408766 28412766 chr2 42130400 42132400 chr8 65442800 65449800 chr17 46476950 46479950 chr8 77774266 77777266 chr17 76983650 76984650 chr9 8845600 8846600 chr18 3445175 3446175 chr9 22438400 22440400 chr18 17196600 17199600 chr9 73568350 73571350 chr18 21186800 21187800 chr9 81378850 81380850 chr20 14261712 14266712

Abstract (if available)

Abstract Epigenetics is the study of changes in gene expression that occur in cells without alterations to DNA sequence. Epigenetic modifications are critical components of eukaryotic gene regulation and chromatin organization. Different epigenetic mechanisms, including the post-translational modifications of DNA-associated histone proteins play a role in the activation or repression of genes. ❧ One of my research goals was to define the epigenetic signature of cultured human embryonic stem cells (hESCs) and to determine how their epigenomes change during lineage commitment. Pluripotent hESCs are capable of self-renewal and have the capacity to differentiate into any lineage of the embryo. However, hESCs grown in culture are heterogeneous in nature, consisting of a mixture of pluripotent to differentiated cells, making investigation of pluripotent hESCs difficult. Therefore precise definition of pluripotent cells present in culture is critical in order to use these cells for future stem cell based therapies. Using a FACS based approach, I demonstrated that I was able to selectively isolate sub-populations from the bulk culture that expressed high levels of pluripotency factors (P2) and those that lacked these factors (P5). For both populations, I performed high resolution ChIP-sequencing for 2 different histone modifications, indicative of transcriptionally active regions (H3K4me3) and repressed regions (H3K27me3) in order to define, compare and contrast their epigenomes. In the widely used female H9 hESC line, I found that the X-chromosome of the P5 population was enriched for H3K27me3 but was not in the P2 population. These findings strongly suggest that P2 represents the more naïve pluripotent stem cells, whereas, P5 has committed to differentiate, consistent with X-chromosome inactivation (Xi). In a separate female hESC line, HES3, I discovered that low passage cells (LP) are devoid of H3K27me3 on the X-chromosome but high passage cells (HP) are enriched for H3K27me3 on the X-chromosome. These findings indicated that extended passage length of hESCs in culture can have a dramatic effect on their epigenetic signature. My detailed analysis of these data sets revealed many novel findings. In LP P2 cells, I defined for the first time, the presence of a non-canonical H3K4me3 profile which is characterized by lowly enriched H3K4me3 domains many kilobases long, and spanning protein families such as zinc finger, keratin, olfactory and extra cellular matrix protein families. The function of these long domains in hESCs are unknown and previously undefined. I have also detected many stochastic quantitative differences in H3K4me3 and/or H3K27me3 between P2 and P5 that are not conserved between cell types, suggesting that embryonic stem cells are epigenetically plastic. However, more importantly, I have identified a core subset of genes, promoters and regulatory regions that contain quantitative epigenetic differences between P2 and P5 and correlate with gene expression changes. These conserved regions may play a critical role in early differentiation or maintenance of pluripotency. Overall, I have defined a unique epigenetic signature of purified pluripotent stem cells and identified conserved epigenetic changes that likely play an important role in the maintenance of pluripotent state or play a role in the commitment to differentiation (Chapter 1). ❧ My second research goal was to investigate the role of the PR-SET7 H4K20 mono-methyltransferase (H4K20me1) in the transcriptional regulation of specific genes. Although PR-SET7-mediated H4K20me1 was previously shown to be involved in several DNA-templated processes including chromatin compaction, DNA damage response, DNA replication and cell cycle progression, the role of H420me1 in transcriptional regulation remains controversial. Initial studies showed that H4K20me1 functioned as a repressor but newer studies suggested a role in activation. Using conventional molecular biology techniques, I found that PR-SET7 and H4K20me1 predominantly functions as a transcriptional repressor of specific sets of genes. Consistent with this, my bioinformatics analysis indicated that H4K20me1 associated genes are largely devoid of acetylated histones

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University of Southern California Dissertations and Theses

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Asset Metadata

Creator Veerappan, Chendhore Sai (author)

Core Title Epigenetic plasticity of cultured female human embryonic stem cells and regulation of gene expression and chromatin by PR-SET7 mediated H4K20me1

School Keck School of Medicine

Degree Doctor of Philosophy

Degree Program Genetic, Molecular and Cellular Biology

Publication Date 11/13/2012

Defense Date 10/16/2012

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag embryonic stem cells,epigenetics,H3K27me3,H4K4me3,heterogeneity,OAI-PMH Harvest,X-chromosome inactivation

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Rice, Judd C. (committee chair), An, Woojin (committee member), Laird, Peter W. (committee member), Stallcup, Michael R. (committee member), Watanabe, Richard M. (committee member)

Creator Email cveerappan@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c3-112300

Unique identifier UC11289082

Identifier usctheses-c3-112300 (legacy record id)

Legacy Identifier etd-VeerappanC-1289.pdf

Dmrecord 112300

Document Type Dissertation

Rights Veerappan, Chendhore Sai

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA