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The role of Hic-5 in glucocorticoid receptor binding to chromatin

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The role of Hic-5 in glucocorticoid receptor binding to chromatin

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THE ROLE OF HIC-5 IN GLUCOCORTICOID RECEPTOR
BINDING TO CHROMATIN

by
Brian Hae Kang Lee

______________________________________________________________________

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
(CANCER BIOLOGY AND GENOMICS)

May 2018

ii
DEDICATION
To my wife, Anne-Sophie Lee,
for her love, support, and sacrifice through the years.

iii
ACKNOWLEDGEMENTS
First of all, I would like to thank my mentor, Dr. Michael Stallcup, for his guidance and
support through this journey. I am sincerely and deeply grateful for his wisdom, patience and
endless encouragement throughout my dissertation research. His scholarly advice, scientific
approach, and keen understanding of my strengths and weaknesses has helped me to grow as
a scientist.
Sincere words of thanks go to my committee members, Dr. Baruch Frenkel and Dr.
Ruchi Bajpai, for their guidance, suggestions, and support during my thesis research. I also
thank my qualifying committee members, Dr. Peggy Farnham, Dr. Gerry Coetzee, Dr. Woojin
An, Dr. Judd Rice, and once again Dr. Baruch Frenkel, for guiding me in forging the path of my
research project. I thank Dr. Yibu Chen, Dr. Meng Li, Dr. Suhn Rhie, Dr. Kim Siegmund, and
Phoebe Guo for advice on bioinformatics analysis.
With great appreciation I acknowledge the support and friendship from members of the
Stallcup lab: Coralie Poulard, Daniel Gerke, Danielle Bittencourt, Rajas Chodanakar, Dai-Ying
Wu, and Chen-Yin Ou. They have helped me overcome technical difficulties and failed attempts,
as well as enjoy the thrill of successful experiments.
I also extend my heartfelt thanks to my family and close friends for supporting me
through this arduous journey whenever I needed it. I am grateful for my parents, David Kyoung
Hwa and Lisa Jong Hee Lee, for their unconditional love and for instilling in me at young age an
incessant pursuit of learning. I am indebted to my parents and my parents-in-law, Minh Hai and
Pauline Bui, for their love and support especially in caring for my daughters so that I can spend
the time needed in the lab.
I would like to thank my wonderful daughters, Noemie and Avaleen Lee, for the bountiful
joy they have brought to my life. Words cannot express how happy I am that both of them joined

iv
me in this journey. They have made me stronger, better and more fulfilled than I could ever
imagine.
I will forever be grateful to my wife, Sophie Lee, for her love, support and sacrifice
throughout my dissertation. My thesis research and, moreover, my growth as a person, would
not have been possible without her encouragement and her continual reminder of putting
everything into perspective. She is the best thing that has ever happened to me.
Finally, I thank God for providing me with the strength, determination and abundant
blessings that helped me complete this journey.

v
TABLE OF CONTENTS
DEDICATION ............................................................................................................................... ii
ACKNOWLEDGEMENTS ............................................................................................................ iii
LIST OF TABLES ......................................................................................................................... vi
LIST OF FIGURES ...................................................................................................................... vii
LIST OF FIGURES ...................................................................................................................... ix
CHAPTER 1 Introduction and Background ................................................................................... 1
CHAPTER 2 Mechanism of action by Hic-5 on the block gene class ……………………….......... 9
2.1 Introduction …….......................................................................................................... 9
2.2 Materials and Methods .............................................................................................. 12
2.3 Results ...................................................................................................................... 22
2.4 Discussion ................................................................................................................ 64
CHAPTER 3 Characteristics that distinguish the block gene class ……………........................... 72
3.1 Introduction ...…........................................................................................................ 72
3.2 Materials and Methods .............................................................................................. 75
3.3 Results ...................................................................................................................... 78
3.4 Discussion .............................................................................................................. 107
CHAPTER 4: Conclusion ...………............................................................................................ 113
REFERENCES ......................................................................................................................... 119

vi
LIST OF TABLES
Table 3.1. Total GR peaks (or GR binding regions, GBR) identified for each condition
using the MACS2+IDR method ……........................................................................................... 79

Table 3.2. Total open chromatin regions from ATAC-seq analysis ............................................. 97

vii
LIST OF FIGURES
Figure 2.1. Comparisons from RNA-seq data used to determine the three gene classes and
the genes within each class that depend on CHD9 or BRM for dex-regulated expression …….. 17

Figure 2.2. Dynamic GR-GBR interaction model ………………………………………………........ 23

Figure 2.3. siRNA depletion of chromatin remodelers (CR) and Hic-5 ………………………....... 27

Figure 2.4. CHD9 and BRM are selectively required for dex-induced expression of
representative block genes ……………….…………………………..………………………………. 29

Figure 2.5. Temporal profile of dex-induced gene expression in cells depleted of Hic-5,
chromatin remodelers, or both …………………………………………………………………...…… 32

Figure 2.6. Temporal profile of dex-induced gene expression in cells depleted of Hic-5,
chromatin remodelers, or both, using a second siRNA for CHD9 and BRM …………………….. 34

Figure 2.7. Genome-wide selective requirement of CHD9 and BRM for dex-regulated
expression by block genes versus ind and mod genes ……………………………………………. 37

Figure 2.8. Alternative overlapping strategy for identifying ind and mod genes dependent
on CHD9 or BRM ……………………………………………………………………………………..... 40

Figure 2.9. Genes dependent on CHD9 and BRM for dex-regulated expression, defined
with more stringent parameters ………………………………………………………………...…….. 42

Figure 2.10. Genome-wide effect of CHD9 and BRM depletion on dex-regulated gene
expression …………………………………………………………………………………………….... 46

Figure 2.11. Gene ontology analysis for block genes and for the combined ind and
mod genes ……………………………………………………………………………………………… 50

Figure 2.12. CHD9 and BRM are required for GR occupancy at GBR of representative
block genes .………………………………………………………………………………………….… 53

viii

Figure 2.13. CHD9 and BRM are selectively required for chromatin remodeling at GBR
of representative block genes ………..……………………………………………………….………. 57

Figure 2.14. Hic-5 effect on the interaction between GR and chromatin remodelers …………... 61

Figure 2.15. Model illustrating the mechanism of Hic-5 action on dex-induced block genes ..… 67

Figure 3.1. Identifying Hic-5 blocked GBR and non-blocked GBR …………………………...…… 81

Figure 3.2. Hic-5 blocked GBR require CHD9 and BRM …………………………………………... 84

Figure 3.3. Differential GR peak analysis shows Hic-5 blocked GBR require CHD9
and BRM ………………………………………………………………………………………………... 87

Figure 3.4. ChIP-seq GR occupancy near representative genes for each class ……………....... 89

Figure 3.5. Hic-5 blocked GBR are preferentially enriched near the block genes ………………. 94

Figure 3.6. Chromatin at Hic-5 blocked GBR is less accessible than at non-blocked GBR ….... 98

Figure 3.7. ETS1 motif is enriched at Hic-5 blocked GBR and GBR near the block genes …... 101

Figure 3.8. ETS1 motif is enriched at Hic-5 blocked GBR that become newly chromatin-
accessible when Hic-5 is depleted ............................................................................................ 105

ix
ABSTRACT

The steroid hormone-activated glucocorticoid receptor (GR) regulates cellular stress
pathways by binding to genomic regulatory elements of target genes and recruiting coregulator
proteins to remodel chromatin and regulate transcription complex assembly. The coregulator
hydrogen peroxide-inducible clone 5 (Hic-5) is required for glucocorticoid regulation of some
genes but not others and blocks the regulation of a third gene set by inhibiting GR binding. How
Hic-5 exerts these gene-specific effects and specifically how it blocks GR binding to the GR
binding regions (GBR) of some genes but not others is unclear. Here I report that site-specific
blocking of GR binding is due to gene-specific requirements for ATP-dependent chromatin
remodeling enzymes. By depletion of 11 different chromatin remodelers, we found that ATPases
chromodomain helicase DNA-binding protein 9 (CHD9) and Brahma homologue (BRM, a
product of the SMARCA2 gene) are required for GC-regulated expression of the blocked genes
but not for other GC-regulated genes. Hic-5 selectively inhibits GR interaction with CHD9 and
BRM, thereby blocking chromatin remodeling and robust GR binding at GR-binding sites
associated with blocked genes. Furthermore, I identify specific differences in chromatin
conformation, chromatin remodeler requirements, and local DNA sequence motifs that
distinguish GR binding regions (GBR) at Hic-5 blocked genes from GBR at other GC-regulated
genes. Genome-wide assessment of GR occupancy with ChIP-seq shows that blocked GBR
generally require CHD9 and BRM for GR occupancy in contrast to GBR that are not blocked by
Hic-5 and that Hic-5 blocked GBR are enriched near Hic-5 blocked GR target genes but not
near GR target genes that are not blocked by Hic-5. Hic-5 blocked GBR are in a closed
conformation prior to Hic-5 depletion, and require Hic-5 depletion and glucocorticoid treatment
to create an open conformation necessary for GR occupancy. Additionally, ETS1 transcription
factor binding motif was enriched near blocked GBR and blocked genes but not near non-

x
blocked GBR or non-blocked GR target genes. Thus, Hic-5 regulates GR binding site selection
by a novel mechanism, exploiting gene-specific requirements to selectively influence DNA
occupancy and gene regulation by a transcription factor.

1
Chapter 1
Introduction and Background

The human body is composed of various cells that carry the same basic genetic
information. The differentiating factor between the various cell types is which genes are being
expressed to carry out the specialized function for each cell. Therefore, understanding how
genes are transcribed is important and fundamental to understanding the functions of the
human body. Gene transcription involves the actions of several coregulator proteins that control
gene expression by contributing to the transcriptional machinery. The following chapters will
focus on regulating gene expression in the context of a particular coregulator,hydrogen
peroxide-inducible clone-5 (Hic-5, also known as TGFB1I1). In addition to acting as a
conventional coregulator, Hic-5 has a unique role in influencing DNA occupancy of a
transcription factor. By understanding the mechanism by which Hic-5 influences transcription I
will be able to uncover the intricacies of gene regulation and hence be able to control cellular
function necessary to combat diseased cells.

Glucocorticoid receptor
The model system I will use to study transcriptional regulation is the glucocorticoid
receptor. The glucocorticoid receptor (GR, NR3C1) is a member of the nuclear receptor family
of ligand activated transcription factors. GR has a structure characteristic of other nuclear
receptors with a N-terminal transcriptional activation domain, a central DNA binding domain,
and a C-terminal ligand binding domain (Kumar and Thompson, 2005). GR regulates diverse
physiological programs including the immune, adipocyte, cardiovascular, musculoskeletal,
respiratory, and hepatic system (Kadmiel and Cidlowski, 2013). For example, synthetic
glucocorticoids (GC), such as dexamethasone (dex), are widely prescribed as anti-inflammatory
drugs. However, one of the major side effects of dexamethasone is that it can suppress bone

2
formation and thereby induce osteoporosis by inducing osteoblast apoptosis, reducing
osteoblast differentiation, and stimulating bone resorbing osteoclasts (Frenkel et al., 2015).
GR mediates the physiological and pharmacological actions of the glucocorticoid
hormone in cells by regulating the activation and repression of target genes (Busillo and
Cidlowski, 2013). Upon binding of GC to the GR ligand binding domain, GR undergoes a
conformational change that allows the receptor to be dissociated from heat shock protein 90
and promotes the translocation of GR into the nucleus. Transcriptional regulation involves the
binding of the GR to specific GR binding regions (GBR) in the chromatin and the recruitment of
numerous coregulator proteins that remodel the chromatin landscape and facilitate the
assembly of an active transcription complex at the transcription start site.

Coregulators
Coregulators are a group of proteins that are recruited to nuclear receptors and other
DNA bound transcription factors that can either activate or repress the rate of transcription of
targeted genes. Hundreds of coregulator proteins have been identified to be associated with
nuclear receptors (Lonard and O'Malley, 2012). The role of some coregulator proteins is to link
the nuclear receptor, such as GR, to the basal transcriptional machinery while others are
involved in reading and modifying histones and remodeling chromatin (Bittencourt et al., 2012;
Engel and Yamamoto, 2011; Fryer and Archer, 1998; Kim et al., 2003; Yang et al., 2000).
Coregulator recruitment to nuclear receptors and other sequence-specific transcription factors
occurs in a gene-specific manner resulting in specific changes in gene regulation. An individual
coregulator protein can act as both a coactivator or a corepressor for transcription. For example,
lysine methyltransferase G9a has been shown to function as both a coactivator and a
corepressor (Bittencourt et al., 2012; Lee et al., 2006). Furthermore, different target genes of the
same transcription factor have different coregulator requirements (Chodankar et al., 2014; Wu
et al., 2014). However, the underlying mechanism directing the gene-specific coregulator

3
requirements is mostly unknown. The unique regulatory environment for each gene may specify
which coregulators are required, including the local chromatin conformation, the DNA sequence
at the transcription factor binding site, and presence of other transcription factors and
coregulators at or near the transcription factor binding site. One of the goals of this study is to
explore the gene-specific requirements of a specific coregulator protein, Hic-5.

Coregulators that modify or remodel chromatin
Many coregulators possess chromatin remodeling enzymatic activity. Coregulators can
influence chromatin structure and activity either modifying histones or by facilitating ATP-
dependent chromatin remodeling. DNA is compacted into the nucleus by wrapping 147 base-
pair of DNA around nucleosomes composed of an octamer of histone proteins with two each of
the following histones: H2A, H2B, H3, and H4. Linker histone H1 found on the DNA between
nucleosomes helps to form higher order chromatin structures and chromatin stability to further
compact the DNA in the nucleus. Histone tails protruding out of the nucleosomes can undergo
post-translational modifications including acetylation, methylation, phosphorylation and
ubiquitination (Prakash and Fournier, 2017). There are many coregulators that are capable of
covalently modifying histone tails or reading the modified histone tails including arginine
methyltransferase CARM1 (Schurter et al., 2001) and lysine methyltransferase G9a (Tachibana
et al., 2001).
While histone modifying proteins alter histone tails, ATP-dependent chromatin
remodelers are needed for nucleosome dynamics. By hydrolyzing ATP, chromatin remodelers
can slide nucleosomes, eject nucleosomes, unwrap the DNA surround the nucleosome, or alter
the composition of the nucleosome by histone dimer exchange or ejection (Clapier and Cairns,
2009). All ATP-dependent chromatin remodelers share a conserved ATPase domain. However,
the flanking domains separate the ATPases into four distinct families of ATP-dependent
chromatin remodeler enzymes: switching defective/sucrose nonfermenting (SWI/SNF), imitation

4
switch (ISWI), chromodomain, helicase, DNA binding (CHD), and the inositol requiring 80
(INO80) family (Clapier and Cairns, 2009). Each of the four families have specialized chromatin
remodeling activities. Numerous ATP-dependent chromatin remodelers, including BRG1, BRM,
and SNF2H, have been implicated in transcriptional regulation of nuclear receptors (Burd et al.,
2012; Fryer and Archer, 1998).

ATP-dependent chromatin remodelers and GR binding
Ligand activated GR can interact with the DNA in a variety of distinct ways. The classical
method of GR binding involves direct binding of GR as a homodimer to the DNA at canonical
GR binding sequences composed of 15 base pairs with pseudopalindromic 6 base pair half-
sites separated by a 3 base pair separator (Strähle et al., 1987). Genome-wide analysis of GR
binding sites through ChIP-seq with GR antibody reveals that although most of the GBRs
contain the canonical GR binding sequence, there are still some GBRs that do not exhibit the
canonical 15bp sequence. GR binding to non-canonical sites may occur at negative GR
response elements with a DNA sequence resembling the canonical GR binding sequence with
slight differences (Surjit et al., 2011). GR can also bind as a monomer to 6bp half-sites and still
target genes (Schiller et al., 2014). GR binding to composite sites with nearby DNA bound
transcription factors can also modulate gene expression (Ratman et al., 2013). Finally, GR may
also tether to other transcription factors to indirectly regulate gene expression. Most of the
tethered GR binding has been linked to transcriptional repression such as when GR is bound to
NF-KB and API (Kassel and Herrlich, 2007). However, transcriptional activation can occur at
GR tethered sites such as when GR is tethered to STAT5 (Engblom et al., 2007).
Chromatin structure plays a major role in dictating GR binding to the DNA. Open or
chromatin accessible regions allow the recruitment of transcription factors, including GR.
Previous studies using the MMTV promoter as a model system showed that GR was able to
bind relatively inaccessible chromatin by triggering localized chromatin remodeling with ATP-

5
dependent chromatin remodelers (Fletcher et al., 2002). More recently, genome-wide studies of
GR binding and chromatin accessibility, as measured by DNAse-I hypersensitivity, have shown
that GR binding mainly occurs at chromatin accessible sites that existed prior to hormone
treatment and only a few sites (~5%) are created de novo by GR (John et al., 2011). However,
upon GR activation the preexisting accessible GR binding sites can undergo additional
chromatin remodeling (Burd et al., 2012).
The conventional transcription factor binding model suggests a static interaction
between a transcription factor and the DNA where upon recruitment to the DNA, the
transcription factor resides at the binding site for a prolonged period. However, recent studies
have shown that GR and other transcription factors interact with the DNA binding site in a
dynamic on and off manner (Voss and Hager, 2014). Techniques using fluorescent recovery
after photobleaching (FRAP) mainly from Dr. Gordon Hager’s lab at National Institutes of Health
reveal that GR transiently interacts with the DNA (McNally et al., 2000). Furthermore, the
transient interaction between GR and the DNA leads to the recruitment of ATP-dependent
chromatin remodeler enzymes which open up the chromatin conformation at the GBR, allowing
the site to become more accessible.
GC-induced binding of GR to GBR is a cooperative process between GR and chromatin
remodelers, in which GR recognizes its specific DNA binding motif and recruits ATP-dependent
chromatin remodeling enzymes that open up chromatin structure (Burd and Archer, 2013; Fryer
and Archer, 1998; Nagaich et al., 2004). Furthermore, GR interacts in a rapid dynamic on-and-
off fashion with its specific DNA motifs (McNally et al., 2000); and experimental techniques such
as chromatin immunoprecipitation which are used to measure the strength of association of GR
with a specific GBR (often referred to as occupancy) are simply capturing a snapshot of the
steady-state dynamic interaction between GR and a given GBR. In view of the dynamic and
cooperative nature of this process, I propose that the initial GR interaction with the GBR is

6
weak; but this weak GR-GBR interaction allows GR to recruit chromatin remodelers, which open
up the chromatin and thereby allow a more robust but still dynamic GR occupancy of the site.

Hic-5 alters chromatin structure and GR binding
An interesting coregulator protein known as Hic-5 selectively affects GC-stimulated gene
regulation by GR. Hic-5 is a member of the paxillin family of molecular adaptor proteins with two
types of protein interaction domains: four LD (leucine and aspartate-containing) motifs at the N
terminus and four Lin11, Isl-1 and Mec-3 (LIM) domains, each composed of two adjacent zinc-
fingers, on the C terminus (Bach, 2000; Brown et al., 1998). Studies have shown that Hic-5 is
highly expressed in mesenchymal, fibroblastic and osteoblastic cell lines, while exhibiting low
expression in cell lines of epithelial origin (Brunskill et al., 2001; Pignatelli et al., 2012;
Shibanuma et al., 2012; Tumbarello and Turner, 2007). In the cytosol Hic-5 has been widely
studied as an adapter protein at focal adhesion complexes (Kim-Kaneyama et al., 2012). As a
focal adhesion protein Hic-5 has been implication in tumor progression including epithelial to
mesenchymal transition and invadopodia formation to promote cell invasion and metastasis
(Deakin and Turner, 2011; Goreczny et al., 2017). As a matter of fact, a number of studies have
suggested Hic-5 has a role in different types of cancers including breast, prostate and
melanoma (Deakin et al., 2012; Noguchi et al., 2012). In the nucleus Hic-5 serves as a
coregulator for a variety of transcription factors including GR (Aghajanova et al., 2009; Drori et
al., 2005; Leach et al., 2014; Li et al., 2011; Shibanuma et al., 2004; Wang et al., 2008; Yang et
al., 2000). As a coregulator of nuclear receptors, Hic-5 has been associated with many
physiological and disease functions including endometriosis through the progesterone receptor
(Aghajanova et al., 2009), epithelial cell differentiation by affecting Peroxisome proliferator
activated receptor g (PPARg) transcriptional activity (Drori et al., 2005), and prostate
tumorigenesis and castrate responsiveness through the androgen receptor (Li et al., 2011).
The Stallcup lab has recently shown that in U2OS cells endogenous Hic-5 modulates

7
GC-regulated gene transcription by GR in a highly gene-specific manner, functioning as
coactivator for some GR target genes and corepressor for others. GC-regulated genes were
categorized into three different classes with respect to their dependence on Hic-5: Hic-5
independent (ind) genes defined as GC-regulated genes where Hic-5 depletion has no effect;
Hic-5 modulated (mod) genes defined as GC-regulated genes where the depletion of Hic-5
alters the magnitude of activation or repression by GC; and Hic-5 blocked (block) genes
described as genes that are not regulated by GC in the presence of Hic-5 but become robustly
GC-regulated after Hic-5 depletion (Chodankar et al., 2014). The effects of Hic-5 depletion were
observed at both mRNA and pre-mRNA levels of mod and block genes, demonstrating that the
mechanism of Hic-5 action occurs at the transcriptional level. Mechanistic examination of
selected GC-induced mod genes showed that Hic-5 is recruited to GR binding regions (GBR) by
its interaction with GR and acts at late stages of transcription complex assembly, facilitating
recruitment of the Mediator complex and RNA polymerase II (Chodankar et al., 2014). In
contrast, examination of three selected block genes indicated that Hic-5 prevented
transcriptional activation by impeding GC-induced chromatin remodeling and robust GR
occupancy at GBR associated with the block genes (Chodankar et al., 2014). Additionally, in
GR chromatin immunoprecipitation sequencing analysis Hic-5 depletion almost doubled the
number of GR-occupied sites in the genome (Chodankar et al., 2015).
Since coregulators generally have been shown to facilitate steps of transcription
complex assembly that occur subsequent to transcription factor binding to DNA, the inhibition by
Hic-5 of GR occupancy at GBR that control GC regulation of a specific set of genes is an
unexpected and unique observation. I explore the hypothesis that transcription factor occupancy
and chromatin remodeling are co-dependent processes and that Hic-5 influences GR
occupancy by interfering with the dynamic interaction of GR with chromatin remodeling
complexes. The recruitment of chromatin remodelers by GR is thus required for chromatin
remodeling which in turn facilitates robust GR occupancy of the GBR and subsequent

8
transcription complex assembly on the transcription start site of the associated GC-regulated
gene. My thesis research also addresses the gene-specific actions of Hic-5. I determine the
mechanism and the gene-specific characteristics that allows Hic-5 to block GR binding and
transcriptional regulation at the block class of GR target genes while allowing robust GR binding
and GC-regulated transcription at other GR target genes (the ind and mod classes). My hope is
that the finding in this dissertation will elucidate new mechanisms that control transcription factor
binding site selection and contribute to the gene-specific actions of coregulators.

9
Chapter 2
Mechanism of action by Hic-5 on the block gene class

2.1 Introduction
Transcription factors regulate gene expression by binding to specific regulatory DNA
sequences, where they can either activate or repress transcription of the associated gene(s).
The regulatory process directed by the transcription factor involves recruitment of numerous
coregulator proteins that remodel the chromatin landscape around the transcription factor
binding site and the transcription start site of the regulated gene and regulate the assembly of
an active transcription complex at the transcription start site. Each coregulator contributes
specific molecular functions to accomplish these complex processes in a presumably
coordinated fashion, resulting in increased or decreased production of mRNA encoded by the
gene (Glass and Rosenfeld, 2000; Lonard and O'Malley, 2012; Rosenfeld et al., 2006). Many
coregulators act in a gene-specific manner, i.e. they are required for the regulation of a subset
of the genes regulated by any given transcription factor. Furthermore, a single coregulator can
have different effects (i.e. activation or repression) and act by different mechanisms on different
target genes, even for genes regulated by a single transcription factor within a single cell type
(Bittencourt et al., 2012; Chodankar et al., 2014; Rogatsky et al., 2002; Won Jeong et al., 2012;
Wu et al., 2014; Yang et al., 2006). However, the mechanisms that specify the action of a
coregulator on a given target gene are mostly unknown. In this chapter I report a specific
mechanism that directs the gene-specific actions of the protein Hydrogen peroxide-inducible
clone-5 (Hic-5, also known as TGFB1I1) as a coregulator for the glucocorticoid receptor (GR,
NR3C1). GR, a member of the nuclear receptor family of ligand activated transcription factors,
regulates diverse physiological programs including inflammation and metabolism of glucose,
lipids, and proteins by activating and repressing transcription of specific genes. GR is activated

10
by binding of the natural glucocorticoid (GC) hormone cortisol or various synthetic analogues,
which are widely used in treatment of many types of inflammatory diseases and cancer (Biddie
et al., 2012).
Hic-5 is a member of the paxillin family of molecular adaptor proteins, characterized by
two types of protein interaction domains: four LD (leucine and aspartate-containing) motifs at
the N terminus and four Lin11, Isl-1 and Mec-3 (LIM) domains, each composed of two adjacent
zinc-fingers, on the C terminus (Bach, 2000; Brown et al., 1998). In the cytosol Hic-5 has been
widely studied as an adapter protein at focal adhesion complexes (Kim-Kaneyama et al., 2012).
In the nucleus Hic-5 serves as a coregulator for a variety of transcription factors including GR
(Aghajanova et al., 2009; Drori et al., 2005; Leach et al., 2014; Li et al., 2011; Shibanuma et al.,
2004; Wang et al., 2008; Yang et al., 2000). As a coregulator of nuclear receptors, Hic-5 has
been associated with many physiological and disease functions including endometriosis through
the progesterone receptor (Aghajanova et al., 2009), epithelial cell differentiation by affecting
Peroxisome proliferator activated receptor g (PPARg) transcriptional activity (Drori et al., 2005),
and prostate tumorigenesis and castrate responsiveness through the androgen receptor (Li et
al., 2011). A previous study showed that endogenous Hic-5 modulates GC-regulated gene
transcription by GR in a highly gene-specific manner, functioning as coactivator for some GR
target genes and corepressor for others. GC-regulated genes were categorized into three
different classes with respect to their dependence on Hic-5: Hic-5 independent (ind) genes are
regulated by GC independently of Hic-5 depletion; Hic-5 modulated (mod) genes are regulated
by GC in the presence of Hic-5, but depletion of Hic-5 alters the magnitude of activation or
repression by GC; Hic-5 blocked (block) genes are not regulated by GC until Hic-5 is depleted
from the cells (Chodankar et al., 2014). The effects of Hic-5 depletion were observed at both
mRNA and pre-mRNA levels of mod and block genes, demonstrating that the mechanism of
Hic-5 action occurs at the transcriptional level. These same Hic-5-influenced classes of genes
were also observed in studies with estrogen receptor a and androgen receptor (Chodankar et

11
al., 2015; Leach et al., 2014). Mechanistic examination of selected GC-induced mod genes
showed that Hic-5 is recruited to GR binding regions (GBR) by its interaction with GR and acts
at late stages of transcription complex assembly, facilitating recruitment of the Mediator complex
and RNA polymerase II (Chodankar et al., 2014). In contrast, examination of three selected
block genes indicated that Hic-5 prevented transcriptional activation by impeding GC-induced
chromatin remodeling and robust GR occupancy at GBR associated with the block genes
(Chodankar et al., 2014). Additionally, in GR chromatin immunoprecipitation sequencing
analysis Hic-5 depletion almost doubled the number of GR-occupied sites in the genome
(Chodankar et al., 2015).
Since coregulators generally have been shown to facilitate steps of transcription
complex assembly that occur subsequent to transcription factor binding to DNA, the inhibition by
Hic-5 of GR occupancy at GBR that control GC regulation of a specific set of genes is an
unexpected and unique observation. In this chapter I explore the hypothesis that transcription
factor occupancy and chromatin remodeling are co-dependent processes and that Hic-5
influences GR occupancy by interfering with the dynamic interaction of GR with chromatin
remodeling complexes; the recruitment of chromatin remodelers by GR is thus required for
chromatin remodeling which in turn facilitates robust GR occupancy of the GBR and subsequent
transcription complex assembly on the transcription start site of the associated GC-regulated
gene. I also address the gene-specific actions of Hic-5, i.e. the mechanism that allows Hic-5 to
block GR binding and transcriptional regulation at the block class of GR target genes while
allowing robust GR binding and GC-regulated transcription at other GR target genes (the ind
and mod classes). The results of this study will elucidate new mechanisms that control
transcription factor binding site selection and contribute to the gene-specific actions of
coregulators.

12
2.2 Materials and Methods
Cell culture and siRNA transfection
U2OS osteosarcoma cells stably expressing wild-type GRα (U2OS-GRα) were
maintained as described (Chodankar et al., 2014). Actions of GC are well known in most cell
types including osteogenic lineages (Pockwinse et al., 1995; Shalhoub et al., 1992). However,
GR is expressed at extremely low levels in the parent U2OS cell line, and therefore the line
used here was derived by introduction of a transgene (Rogatsky et al., 1997). U2OS-GRα cells
express levels of GR that are equivalent to levels expressed naturally in many cell lines and
primary cells. Cells were grown in medium supplemented with 5% (vol/vol) FBS and transfected
with siRNA using Lipofectamine RNAiMAX (Invitrogen). For double depletion of chromatin
remodeler and Hic-5, equivalent amounts siRNA for a chromatin remodeler and Hic-5 (siHic5)
were added. For single protein depletions, equivalent amounts of siRNA for the targeted protein
and nonspecific control siRNA (siNS) were used such that the total volume and mass of siRNA
was consistent. 48 h after siRNA transfection the cells were either treated for the indicated
length of time with 100 nM dex (Sigma) or an equivalent amount of ethanol as control. siRNA
sequences for siNS and siHic-5 were previously described (Chodankar et al., 2014). siRNAs for
chromatin remodelers were designed and purchased through MISSION predesigned siRNA
(Sigma). The sense sequences are: siBRG1 (5’-GGAAUACCUCAAUAGCAUU-3’), siBRM (5’-
CCAAAUGAUUGCUCGACGA-3’), siSNF2h (5’-CAACAGAUAUGCAUCUAGU-3’), siINO80 (5’-
GCAUGAAUUGGUUGGCCAA-3’), siEP400 (5’-CUGAUGAGGAGUUUCAACA-3’), siCHD1 (5’-
CUCAGUACCAUGAUCAUCA-3’), siCHD4 (5’-CAAACAGGAGCUUGAUGAU-3’), siCHD6 (5’-
CAAACUUCUGGAGGGUCU-3’), siCHD7 (5’-GGACUUUGCACGUAGCACA-3’), siCHD8 (5’-
CAGAAUCAUUCAGGUCUAU-3’), siCHD9 (5’-CGAAUUGAUGGCAGAGUCA-3’). siRNA
depletion was verified by immunoblot. Depletion of BRM and CHD9 were validated with a

13
second siRNA with sense sequences as follows: siCHD9#2 (5’-AAGUAUUUGAUGGAGUU-3’),
siBRM#2 (5’-CCAAAUGAUUGCUCGACGA-3’).

Immunoblot
48 h after transfection with siRNA, the cells were washed three times with PBS and
lysed for 10 min with RIPA lysis buffer containing protease inhibitor cocktail (Roche) at 4ºC. Cell
lysates were centrifuged, and the supernatant was resolved by electrophoreses in 4–15% Mini-
PROTEAN TGX precast polyacrylamide gradient protein gels (Bio-Rad). The following primary
antibodies were used for immunoblotting: GAPDH (Sigma G9545), Hic-5 (BD Transduction Lab
611165), GR (Santa Cruz Biotechnology SC8992), BRG1 (Abcam EPNCIR11A), BRM (Abcam
ab15597), INO80 (Abcam ab118787), SNF2h (Abcam ab72499), p400 (Abcam ab5201), CHD1
(Bethyl Laboratories A301218A), CHD4 (Abcam ab72418), CHD6 (Bethyl Laboratories
A301221A), CHD7 (Cell Signaling Technologies 6505S), CHD8 (Cell Signaling Technology
11891S), and CHD9 (Novus Biologicals NB100-60419). Secondary antibodies used were goat
anti-rabbit (Promega W4011) and goat anti-mouse (Promega W4021).

Quantitative RT-PCR
Total RNA was isolated from cells using TRIzol reagent (Thermo Scientific) and reverse
transcribed using iScript cDNA synthesis kit (Bio-Rad). The resulting cDNA was analyzed by
quantitative PCR amplification with the LightCycler 480 SYBR Green I Master (Roche) on the
LightCycler 480 system (Roche). The following primers used to detect mRNA expression with
quantitative PCR were described previously: RP1L1, HOXD1, IGFBP1, MSX2, and SCNN1A
(Chodankar et al., 2014); and GRAMD4 (Chodankar et al., 2015). Other primers used were as
follows: TIPARP (forward primer: 5’-TCCGCTCCTGTTTTATACTGC-3’; reverse primer: 5’-
AGTTTGCTGAAGTGACCCC-3’), and SLN (forward primer: 5’-CAAGCCGCTGTGAAAATGG-
3’; reverse primer: 5’-GAGCATCTCAGTCAATCCCAG-3’).

14

RNA-sequencing analysis
U2OS-GRα cells transfected with combinations of siRNA were treated with either
ethanol or 100 nM dex for 8 hours. 12 different conditions were examined: control (siNS/siNS) ±
dex; Hic-5 only depletion (siHic5/siNS) ± dex; CHD9 only depletion (siCHD9/siNS) ± dex; CHD9
and Hic-5 double depletion (siCHD9/siHic5) ± dex; BRM only depletion (siBRM/siNS) ± dex; and
BRM and Hic-5 double depletion (siBRM/siHic5) ± dex; Total RNA was extracted using TRIzol
(Thermo Scientific) and 3 biological replicates were performed on different days. RNA samples
were reverse transcribed using iScript cDNA synthesis kit (Bio-Rad); quality of the cDNA was
assessed using Agilent Technologies 2100 Bioanalyzer, and quantitative RT-PCR analysis was
performed for selected GR target genes. A total of 36 high quality samples (12 conditions x 3
replicates each) were submitted to the Next-Generation Sequencing Core at the University of
Southern California Norris Comprehensive Cancer Center for library preparation and
sequencing. Single-end 75 bp RNA-sequencing data were generated for the samples using
Illumina NEXTseq 500. Sequencing results produced 36-58 million raw reads per sample. After
trimming the raw reads for quality and adapter sequence, the samples were mapped using
TopHat 2.1.1 against the GRCh38/hg38 human reference genome (Trapnell et al., 2010).
Mapped reads were quantified to known UCSC genes using the GenomicAlignments R package
(Lawrence et al., 2013). Gene expression levels were normalized with the upper quantile (UQ)
method and lowly expressing genes were excluded such that genes with more than one count
per million in at least 3 samples were analyzed (Bullard et al., 2010). I implemented the remove
unwanted variation (RUV) strategy to account for unknown nuisance technical effects between
samples (Risso et al., 2014). Differentially expressed genes were identified with edgeR using a
1.3-fold change in expression (log
2
= 0.4) and false discovery rate adjusted p-value ≤ 0.05 as
cutoffs (Robinson et al., 2010). RNA-sequencing data was submitted to Gene Expression

15
Omnibus (GEO) under the accession number GSE93871. Gene Ontology was used for
functional annotation of the gene classes (Ashburner et al., 2000;
The Gene Ontology Consortium, 2017).

Algorithms for defining ind, mod, and block gene classes and genes that require CHD9 or
BRM
Assignment of genes to the block, ind, and mod classes involved a previously developed
strategy (Wu et al., 2014), using three different comparisons from the RNA sequencing data to
define the following gene sets from the RNA sequencing data: dex-regulated genes in control
cells transfected with non-specific siRNA (Figure 2.1A, comparison I), dex-regulated genes in
Hic-5 depleted cells (Figure 2.1A, comparison II), and genes with mRNA levels that were
significantly different between the control dex-treated cells and Hic-5 depleted dex-treated cells
(Figure 2.1A, comparison III). Except where otherwise indicated, significant differences in
mRNA levels were defined with a 1.3-fold change cutoff and FDR adjusted p-value ≤ 0.05.
Specific regions of overlap among the gene sets derived from these three comparisons defined
the block, ind, and mod gene sets: the ind genes (Figure 2.1B, blue region) were dex regulated
in control cells (included in set I) and in Hic-5 depleted cells (included in set II) and had dex-
treated mRNA levels that were not significantly different in control and Hic-5-depleted cells
(excluded from set III); mod genes (Figure 2.1B, green regions) were defined by the intersection
of set I (dex-regulated in control cells), and set III (different dex-treated mRNA levels in control
versus Hic-5 depleted cells); the block genes (Figure 2.1B, red region) were included in sets II
and III but not in set I, i.e. they were dex-regulated only after Hic-5 depletion and had different
dex-treated mRNA levels in control versus Hic-5 depleted cells.
The number of genes in each of the three classes (block, ind, and mod) that required
CHD9 or BRM for dex-regulated expression was defined by overlapping each of the three gene
classes (block, ind, and mod) with two other comparisons from the RNA sequencing data.

16
Specifically, each gene class (block, ind, and mod) was overlapped with sets a and b or with
sets c and d (Figure 2.1C, D). Sets a and b were derived by comparing mRNA levels in cells
depleted of Hic-5 alone versus cells doubly depleted of Hic-5 and CHD9 or Hic-5 and BRM
(Figure 2.1C, left panel). Sets c and d were derived by comparing mRNA levels in cells depleted
of CHD9 or BRM alone versus control cells with no depletions (Figure 2.1C, right panel).
Although overlapping these comparisons identified several gene subsets of the block, ind, and
mod gene classes (Figure 2.1D, sectors i-iv), the genes of primary interest were those for which
significant regulation by dex was entirely dependent on CHD9 or BRM, i.e. genes that were
excluded from set a or c and included in set b or d (Figure 2.1D, sector iii).
Since block genes are only dex-regulated when Hic-5 is depleted, genes in the block
class were overlapped with the sets a and b. Again, except where otherwise specified we used
1.3-fold change in expression and FDR adjusted p-value ≤ 0.05 as cutoffs for the analysis.
Similar analyses were conducted with the ind and mod gene classes to determine the genes
that were dependent on CHD9 and/or BRM for dex-regulated expression. Since the dex
regulation of ind genes is independent of the presence or absence of Hic-5, the ind genes can
be overlapped with results from either the double depletion of chromatin remodeler and Hic-5
(Figure 2.1C, gene sets a and b) or the chromatin remodeler depletion only (Figure 2.1C, gene
sets c and d). Since many mod genes (138 out of 364 genes from Figure 2.7A) were no longer
dex-regulated upon Hic-5 depletion (Figure 2.1B, green region excluded from set II), genes in
this class were best analyzed by single depletion of CHD9 or BRM (without depleting Hic-5), so
that all mod genes were included in the analysis. Therefore, mod genes were overlapped with
sets c and d (Figure 2.1D). However, to test reproducibility, I analyzed the dependence of ind
and mod genes on CHD9 and BRM both in cells containing Hic-5 (Figure 2.1D) and in cells
lacking Hic-5 (Figure 2.1E).

17
Figure 2.1. Comparisons from RNA-seq data used to determine the three gene classes
and the genes within each class that depend on CHD9 or BRM for dex-regulated
expression. (A) Diagram shows which samples were compared to define the three gene sets
(comparison I, II, III) that were overlapped to identify the block, ind, and mod genes. (B) The set
of block (red), ind (blue), and mod (green) genes were determined by overlapping three gene
sets: set of genes from comparison I, set of genes from comparison II, and the set of genes
from comparison III (Figure 2.7A), according to the algorithms described in Materials and
Methods. This strategy for overlapping of gene sets was used to generate the data in Figure
2.7A. Differentially expressed genes in each of the three comparisons were identified using
edgeR with FDR adjusted p-value threshold of 0.05 and a 1.3 fold change cutoff. Theoretical
examples of activated and repressed gene expression profiles are provided as bar graphs for
each gene class. (C) Diagrams show which samples were compared to define gene sets that
were overlapped with the block, ind, or mod gene sets to identify genes that require CHD9 or
BRM for their dex-regulated expression, as described in the algorithms in Materials and
Methods: a and b compare mRNA levels in cells depleted of Hic-5 which contain or are depleted
of CHD9 or BRM; c and d compare mRNA levels in cells containing Hic-5 and either containing
or depleted of CHD9 or BRM. (D) Diagrams show which gene sets were overlapped to
determine the block, ind, and mod genes that require CHD9 or BRM for their dex regulated
expression, as described in the algorithms in Experimental Procedures. The strategies for
overlapping of gene sets shown here were used to generate the data in Figure 2.7B-D. (E) As
an alternative to the overlapping set strategy specified in C above, ind genes or the subset of
mod genes that were still dex-regulated after depletion of Hic-5 (226 genes in the central sector
of Figure 2.7A) were overlapped with sets a and b described in C above. This strategy indicates
the effect of depleting CHD9 or BRM in cells that are also depleted of Hic-5. Dark regions
identify the number and percent of genes from the ind genes or from the mod genes subset that
are dependent on CHD9 or BRM for dex-regulated expression.

18
Figure 2.1

A

B

19
Figure 2.1 continued

C

D

E

20
For the box plot showing the quantitative effects of depleting CHD9 and BRM on the
expression of block, ind, and mod genes the following formula was used:

𝑌 = (𝑋−𝑍)/𝑋 ´ 100

where Y = % decrease in log
2
fold change after CHD9 or BRM depletion; X = log
2
fold change
cause by dex in cells containing CHD9 or BRM; Z = log
2
fold change caused by dex in cells
depleted of CHD9 or BRM.

Chromatin immunoprecipitation
These experiments were performed as previously described (Chodankar et al., 2014)
with slight modifications. Briefly, U2OS-GRα cells grown on 15-cm dishes were transfected with
the appropriate siRNAs. After 48 h, the cells were treated with 100 nM dex or equivalent
amounts of ethanol for 1 h before crosslinking with 1% (v/v) formaldehyde for 10 mins at room
temperature and extracting chromatin from the harvested cells. Chromatin was sonicated 20-30
min (30s on/off cycles) with the Biorupter (Diagenode) at 4ºC to produce DNA fragment size of
400-600 bp. Immunoprecipitation of the sonicated chromatin samples was conducted with a
cocktail of GR antibodies: H300 (6 µg, Santa Cruz Biotechnology), PA1-511A (2 µg, Thermo
Scientific), D6H2L (2 µg, Cell Signaling Technology). Protein G Sepharose magnetic beads (GE
Healthcare) were used to isolate the immune complexes with the crosslinked DNA. Once the
DNA was purified, quantitative PCR amplification with the LightCycler 480 SYBR Green I Master
(Roche) on the LightCycler 480 system (Roche) was performed with primers for the GBRs at the
genes of interest. The following are the primer sequences for the GBRs used in this study:
RP1L1, IGFBP1, MSX2, and SCNN1A (Chodankar et al., 2014); GRAMD4 (Chodankar et al.,
2015); and SLN (forward primer: 5’-CAGGCTACCCATCACACTTCTTT-3’; reverse primer: 5’-
TCAAGGTCACCATTAAAGTGCAAGA-3’).

21
FAIRE
The protocol used for Formaldehyde Assisted Isolation of Regulatory Elements (FAIRE)
experiment was previously described (Simon et al., 2012). Cells were treated as in the
chromatin immunoprecipitation experiments. Once free DNA was purified from crosslinked,
sonicated chromatin by phenol extraction, quantitative PCR primers for GBRs at the genes of
interest as mentioned for chromatin immunoprecipitation were used to assess chromatin
accessibility.

Proximity Ligation Assay
The proximity ligation assay (PLA) technology developed by Olink Bioscience (Sweden)
allows the visualization of protein-protein interactions in situ (Söderberg et al., 2006). U2OS-
GRα cells were grown on coverslips in 12-well plates and transfected with siRNA for 48 h
followed by 1 h dex or ethanol treatment. The cells were fixed with methanol for 2 min and
treated according to the PLA probe protocol in the manufacturer instructions (Olink Bioscience).
Samples were first saturated with blocking solution and then incubated with two primary
antibodies of differing species that bind to their respective potentially interacting proteins for 1 h
in a 37°C humidified chamber. Next, secondary antibodies conjugated with complementary
oligonucleotides serving as PLA probes were added for 1 h in a 37°C humidified chamber.
Ligation and rolling-circle amplification were performed for 100 min at 37°C. During the
amplification step fluorescently labeled oligonucleotides hybridized to the amplified product. The
coverslips were then dried and mounted using Duolink II Mounting Medium (Sigma) with DAPI
for nuclei staining. Slides were analyzed using a fluorescence microscope. Each PLA
fluorescent dot represents one bimolecular protein interaction. ImageJ version 1.49
(https://imagej.nih.gov/ij/) was used to quantify the fluorescent dots in the nucleus. For each
sample interactions were counted for at least 400 cells.

22
Statistical Analysis
Statistical analysis of quantitative RT-PCR, chromatin immunoprecipitation with
quantitative PCR, FAIRE with quantitative PCR, and quantification of PLA interactions were
performed using paired t-test. The number of biological replicates (n) and p-values for each
experiment are indicated in the Figure legends. Boxplots were generated using R. The
horizontal center lines indicate the median with the upper and lower box limits denoting the 25
th

and 75
th
percentile, respectively. The whiskers extend 1.5 times the interquartile range from the
25th and 75th percentiles; and outliers are represented by dots. P-values indicating statistical
significance between the data sets in the boxplots were obtained by using the Mann-Whitney U
test. RNA sequencing data was analyzed using EdgeR. Differentially expressed genes were
obtained using 1.3 fold change (log
2
= 0.4) and 0.05 FDR adjusted p value cutoff. For higher
stringency, 1.5 or 2.0 fold change and 0.01 FDR adjusted p values were used for the cutoff.

2.3 Results
Hypothesis: A dynamic model for explaining differential effects of Hic-5 on GR binding to
different classes of GBR
The chromatin conformation at GBR is dramatically altered to a more open state in
response to GC-treatment of cells (Chodankar et al., 2014), e.g. by creating a nucleosome-free
region at the GBR (Figure 2.2). GC-induced binding of GR to GBR is a cooperative process
between GR and chromatin remodelers, in which GR recognizes its specific DNA binding motif
and recruits ATP-dependent chromatin remodeling enzymes that open up chromatin structure
(Burd and Archer, 2013; Fryer and Archer, 1998; Nagaich et al., 2004). Furthermore, GR
interacts in a rapid dynamic on-and-off fashion with its specific DNA motifs (McNally et al.,
2000); and experimental techniques such as chromatin immunoprecipitation which are used to
measure the strength of association of GR with a specific GBR (often referred to as occupancy)

23
Figure 2.2. Dynamic GR-GBR interaction model. Before dex treatment GBR is in a relatively
closed chromatin conformation, and gene transcription is silent (or at a basal level). Upon
binding dex, GR recognizes and initially interacts weakly with the GBR in a dynamic on/off
relationship. GR recruits chromatin remodeling enzymes that subsequently remodel chromatin
at the GBR to a more open state, thereby allowing a more robust but still dynamic GR
occupancy, recruitment of coregulators, and recruitment of transcription machinery including
RNA polymerase II (Pol II) to the transcription start site, resulting in enhanced expression of the
target gene.

24
Figure 2.2

25
are simply capturing a snapshot of the steady-state dynamic interaction between GR and a
given GBR. In view of the dynamic and cooperative nature of this process, I propose that the
initial GR interaction with the GBR is weak; but this weak GR-GBR interaction allows GR to
recruit chromatin remodelers, which open up the chromatin and thereby allow a more robust but
still dynamic GR occupancy of the site (Figure 2.2).
In cells, Hic-5 selectively prevents robust GC-induced GR occupancy at GBR of block
genes and the accompanying transition to a more open chromatin conformation (Chodankar et
al., 2014). I therefore further hypothesize that, while GR can interact weakly with GBR of block
genes in the presence of Hic-5, Hic-5 inhibits the dynamic and cooperative chromatin
remodeling process by GR and chromatin remodelers at GBR of block genes, thus maintaining
a chromatin conformation at the GBR of block genes that is not permissive for the robust GR
interaction with the GBR required for establishing an active transcription complex. In addition, to
explain why Hic-5 prevents chromatin remodeling and robust GR binding at block genes, but not
at mod and ind genes, I recall a previous report that different DNase hypersensitive sites (which
frequently overlap with transcription factor binding sites) require different combinations of
chromatin remodeling complexes (Morris et al., 2014). In light of these findings I propose that
different chromatin remodeling complexes are required for GR binding at block genes than at
mod and ind genes. In the experiments described below, I test these hypotheses.

CHD9 and BRM chromatin remodelers selectively facilitate GC-regulated expression of
representative GR target genes from the block gene class
Using the U2OS-GRα cell line that previously characterized the actions of Hic-5
(Chodankar et al., 2014), I first determined whether specific chromatin remodeling enzymes are
selectively required for GC-induced expression of representative genes from the block gene
class. Eleven ATP-dependent chromatin remodeling enzymes previously shown to be involved
in transcriptional activation by GR or other transcription factors (Bao and Shen, 2007; Clapier

26
and Cairns, 2009; Engel and Yamamoto, 2011; Fryer and Archer, 1998; Gaspar-Maia et al.,
2009; Lutz et al., 2006; Marom et al., 2006; Menon et al., 2010; Pradhan et al., 2016; Schnetz et
al., 2009; Sparmann et al., 2013; Wiechens et al., 2016) were individually depleted by siRNA
transfection. Since Hic-5 inhibits expression of block genes, it was depleted simultaneously with
each chromatin remodeler. Immunoblots verified successful depletion of the chromatin
remodeling enzymes and Hic-5 (Figure 2.3). Depletion of the chromatin remodelers did not
affect the expression of GR or Hic-5, and depletion of Hic-5 did not alter the expression of GR or
the chromatin remodelers (Figure 2.3). Cells were treated with synthetic GC agonist
dexamethasone (dex) or an equivalent volume of the vehicle ethanol for 4 h, and GC-induced
expression of representative block and ind genes identified in a previous study (Chodankar et
al., 2014) was measured using quantitative RT-PCR (Figure 2.4). As previously shown
(Chodankar et al., 2014), depletion of Hic-5 alone resulted in dex-regulated expression of block
genes RP1L1 and HOXD1, whereas these genes were not induced by dex in the non-specific
siRNA control sample (Figure 2.4A). For most of the chromatin remodelers, the double depletion
of a chromatin remodeler and Hic-5 did not eliminate dex-induced expression of the block
genes. However, the double depletion of CHD9 (Chromodomain Helicase DNA binding protein
9) and Hic-5 or of BRM (Brahma homologue, product of the SMARCA2 gene) and Hic-5
eliminated a significant increase in mRNA for the block genes RP1L1 and HOXD1 upon dex
treatment, indicating that CHD9 and BRM are required for dex-regulated expression of these
two block genes. In contrast, dex treatment induced expression of the ind genes IGFBP1 and
MSX2 in the presence and absence of Hic-5, and depletion of each of the eleven chromatin
remodelers along with Hic-5 had little or no effect on their dex-regulated expression (Figure
2.4B).
Similar results were observed when expression of genes was examined at 2, 4, and 8 h
of dex treatment. Dex-induced expression of block genes RP1L1, GRAMD4, and HOXD1 was

27
Figure 2.3. siRNA depletion of chromatin remodelers (CR) and Hic-5. Immunoblots for the
indicated chromatin remodelers, GR and Hic-5 are shown with GAPDH as an internal control.

28
Figure 2.3

29
Figure 2.4. CHD9 and BRM are selectively required for dex-induced expression of
representative block genes. Cells were transfected with the indicated siRNA combinations,
and after 48 h relative mRNA levels for the indicated block genes (A) and ind genes (B) were
measured by quantitative RT-PCR following 4 h of ethanol (etoh) or dex treatment. Relative
mRNA levels for each gene were normalized to GAPDH mRNA levels. Data shown is the mean
± SD of 3 biological replicates. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant; paired
t-test).

30
Figure 2.4

A
B

31
observed after depletion of Hic-5 alone, but was eliminated by double depletion of Hic-5 and
CHD9 or of Hic-5 and BRM (Figure 2.5A). There was no dex-induced expression of the three
block genes when Hic-5 was present in cells and BRM only or CHD9 only was depleted. In
contrast, the temporal expression profiles of three representative ind genes, IGFBP1, MSX2 and
TIPARP were unaffected by the depletion of Hic-5, CHD9 or BRM individually or in
combinations (Figure 2.5B). Similarly, CHD9 and BRM were not required for dex-induced
expression of two representative mod genes SCNN1A and SLN (Figure 2.5C). As shown
previously (Chodankar et al., 2014) these genes were activated by dex in the presence but not
in the absence of Hic-5. When a second siRNA for CHD9 and BRM was used, similar results
were obtained on these representative block, ind, and mod genes (Figure 2.6A-D), thus
eliminating concerns about off-target effects of the first set of siRNAs used. Therefore, CHD9
and BRM chromatin remodelers are necessary for the dex-induced expression of the block
genes, but not the ind and mod genes.

Genome-wide analysis of CHD9 and BRM requirements for dex-regulated expression of
block, ind, and mod GR target genes
Since CHD9 and BRM were selectively required for dex-induced expression of
representative genes from the block gene class, I employed RNA sequencing to explore
whether or not this association applies genome-wide. RNA was prepared from cells transfected
with six different siRNA combinations: non-specific siRNA control (siNS/siNS), depletion of Hic-5
(siHic5/siNS), depletion of CHD9 (siCHD9/siNS), double depletion of Hic-5 and CHD9
(siCHD9/siHic5), depletion of BRM (siBRM/siNS), and double depletion of Hic-5 and BRM
(siBRM/siHic5). Cells in each category were treated with either 100 nM dex or ethanol for 8
hours, making a total of 12 conditions, and three biological replicates were performed on
different days. The differentially expressed genes reported here are defined as genes with at
least a 1.3-fold change in expression and false discovery rate (FDR) adjusted p-value ≤ 0.05.

32
Figure 2.5. Temporal profile of dex-induced gene expression in cells depleted of Hic-5,
chromatin remodelers, or both. Cells were transfected with the indicated combinations of
siRNAs against Hic-5, CHD9, and BRM, and non-specific siRNA (siNS) was used as control.
Quantitative RT-PCR data for the indicated mRNAs from representative genes in the block (A),
ind (B) and mod (C) gene classes is shown as mean ± SD for 3 biological replicates conducted
on different days.

33
Figure 2.5

A

B

C

34
Figure 2.6. Temporal profile of dex-induced gene expression in cells depleted of Hic-5,
chromatin remodelers, or both, using a second siRNA for CHD9 and BRM. (A-C)
Experiments were conducted as in Figure 2.5, but with a second set of siRNAs directed against
CHD9 and BRM. (D) siRNA depletion of chromatin remodelers and Hic-5. Immunoblots for the
indicated chromatin remodelers, GR and Hic-5 are shown with GAPDH as an internal control.

35
Figure 2.6

A

B

C

D

36
These relatively low-stringency values were chosen to include reasonably large numbers of
genes in the block, ind and mod gene classes and thus provide more statistical power for
subsequent analysis of CHD9 and BRM effects on these gene classes. Assignment of genes to
the block, ind, and mod classes involved a previously developed algorithm (Wu et al., 2014)
described in detail in Experimental Procedures. In total there were 105 ind genes (Figure 2.7A,
blue region), 364 mod genes (Figure 2.7A, green region), and 534 block genes (Figure 2.7A,
red region). These categories include genes that were upregulated or downregulated by dex
and where Hic-5 could have a positive or negative effect, as illustrated by the hypothetical
examples for each of the three gene classes in Figure 2.1B.
Next I determined the number of genes in each class that required CHD9 or BRM for
dex-regulated expression. Briefly, the set of genes in each gene class (block, ind, and mod) was
overlapped with two other gene sets derived from the samples depleted or not depleted of
CHD9 or BRM, defined as illustrated in Figure 2.1C-E, and explained in detail in Experimental
Procedures. Although this procedure identified several subsets of genes (Figure 2.1D, E;
sectors i-iv), the genes of primary interest were genes in each class for which the dex-regulated
expression was entirely dependent on CHD9 or BRM (Figure 2.1D, E; sector iii), i.e. genes in
the block, ind or mod gene sets that were no longer dex-regulated after depletion of CHD9 or
BRM.
Since block genes are only dex-regulated in cells depleted of Hic-5, I used data from
cells that were depleted of Hic-5 alone or doubly depleted of Hic-5 and CHD9 or BRM to identify
block genes that require CHD9 or BRM (Figure 2.1C, D). Using this algorithm (explained in
detail in Experimental Procedures), I identified 296 out of 534 block genes (55%) that require
CHD9 for dex-regulated expression (Figure 2.7B; left panel) and 292 out of the 534 block genes
(55%) that require BRM for dex-regulated expression (Figure 2.7C, left panel). More than half of
the block genes that required CHD9 were also dependent on BRM for dex-induced expression,
and vice-versa (Figure 2.7D; left panel). Altogether, 78% (414/534 genes) of the block gene

37
Figure 2.7 Genome-wide selective requirement of CHD9 and BRM for dex-regulated
expression by block genes versus ind and mod genes. Genome-wide RNA sequencing
analysis was performed to evaluate the genes dependent on CHD9 and BRM for dex-regulated
expression. (A) The numbers of block (red), ind (blue), and mod (green) genes were determined
by overlapping three gene sets (Figure 2.1A), according to the algorithms described in Materials
and Methods. (B) Block, ind, and mod genes that require CHD9 for dex-regulated expression.
The block gene set was overlapped with gene sets a and b (Figure 2.1C). The ind and mod
gene sets were each overlapped with gene sets c and d (Figure 2.1C). Overlapping was
performed according to the algorithms in Materials and Methods. Dark-colored regions show
number and percentage of genes in each class (block, ind and mod) that are dependent on
CHD9 for dex-regulated expression. (C) Block, ind, and mod genes that require BRM for dex-
regulated expression. BRM-dependent genes in each class were determined as in B, using data
from cells with depletion of BRM instead of CHD9. (D) Pie charts showing percent of genes from
each class that are dependent on CHD9 and/or BRM.

38
B

C

D
A

Figure 2.7

39
class were dependent on CHD9 and/or BRM for dex-induced expression, and 33% of block
genes required both CHD9 and BRM (Figure 2.7D; left panel).
Similar analyses were conducted with the ind and mod gene classes to determine the
genes that were dependent on CHD9 and/or BRM for dex-regulated expression. The effect of
depleting each chromatin remodeler was determined in cells containing Hic-5 and in cells
depleted of Hic-5. When the chromatin remodeler was depleted in cells containing Hic-5, 14 out
of 105 ind genes (13%) were dependent on CHD9, and 12 genes (11%) were dependent on
BRM (Figure 2.7B, C; center panels). There were 2 genes (2%) regulated by both CHD9 and
BRM, resulting in a total of 24 genes (23%) of the ind genes that required CHD9 or BRM or both
for dex-induced expression (Figure 2.7D, center panel). When this analysis was performed in
cells lacking Hic-5, 35% of the ind genes were dependent on CHD9 and/or BRM for dex-
regulated expression (Figure 2.8A-C, left panels).
When mod genes were analyzed by single depletion of CHD9 or BRM in cells containing
Hic-5, 80 out of 364 mod genes (22%) were dependent on CHD9, and 63 (17%) were
dependent on BRM for dex-regulated expression (Figure 2.7B, C; right panels). In total, 119
genes (33%) of the mod class were dependent on CHD9 or BRM or both, and only 7% required
both CHD9 and BRM (Figure 2.7D; right panel). Similar results were found when the effect of
depleting CHD9 or BRM on mod gene regulation by dex was determined in cells lacking Hic-5
(Figure 2.8A-C; right panels). Since 138 out of the 364 mod genes were no longer regulated by
dex in cells depleted of Hic-5 (Figure 2.7A), I analyzed the remaining 226 mod genes that were
still dex regulated in cells depleted of Hic-5. In this analysis 32% of the Hic-5 regulated mod
genes were dependent on CHD9 and/or BRM for dex-regulated expression (Figure 2.8A-C, right
panels).
Thus, the great majority of the block genes (78%) required CHD9, BRM, or both for their
dex-regulated expression, while relatively minor fractions of the ind and mod genes (23-35%)
required these chromatin remodelers; the comparison of gene classes was even more striking

40
Figure 2.8 Alternative overlapping strategy for identifying ind and mod genes dependent
on CHD9 or BRM. (A-B) As an alternative to the overlapping set strategy specified in Figure
2.7, ind genes or the subset of mod genes that were still dex-regulated after depletion of Hic-5
(226 genes in the central sector of Figure 2.7A) were overlapped with sets a and b described in
Materials and Methods. This strategy indicates the effect of depleting CHD9 or BRM in cells that
are also depleted of Hic-5. Dark regions identify the number and percent of genes from the ind
genes or from the mod genes subset that are dependent on CHD9 (A) or BRM (B) for dex-
regulated expression. (C) Pie chart summarizing the percent of ind genes and of the mod gene
subset that are dependent on CHD9, BRM, or both, derived from the data in (A) and (B) above.

41
Figure 2.8
A

B

C

42
Figure 2.9. Genes dependent on CHD9 and BRM for dex-regulated expression, defined
with more stringent parameters. The analyses in Figure 2.7B-D were repeated, but more
stringent fold change and FDR cutoffs were used for comparisons b and d (from Figure 2.1C),
i.e. the effects of CHD9 or BRM depletion. The genes from the block, ind and mod gene classes
that required CHD9 and/or BRM for their dex-regulated expression were determined using FDR
< 0.01 and fold change ³ 1.5 or £ -1.5 (A-B), or FDR < 0.01 and fold change ³ 2.0 or £ -2.0 (C-
D) for comparisons b and d.

43
Figure 2.9

A

B

44
Figure 2.9 continued

C

D

45
when considering the genes that required both CHD9 and BRM (33% for block, 2-9% for ind
and mod) (Figures 2.7D and 2.8C). While these analyses were done with 1.3-fold change in
expression and FDR adjusted p-value ≤ 0.05 as cutoff values, the large difference in the percent
of block genes that required CHD9 and/or BRM compared with the ind or mod genes was
preserved when more stringent parameters were used to identify genes that were significantly
affected by depletion of CHD9 or BRM (Figure 2.9).
To assess the overall genome-wide magnitude of the effect of CHD9 and BRM depletion
on the dex-regulated expression of all genes in each of the three classes, I compared the log
2

fold change in mRNA levels caused by dex treatment in cells containing (Figure 2.10A-C, red
bars) or depleted (Figure 2.10A-C, blue bars) of each chromatin remodeler. The depletion of
CHD9 or BRM caused a large decrease in the effect of dex on the expression of the great
majority of the block genes, as indicated by the large amount of red visible in Figure 2.10A. This
trend was evident for block genes that were activated (Figure 2.10A, left side) or repressed
(Figure 2.10A, right side) by dex. In contrast, depletion of CHD9 or BRM caused a much more
modest decrease in dex regulation of the ind and mod genes, as indicated by the much lower
amount of red visible in Figures 2.10B-C. In fact, depletion of CHD9 or BRM caused little or no
change in dex regulation for a large percentage of the ind and mod genes (very little difference
in the height of red and blue bars). To analyze this genome-wide data quantitatively and
statistically, I calculated the percent decrease in the dex regulation of each gene (see formula in
Experimental Procedures) caused by depletion of CHD9 or BRM, and displayed this data as a
box plot showing the genes in quartiles for each gene class (Figure 2.10D). For the block genes,
the median decrease in dex regulation was 52.9% for CHD9 depletion and 59.6% for BRM
depletion. In contrast, the median decrease in dex regulation for ind genes was 0.04% and
15.1% for CHD9 and BRM depletion, respectively; and the median decrease in dex regulation
for mod genes was 12.5% and 18.9% for CHD9 and BRM depletion, respectively. Furthermore,

46
Figure 2.10. Genome-wide effect of CHD9 and BRM depletion on dex-regulated gene
expression. The log
2
fold change in mRNA levels from dex treatment is shown for cells
containing (red bars) or depleted of (blue bars) CHD9 or BRM for all genes from the block (A),
ind (B), and mod (C) gene classes (from Figure 2.7A). Red bars are arranged (left to right) from
most positive to most negative log
2
fold change caused by dex treatment, and blue bars for the
same gene are superimposed on the red bars. Thus, genes that are positively regulated by dex
when neither CHD9 or BRM is depleted are shown on the left, and genes that are negatively
regulated by dex are shown on the right. Fold change values were determined in cells
transfected with the indicated siRNAs. (E) The decrease in dex regulation caused by depletion
of CHD9 (left panel) or BRM (right panel) is shown as the distribution of the depletion effects for
all block, ind and mod genes, using a box plot that divides genes for each class into quartiles.
The y-axis values represent the percent decrease in dex effect, calculated with the formula
specified in Materials and Methods. Median decrease caused by depletion of CHD9 or BRM is
indicated by thick horizontal lines with median values shown above the plot. p values were
obtained by comparing ind or mod class to the block class using the Mann-Whitney U test.

47
Figure 2.10

A

B

48
Figure 2.10 continued

C

D

49
the difference in the distribution of values between the block genes and each of the other two
gene classes was highly significant (Figure 2.10D).
Gene ontology analysis (Ashburner et al., 2000; The Gene Ontology Consortium, 2017)
of block genes versus a combination of ind and mod genes indicated some common pathways
(e.g. developmental and cell differentiation pathways), but also some distinct physiological
pathways. The block class was enriched for genes involved in localization of cells and cell
contents and for genes involved in phosphate metabolism. In contrast, the combined ind and
mod classes were enriched for genes involved in angiogenesis, apoptosis, and pathways
involved in oxygen and mitogen activated protein kinase signaling (Figure 2.11).

CHD9 and BRM are required for GR occupancy at GBR associated with block genes
To explore the mechanism by which CHD9 and BRM contribute to dex-regulated
transcription of the block genes, and since I propose that efficient GR binding to DNA is a
cooperative process between GR and chromatin remodelers (Figure 2.2), I examined the effect
of CHD9 and BRM on the binding of GR to GBR associated with the representative block genes
RP1L1 and GRAMD4 discussed above. Since there is no data to indicate which GBR controls
each dex-regulated gene, I focused on the GR binding site which was most closely associated
with each GR target gene in terms of linear genomic distance (Chodankar et al., 2014; Schiller
et al., 2014). Supporting this choice is a previous finding in our lab that Hic-5 blocks dex-
induced expression of these block genes and also prevents robust dex-induced GR binding and
chromatin remodeling at the GBRs closest to these genes (Chodankar et al., 2014); this
phenotypic correlation supports the conclusion that the closest GBR is involved in regulation of
the representative genes examined here. As stated earlier in my hypothesis, I propose that
there is an initial weak interaction between GR and GBRs, but robust GR occupancy requires
recruitment of chromatin remodelers by GR and resulting opening of the chromatin conformation

50
Figure 2.11 Gene ontology analysis for block genes and for the combined ind and mod
genes. The analysis was conducted using the web-based Gene Ontology Consortium software
(Ashburner et al., 2000; The Gene Ontology Consortium, 2017). The 10 categories with the
lowest p values are shown for each gene set. Enriched gene categories that are distinct in the
block genes versus the combined ind and mod genes (and vice-versa) are indicated by black bars.

51
Figure 2.11

52
(e.g. by creating a nucleosome free region at the GBR) (Figure 2.2). I further propose that Hic-5
interferes with this chromatin remodeling process at GBR of block genes.
Consistent with my model, I observed weak dex-induced GR binding at the GBR of the
two representative dex-induced block genes in cells containing Hic-5, and robust dex-induced
GR occupancy depended on depletion of Hic-5, as reported previously (Chodankar et al., 2014).
GR occupancy was mostly eliminated after the double depletion of CHD9 and Hic-5 or BRM and
Hic-5 (Figure 2.12A). The GR binding level for the double depletions was essentially the same
as the low level of GR binding observed when none of the proteins were depleted, suggesting
that both CHD9 and BRM are required for the robust GR binding observed when Hic-5 is
depleted. In contrast to CHD9 and BRM, the double depletion of Hic-5 and BRG1 (Brahma-
related gene 1, product of the SMARCA4 gene), a chromatin remodeler which was not required
for dex-induced expression of the representative block genes (Figure 2.4A), had no significant
effect on GR occupancy at the GBR associated with the two block genes (Figure 2.12A). In a
similar chromatin immunoprecipitation analysis of GBRs associated with two representative ind
genes (MSX2 and IGFBP1) and two representative mod genes (SCNN1A and SLN), single
depletion of CHD9, BRM or BRG1 or double depletion of Hic-5 along with each chromatin
remodeler had little or no effect on dex-induced GR binding (Figure 2.12B, C); there was a small
but marginally significant decrease in GR binding to the MSX2 GBR after double depletion of
Hic-5 and CHD9, but there were no other significant decreases in GR binding to the ind or mod
genes when any of the three chromatin remodelers was depleted. A region where GR does not
bind was examined as a negative control (Figure 2.12D). Thus, CHD9 and BRM, but not BRG1,
were selectively required for GR binding to GBR associated with block genes.

53
Figure 2.12. CHD9 and BRM are required for GR occupancy at GBR of representative block
genes. Cells depleted of Hic-5 or a chromatin remodeler only (CHD9, BRM or BRG1) or doubly
depleted of both a chromatin remodeler and Hic-5 were treated with dex for 1 h. Chromatin
immunoprecipitation with antibodies against GR or normal IgG followed by quantitative PCR at
the associated GBRs was performed for the indicated block genes (A), ind genes (B), and mod
genes (C); GR occupancy at a negative control region is also shown (D). Values shown are mean
± SD for n = 4 biological replicates performed on different days. *, p < 0.05; **, p < 0.01 from a
paired t-test. ns, not significant.

54
Figure 2.12

A

B

55
Figure 2.12 continued

C

D

56
CHD9 and BRM are required for dex-induced chromatin remodeling at GBR of the block
genes
To determine whether CHD9 and BRM are required for the dex-induced increase in
chromatin accessibility at the GBR of the representative block genes, I performed
Formaldehyde Assisted Isolation of Regulatory Elements (FAIRE) followed by quantitative PCR.
As shown previously (Chodankar et al., 2014), dex treatment increased FAIRE signals at the
GBR of representative block genes RP1L1 and GRAMD4 after Hic-5 depletion, but not in the
control cells transfected with non-specific siRNA. Double depletion of CHD9 and Hic-5 or of
BRM and Hic-5 eliminated most of the dex-induced FAIRE signal for these block genes (Figure
2.13A). However, depletion of BRG1 along with Hic-5 did not significantly alter the dex-induced
FAIRE signal at the GBR of the block genes, compared with depletion of Hic-5 alone. In contrast
to the block genes, a robust dex-induced FAIRE signal was observed for the representative ind
and mod genes either in the presence or absence of Hic-5, and depletion of CHD9, BRM or
BRG1 did not significantly alter these FAIRE signals either in the presence of absence of Hic-5
(Figure 2.13B, C), except in the case of the SCNN1A GBR where the double depletion of BRM
and Hic-5 (but not the single depletion of BRM) caused a small but significant decrease in the
dex-induced FAIRE signal. Thus, CHD9 and BRM, but not BRG1 are required for dex-induced
chromatin remodeling activity at the GBR of the block genes, and the requirement of CHD9 and
BRM was specific for the block gene class, but not for ind and mod genes.

Hic-5 selectively inhibits the interaction between GR and block gene-specific chromatin
remodelers
Since Hic-5 inhibited the chromatin remodeling actions of CHD9 and BRM which are
required for GR occupancy of block gene GBRs (Figures 2.12-2.13), and since Hic-5 binds
directly to the hinge region of GR located between the DNA binding domain and the ligand

57
Figure 2.13. CHD9 and BRM are selectively required for chromatin remodeling at GBR of
representative block genes. Cells depleted of Hic-5 or a chromatin remodeler only (CHD9, BRM
or BRG1) or doubly depleted of both a chromatin remodeler and Hic-5 were treated with ethanol
(etoh) or dex for 1 h. Chromatin accessibility as measured by FAIRE followed by quantitative PCR
at the associated GBRs was performed for the indicated block genes (A), ind genes (B), and mod
genes (C). FAIRE signal at a negative control region is also shown for comparison. Values shown
are mean ± SD for n = 3 biological replicates performed on different days. *, p < 0.05; **, p < 0.01
from a paired t-test. ns, not significant.

58
Figure 2.13

A

B

59
Figure 2.13 continued

C

60
binding domain (Yang et al., 2000), I investigated whether or not Hic-5 inhibits the interaction
between GR and CHD9 or BRM using the proximity ligation assay (PLA). PLA detects protein-
protein interactions by using primary antibodies of differing species for each of the putative
protein partners. When the proteins are in proximity of less than 40 nm, secondary antibodies
attached with oligonucleotides guide the formation of circular DNA strands that serve as
templates for rolling-circle amplification. Fluorescent probes that bind to the amplified DNA
generate a fluorescence signal for each bimolecular interaction which appears as a pinpoint
signal against the background of the cell micrograph, which in this case includes DAPI-stained
nuclei (Figures 2.14A-C). The number of signals is quantifiable with image-analysis software
(Figure 2.15A-C). Antibodies for GR, CHD9, BRM, and BRG1 were used to examine the
interactions between GR and chromatin remodelers in cells containing or depleted of Hic-5 and
treated with dex or ethanol. Signals above background were only detected in dex-treated cells
(Figure 2.14 and Figure 2.15), presumably at least in part because GR is only localized to the
nucleus after dex treatment; this result validated the specificity of the contribution of the GR
antibody to the PLA signal. Validation of the antibodies for the chromatin remodelers was
achieved by showing that the dex-induced signals for the GR-chromatin remodeler interactions
were reduced to background by depletion of the relevant chromatin remodeler (Figure 2.14A-C;
bottom right panels). Robust dex-induced signals for the GR-CHD9 and GR-BRM interactions
were observed only after depletion of Hic-5; in contrast, a robust GR-BRG1 interaction was
observed either in the presence or absence of Hic-5. Hence the PLA experiments demonstrate
that Hic-5 inhibits the interaction between GR and the block-gene specific chromatin remodelers
CHD9 and BRM, but does not inhibit GR interaction with other ATP-dependent chromatin
remodelers, such as BRG1.

61
Figure 2.14. Hic-5 effect on the interaction between GR and chromatin remodelers. Cells
were depleted of the specified protein(s) using siRNA and treated with ethanol (etoh) or dex for
1 h. (A-C) PLA of both ethanol- and dex-treated cells was performed to measure the indicated
protein-protein interactions. Red fluorescent dots indicate individual bimolecular interactions
between two molecules. DAPI (blue) staining specifies nuclei. (D) Quantification of PLA signals
within the nuclei of ethanol- and dex-treated cells (number of dots in the nuclei / number of
nuclei) is shown. Values shown are mean ± SD for n = 3 biological replicates performed on
different days. *, p < 0.05; **, p < 0.01 from a paired t-test. ns, not significant. White scale bar =
20 µm.

62
Figure 2.14

A

B

63
Figure 2.14 continued

C

D

64
2.4 Discussion
The dynamic co-dependency of GR occupancy and chromatin remodeling is regulated by
Hic-5
My results demonstrate that the block class of GR target genes is defined by its
requirement for specific chromatin remodelers, CHD9 and BRM, which are not required for most
of the genes in the ind and mod classes of GR target genes. 78% of block genes required at
least one of these two chromatin remodelers, and 33% required both of them. In contrast, only
23-35% of ind and mod genes required BRM or CHD9, and only 2-9% required both (Figure
2.7D and 2.8C). Of course, the specific number of genes assigned to each category depends
upon the stringency of the fold-change and statistical cutoffs chosen; but the dramatic difference
in dependence on BRM and CHD9 for dex-regulated expression of the block versus ind and
mod genes was consistent even when more stringent cutoff values were tested (Figure 2.9).
Several possible explanations can be offered for the 22% of the block genes that did not appear
to require CHD9 or BRM: there may be other chromatin remodelers required for some block
genes that were not tested in this study; the fold-change and statistical cutoffs chosen may have
generated some false positive block genes; or the effects of CHD9 or BRM depletion on some
block genes may have missed the statistical cutoff. Similarly, the fact that 23-35% of the ind and
mod genes demonstrated a requirement for CHD9 or BRM could be due to the low 1.3-fold-
change cut-off chosen. This conclusion is supported by the generally small effect of CHD9 or
BRM depletion on the ind and mod gene classes, compared with the large effect on the block
gene class (Figure 2.10D).
The fact that 33% of the block genes require both CHD9 and BRM for dex-regulated
expression suggests that each chromatin remodeler contributes some unique function to
remodel the chromatin at the GBR associated with these genes and that both of these functions
are required for dex-induced chromatin remodeling and for the resulting robust GR binding and

65
transcriptional regulation. The requirement for multiple ATPases to maintain transcription factor
binding sites has been previously demonstrated (Morris et al., 2014) and suggests that different
types of chromatin remodeling activities cooperate to accomplish chromatin remodeling
associated with transcriptional regulation. In fact, CHD9 and BRM belong to different families of
ATPases, and in vitro assays indicate that they support different types of nucleosome and
chromatin remodeling activities (Bartholomew, 2014; Clapier and Cairns, 2009).
Hic-5 binds to GR (Yang et al., 2000) and was not detectable on any of the block gene
GBRs tested in the absence of dex, but was recruited to the GBRs of ind and mod genes in a
dex-induced manner (Chodankar et al., 2014). This indicates that Hic-5 is recruited to GBRs by
GR. This raises the question of how Hic-5 can prevent robust GR occupancy on block gene
GBRs. My proposal that GR makes initial weak contact with the GBR but that robust occupancy
and chromatin remodeling of GBRs are co-dependent processes (Figure 2.2) resolves this
conundrum. The co-dependency provides an opportunity for regulation, and in this case Hic-5
regulates this process selectively on block genes (Figures 2.12-2.13) by interfering with the
interaction between GR and the two block gene-specific chromatin remodelers, CHD9 and BRM
(Figure 2.14). And when Hic-5 is depleted, CHD9 and BRM are allowed to interact with GR and
these two chromatin remodelers are required for the robust binding of GR and chromatin
remodeling at the GBR of the block genes (Figure 2.15).
My findings that different GR target genes require different chromatin remodelers is
presumably related to a previous report that different DNase hypersensitive sites are maintained
by different chromatin remodelers (Morris et al., 2014); it is well established that GBRs (and
other transcription factor binding sites) are generally marked by DNase hypersensitive sites,
which are often present before GR binding but increase in accessibility after GR binding (John
et al., 2011). My results indicate that the increased accessibility of GBRs induced by dex
requires gene-specific chromatin remodelers, and the dex-dependency of the chromatin
transition indicates a requirement for GR to recruit or activate the chromatin remodelers, thus

66
Figure 2.15. Model illustrating the mechanism of Hic-5 action on dex-induced block genes.
In the presence of Hic-5, interactions between GR and CHD9 and between GR and BRM are
inhibited, preventing dex-induced chromatin remodeling and robust GR occupancy at the GBR.
Hence, expression of the block gene is not dex-regulated. In the absence of Hic-5, GR is able to
recruit block-gene specific chromatin remodelers, CHD9 and BRM, allowing the opening of
chromatin, robust GR occupancy, recruitment of coregulators and RNA polymerase II (Pol II), and
dex-induced expression of the block gene.

67
Figure 2.15

68
supporting the conclusion that GR occupancy and chromatin remodeling are co-dependent
processes. Also consistent with this is the previous demonstration that components of the Swi-
Snf chromatin remodeling complex (for which BRG1 and BRM serve as alternative ATPase
subunits) interact directly with GR and are recruited to many GBRs in a hormone-induced
manner (Burd and Archer, 2013; Engel and Yamamoto, 2011; Fryer and Archer, 1998; Nagaich
et al., 2004). In addition, BRM and GR have previously been shown to regulate each other's
occupancy on GBRs in a gene-specific manner (Engel and Yamamoto, 2011).
Another question suggested by my results is what drives gene specific requirements for
CHD9 and BRM. As with gene-specific coregulator actions in general, we propose that these
come from inherent properties of the chromatinized genes. First, the specific DNA sequence to
which GR binds is known to influence GR conformation and actions (Lefstin and Yamamoto,
1998; Meijsing et al., 2009). Second, the set of additional regulatory elements, which bind
additional transcription factors and coregulators, is specific for each gene. Third, the chromatin
conformation may also be gene-specific. Together, these factors establish a specific regulatory
environment which determines the specific coregulators that are required for activation or
repression of transcription. Protein-protein interactions among all the transcription factors and
coregulators at the site, as well as post-translational modifications made by enzymatic
coregulators present in this regulatory environment, should influence the coregulators that are
required for transcription and the specific actions of the transcription factor and coregulators
(e.g. whether they have positive or negative effects on transcription).

Physiological implications of the gene-specific requirements for and actions of Hic-5
There are two separate aspects of the gene-specific coregulator activity of Hic-5: first, it
affects the GC-regulated expression of some but not all GR target genes; second it acts by
different mechanisms on different GR target genes, as evidenced by its distinctive mechanisms
of action on mod and block genes. For mod genes that require Hic-5 for GC-induced

69
expression, Hic-5 facilitates recruitment of the Mediator complex and RNA polymerase II; in
contrast, Hic-5 prevents GC-induced GR binding and chromatin remodeling at GBR of block
genes (Chodankar et al., 2014). There is accumulating evidence that the gene specific actions
of coregulators may correlate with specific physiological pathways. For example, GC regulate
many different physiological pathways, including anti-inflammatory pathways, developmental
pathways, and metabolism of glucose, lipids and bone. If different coregulators are required for
GC-regulated genes that control these different pathways, then regulation of the amounts or
activities (e.g. by post-translational modifications) of specific coregulators could modulate the
specific physiological response to GC. It is thus interesting to note that gene ontology analysis
indicated that the block gene class is enriched for biological processes that are different from
those enriched in the combined mod and ind genes (Figure 2.11). The considerable number of
block genes that require both CHD9 and BRM supports the notion that CHD9 and BRM affect
similar biological processes. For example, previous studies indicate that BRM and CHD9 may
both be involved in regulating osteogenic genes (Marom et al., 2006; Nguyen et al., 2015).
Since the current study shows that the interaction between GR and these two chromatin
remodelers is regulated by Hic-5, it would be interesting to explore how Hic-5 influences GR
interaction with BRM and CHD9 to regulate osteogenic genes.
Since Hic-5 prevents GR binding to a specific subset of potential GBRs, Hic-5 actually
alters the GR cistrome (the genome-wide set of sites occupied in a given cell type). The GR
cistrome varies in a cell type specific manner (John et al., 2011), but the factors that contribute
to cell type-specific transcription factor binding are only partially understood. Cell type-specific
heterochromatin domains certainly contribute (Becker et al., 2016), and chromatin remodelers
have been shown to influence the locations of hypersensitive sites and transcription factor
binding (John et al., 2011; Morris et al., 2014). Hic-5 represents a new type of mechanism that
contributes to determination of the GR cistrome.

70
Thus, studying the mechanism of Hic-5 action on GR provides a unique opportunity to
advance our understanding on how transcription factor binding site selection is regulated and
the role of coregulators in mediating transcription factor binding and activity. The current study
elucidates a mechanism that explains the block gene-specific actions of Hic-5 on a subset of
GC-regulated genes. However, Hic-5 has been shown to block transcription factor occupancy
and hormone-induced expression of genes by estrogen receptor (Chodankar et al., 2015) and
actually promoted androgen receptor binding to some sites (Leach et al., 2014). Additionally,
coregulators CCAR1 (Cell Cycle And Apoptosis Regulator 1), CCAR2 (Cell Cycle And
Apoptosis Regulator 2), CALCOCO1 (Calcium Binding And Coiled-Coil Domain 1) and ZNF282
(Zinc Finger Protein 282) also block expression of a subset of GC regulated genes in A549
cells, although not as robustly as Hic-5 (Wu et al., 2014). Therefore, although the current study
focuses on GR and Hic-5, it is likely that my model applies to other transcription factors and
coregulators. Transcription factor binding site selection and gene-specific actions of
coregulators are relevant to all transcription factors and, hence, my study broadly contributes to
our understanding of the role of coregulators in transcriptional regulation.
Because Hic-5 modulates (both positively and negatively) the GC-regulated expression
of some GR target genes (mod genes) and blocks the activation or repression of others (block
genes) in response to GC, it has the potential to dramatically influence the outcome of the
response to GC, but only if the amount or activity of Hic-5 is regulated. It is thus interesting to
note that Hic-5 expression is highly cell type- and tissue-specific (Deakin et al., 2012;
Yuminamochi et al., 2003). In addition, phosphorylation of Hic-5 by multiple kinases on multiple
residues of Hic-5 has been reported, with some of these modifications promoting and others
inhibiting interaction between Hic-5 and androgen receptor (Ishino et al., 2000; Maudsley et al.,
2006; Wang et al., 2002). In addition, Hic-5 activity and changes in Hic-5 expression have been
linked to various types of cancer, especially prostate cancer (Deakin et al., 2012; Leach et al.,
2014; Li et al., 2011). In that light it is also relevant to note that enhanced GR activity has been

71
recently implicated in progression of castration resistant prostate cancer (Arora et al., 2013). In
future work it will be very interesting to test whether post-translational modifications of Hic-5 and
the signaling pathways that control them alter Hic-5 effects on androgen receptor- and GR-
regulated gene expression and emerge as novel therapeutic targets in cancer.

72
Chapter 3
Characteristics that distinguish the block gene class

3.1 Introduction
Transcriptional regulation is a major factor regulating cellular activities and thereby in
determining human health and disease. Regulating transcription involves transcription factors
binding to specific regulatory DNA sequences where they can either activate or repress
transcription of the associated genes. Transcription factor binding initiates the recruitment of
numerous coregulator proteins which remodel the chromatin landscape around the transcription
factor binding site and the transcription start site (TSS) of the regulated gene and regulate the
assembly of an active transcription complex at the TSS. Each coregulator contributes specific
molecular functions to accomplish these complex processes in a presumably coordinated
fashion, resulting in increased or decreased production of mRNA encoded by the gene (Glass
and Rosenfeld, 2000; Lonard and O'Malley, 2012; Rosenfeld et al., 2006). Transcription factors
can recruit different set of coregulators for different target genes, and different sets of
coregulators are required for the regulation of different genes by the same transcription factor in
a given cell type. Thus coregulators function in a gene-specific manner and are required for the
regulation of a subset of the genes regulated by any given transcription factor (Bittencourt et al.,
2012; Chodankar et al., 2014; Lonard and O'Malley, 2012; Wu et al., 2014). However, the site-
specific characteristics at the transcription factor binding sites that determine the recruitment of
and/or requirement for specific coregulators are mostly unknown. In this chapter I report site-
specific characteristics of chromatin and DNA sequence that dictate the gene-specific actions of
the protein Hydrogen peroxide-inducible clone-5 (Hic-5, also known as TGFB1I1) as a
coregulator for the glucocorticoid receptor (GR, NR3C1). GR, a member of the nuclear receptor
family of ligand activated transcription factors, regulates diverse physiological programs

73
including inflammation and metabolism of glucose, lipids, and proteins by activating and
repressing transcription of specific genes. GR is activated by binding of the natural
glucocorticoid (GC) hormone cortisol or various synthetic analogues, which are widely used in
treatment of many types of inflammatory diseases and cancer (Biddie et al., 2012).
Hic-5 is a member of the paxillin family of molecular adaptor proteins and has been
widely studied in the cytosol as an adapter protein at focal adhesion complexes (Kim-Kaneyama
et al., 2012). In the nucleus Hic-5 serves as a coregulator for a variety of transcription factors
including GR (Aghajanova et al., 2009; Drori et al., 2005; Leach et al., 2014; Li et al., 2011;
Shibanuma et al., 2004; Wang et al., 2008; Yang et al., 2000). As a coregulator of nuclear
receptors, Hic-5 has been associated with many physiological and disease functions including
endometriosis through the progesterone receptor (Aghajanova et al., 2009), epithelial cell
differentiation by affecting PPARg transcriptional activity (Drori et al., 2005), and prostate
tumorigenesis and castrate responsiveness through the androgen receptor (Li et al., 2011).
Endogenous Hic-5 modulates GC-regulated gene transcription by GR in a highly gene-specific
manner, functioning as coactivator for some GR target genes and corepressor for others.
Previously in chapter 2, I categorized GC-regulated genes into three different classes with
respect to their dependence on Hic-5: Hic-5 independent (ind) genes are regulated by GC
independently of Hic-5; Hic-5 modulated (mod) genes are regulated by GC when Hic-5 is
present in cells, and depletion of Hic-5 alters the magnitude of activation or repression by GC;
Hic-5 blocked (block) genes are not regulated by GC until Hic-5 is depleted from the cells
(Chodankar et al., 2014; Lee and Stallcup, 2017). Examination of selected GC-induced mod
genes showed that Hic-5 is recruited to GR binding regions (GBR) by its interaction with GR and
acts at late stages of transcription complex assembly, facilitating recruitment of the Mediator
complex and RNA polymerase II (Chodankar et al., 2014). In contrast, examination of three
selected block genes indicated that Hic-5 prevented transcriptional activation by impeding GC-

74
induced chromatin remodeling and robust GR occupancy at GBR associated with the block
genes (Chodankar et al., 2014; Lee and Stallcup, 2017).
Chromatin structure plays a major role in GR occupancy. GR and other transcription
factors interact with the DNA binding site in a dynamic on and off manner (McNally et al., 2000;
Voss and Hager, 2014). GC-induced binding of GR to GBR is a cooperative process between
GR and chromatin remodelers, in which GR at first recognizes its specific DNA binding motif
weakly (due to restrictive chromatin conformation) and recruits ATP-dependent chromatin
remodeling enzymes that open up chromatin structure allowing more robust GR occupancy
(Burd and Archer, 2013; Fryer and Archer, 1998; Lee and Stallcup, 2017; Nagaich et al., 2004).
The discovery that Hic-5 prevents transcription of block genes by impeding GR occupancy and
GC-regulated chromatin remodeling at those genes, but not at mod or ind genes, suggests that
different chromatin remodelers may be required for robust GR binding at GBR associated with
block genes versus mod and ind genes. Therefore, in the current chapter, I assessed global GR
binding sites to determine the gene-specific characteristics that specify the actions of Hic-5 to
prevent GR binding and transcriptional regulation at the block class of GR target genes while
allowing robust GR binding and GC-regulated transcription at other GR target genes (the ind
and mod classes). I tested the hypothesis that gene-specific actions of coregulators are
determined by the chromatin environment and DNA sequence motifs specifying potential
binding sites for other transcription factors around the GBR, and that Hic-5 influences GR
occupancy by interfering with the dynamic interaction of GR with specific chromatin remodeling
complexes. I therefore globally identified the sets of Hic-5 blocked and non-blocked GBR and
examined the chromatin remodeler requirements, the chromatin structure and the DNA
sequence (specifying potential binding sites for other proteins) around these two classes of
GBR. My findings address the gene-specific characteristics that control transcription factor
binding site selection and contribute to the gene-specific actions of coregulators.

75
3.2 Materials and Methods
Cell culture and siRNA transfection
U2OS osteosarcoma cells stably expressing wild-type GRα (U2OS-GRα) were a gift
from Dr. Inez Rogatsky (Hospital for Special Surgery, New York, NY). U2OS cells were
originally obtained from ATCC and stably transformed to express GRα (Rogatsky et al., 1997).
The cells were authenticated by Short Tandem Repeat Profiling, tested negative for
mycoplasma, and maintained as described (Chodankar et al., 2014). Cells were grown in
medium supplemented with 5% (vol/vol) FBS and transfected with siRNA using Lipofectamine
RNAiMAX (Invitrogen). For double depletion of chromatin remodeler and Hic-5, equivalent
amounts siRNA for a chromatin remodeler and Hic-5 (siHic5) were added. For single protein
depletions, equivalent amounts of siRNA for the targeted protein and nonspecific control siRNA
(siNS) were used such that the total volume and mass of siRNA was consistent. 48 h after
siRNA transfection the cells were either treated for the indicated length of time with 100 nM dex
(Sigma) or an equivalent amount of ethanol as control. siRNA sequences for siNS, siHic-5,
siBRM, and siCHD9 were previously described (Lee and Stallcup, 2017).

Chromatin Immunoprecipitation followed by high-throughput sequencing
ChIP (Chromatin Immunoprecipitation) experiments were performed as previously
described (Lee and Stallcup, 2017). Briefly, U2OS-GRα cells grown on 15-cm dishes were
transfected with the appropriate siRNAs. After 48 h, the cells were treated with 100 nM dex or
equivalent amounts of ethanol for 1 h before crosslinking with 1% (v/v) formaldehyde for 10 min
at room temperature and extracting chromatin from the harvested cells. Chromatin was
sonicated 20-30 min (30 s on/off cycles) with the Biorupter (Diagenode) at 4ºC to produce DNA
fragment size of 400-600 bp. Immunoprecipitation of the sonicated chromatin samples was
conducted with a cocktail of GR antibodies: H300 (6 µg, Santa Cruz Biotechnology), PA1-511A

76
(2 µg, Thermo Scientific), D6H2L (2 µg, Cell Signaling Technology) in a volume of 1 ml
containing chromatin from 20 x 10
6
cells. Protein G Sepharose magnetic beads (GE Healthcare)
were used to isolate the immune complexes with the crosslinked DNA. Purified DNA from the
ChIP experiments were analyzed for quality using Agilent Technologies 2100 Bioanalyzer.
Samples were submitted to Next-Generation Sequencing Core at the University of Southern
California Norris Comprehensive Cancer Center for library preparation and sequencing. Single-
end 75 bp DNA-sequencing data were generated for the samples using Illumina NEXTseq 500.
Sequencing results produced 36-70 million raw reads per sample. ChIP-seq data was submitted
to Gene Expression Omnibus (GEO) under the accession number GSE109383 within the
GSE109590 superseries. After trimming the raw reads for quality and adapter sequence, the
samples were mapped using BWA against the GRCh38/hg38 human reference genome (Li and
Durbin, 2009). 94-98% of the raw reads were mapped to the genome. Duplicate reads and
reads mapping to mitochondrial DNA were removed. To determine the peaks with the
MACS2+IDR method, mapped reads were analyzed using Model-based Analysis for ChIP-Seq
version 2 (MACS2) (Zhang et al., 2008) and the Irreproducibility Discovery Rate (IDR)
framework developed for ENCODE [https://sites.google.com/site/anshulkundaje/projects/idr].
For peaks identified with the MACS2+DiffBind method, DiffBind (Ross-Innes et al., 2012) was
used to determine the differentially bound peaks from the sets of peaks called by MACS2 in at
least 2 samples from all of the conditions tested. False discovery rate adjusted p-value ≤ 0.01
and the indicated fold-change cut-off for the change in CPM values for GR occupancy were
used along with the DESeq2 option during DiffBind analyses to identify the set of differentially
bound peaks and create the MA plots. Venn diagrams overlapping the different sets of peaks
were created using ChIPpeakAnno (Zhu et al., 2010). Both DiffBind and ChIPpeakAnno are
packages available on Bioconductor (Gentleman et al., 2004; Huber et al., 2015). The
Integrative Genomics Viewer was used to visualize the ChIP-seq data (Robinson et al., 2011).

77
Motif analysis was performed using Hypergeometric Optimization of Motif EnRichment
(HOMER) with a 1 kb window centered at the GBR summit (Heinz et al., 2010).

ATAC-seq
ATAC-seq was performed as previously described (Buenrostro et al., 2013). Briefly,
nuclei were prepared from 50,000 U2OS-GRα cells and resuspended in the transposase
reaction mix (FC-121-1030; Illumina). Transposition reaction was performed at 37 °C for 30 min,
and the samples were purified using a Qiagen PCR MinElute kit (28006; Qiagen). Library
fragments were amplified using Nextera PCR Primers (FC-121-1011; Illumina) and NEBnext
PCR master mix (0541; New England Lab) for a total of 6-8 cycles. The libraries were then
purified and size selected in the 100-450 bp range using Agencourt AMPure XP (A63880;
Beckman Coulter). The size-selected libraries were quantified using the Agilent Bioanalyzer.
Samples were submitted to Next-Generation Sequencing Core at the University of Southern
California Norris Comprehensive Cancer Center for sequencing. Paired-end 41 bp and 76 bp
sequencing data were generated for the samples using Illumina NEXTseq 500. Sequencing
results produced a total of 106-158 million raw reads per sample. ATAC-seq data was submitted
to Gene Expression Omnibus (GEO) under the accession number GSE109589 within the
GSE109590 superseries. Samples were mapped against the GRCh38/hg38 human reference
genome using Bowtie 2 (Langmead and Salzberg, 2012). 96-98% of the raw reads mapped to
the genome. Duplicated reads and reads aligned to mitochondrial DNA were removed.
Accessible regions and peaks were identified using the broad peak calling parameters of
MACS2. Chromatin accessibility profiles were created using the SeqPlots package (Stempor
and Ahringer, 2016) from Bioconductor by assessing the chromatin with ATAC-seq reads per
per million mapped reads (RPM) per 10 nucleotides +/- 1kb from the GBR summit.

78
3.3 Results
Genome-wide analysis of GR binding regions that are blocked or not blocked by Hic-5
Previously in chapter 2, I showed that CHD9 and BRM are required for the binding of GR
to GR binding regions (GBR) associated with 2 representative block genes but not for GBR
associated with 2 representative ind or mod genes (Lee and Stallcup, 2017). In this chapter, I
extended the previous studies by assessing whether the same site-specific chromatin remodeler
requirement applies genome-wide. GR occupancy was assessed by ChIP-seq experiments with
GR antibody, using U2OS-GRa osteosarcoma cells transfected with six different siRNA
combinations: non-specific siRNA control (siNS/siNS), depletion of Hic-5 (siHic5/siNS),
depletion of CHD9 (siCHD9/siNS), double depletion of Hic-5 and CHD9 (siCHD9/siHic5),
depletion of BRM (siBRM/siNS), and double depletion of Hic-5 and BRM (siBRM/siHic5). Cells
in each category were treated with 100 nM dex for 1 hour, and two biological replicates were
performed on different days.
I first identified the sets of GBR that are blocked by Hic-5 (blocked GBR) or not blocked
by Hic-5 (non-blocked GBR). For each condition, GBR were determined using the IDR
framework with MACS2 as the peak caller (MACS2+IDR GBR) (Zhang et al., 2008). The
number of GBR in cells depleted of Hic-5 (9526) was almost 4 times that in cells containing Hic-
5 (2553), indicating that Hic-5 blocks GR binding to specific loci on a genome-wide basis (Table
3.1). When these 2 sets of GBR were overlapped, almost all of the GR peaks detected in cells
containing Hic-5 were also detected in cells depleted of Hic-5 (2471 shared GBR), but there
were 7055 Hic-5 blocked GBR that were called only after depletion of Hic-5 (Figure 3.1A).
In addition to analyzing GBR called by MACS2+IDR, I used a statistical method to
compare quantitatively the GR occupancy at all GBR in cells containing or lacking Hic-5. I
assessed the GR ChIP-seq data using peaks identified with default settings in MACS2 followed
by statistical analysis of the peak intensities through DiffBind (Ross-Innes et al., 2012). My

79
Table 3.1. Total GR peaks (or GR binding regions, GBR) identified for each condition
using the MACS2+IDR method. ChIP-seq with GR antibody was performed on U2OS-GRa
cells transfected with indicated siRNA combinations and treated with dex for 1 hr. Total raw
reads for biological replicate samples are also shown.

80
previous qPCR analysis of a few representative GBR in chapter 2 (Chodankar et al., 2014; Lee
and Stallcup, 2017) revealed that GR ChIP signal increased upon Hic-5 depletion for most GBR,
but the signal increased much more dramatically (more than 3-fold) for block genes. Therefore, I
established a quantitative cut-off of 3-fold along with a statistical cut-off (FDR < 0.01) for
classification of Hic-5 blocked GBR versus non-blocked GBR. GBR were assessed by plotting
the difference in log
2
fold change of GR binding signal between cells depleted of Hic-5 and cells
containing Hic-5 (y-axis) against the average binding intensity across all six experimental
conditions (x-axis) (Figure 3.1B). Blue dots represent GBR that did not satisfy the statistical and
quantitative cut-offs for changes caused by depletion of Hic-5, and blue smears indicate
overrepresentation of blue dots. Red dots represent GBR that were significantly different (FDR <
0.01) and changed at least 3-fold in cells depleted of Hic-5. There was a general increase in GR
binding intensity for all GBR upon Hic-5 depletion, as observed by the large clustering of dots
above the log
2
= 0 fold change line. Essentially all of the 3697 GBR that met the statistical and
fold-change criteria had increased (rather than decreased) occupancy by GR in cells depleted of
Hic-5. By overlapping the set of differentially bound GR peaks (3697 DiffBind GBRs, determined
by MACS2+DiffBind, from Figure 3.1B) with the sets of MACS2+IDR-called GBR (from Figure
3.1A) in cells containing Hic-5 and depleted of Hic-5, I defined 3102 DiffBind Hic-5 blocked GBR
and 2156 DiffBind non-blocked GBR (Figure 3.1C). Almost 90% of the blocked GBR (2787
peaks) were only observed in cells depleted of Hic-5; however, some of the blocked GBR (315
peaks) were called as peaks in cells containing or depleted of Hic-5 but had significantly
different levels of GR occupancy (FDR < 0.01 and 3-fold change cutoff) in cells containing Hic-5
versus cells depleted of Hic-5.

81
Figure 3.1. Identifying Hic-5 blocked GBR and non-blocked GBR. (A) Venn diagram
showing overlap of GR peaks after 1 hr dex treatment in Hic-5 positive cells (siNS/siNS) and
Hic-5 depleted cells (siHic5/siNS). (B) MA plot analysis to identify GBR with statistically altered
level of GR binding between Hic-5 positive cells (siNS/siNS) and Hic-5 depleted cells
(siHic5/siNS). X-axis shows the log
2
of the average GR binding intensity for each GBR (in
counts per million, cpm) from all conditions as mentioned in A. Y-axis shows the log
2
fold-
change in GR binding between siHic5/siNS and siNS/siNS for each GBR. Each red dot
represents a GR peak that is significantly different between siHic5/siNS and siNS/siNS with
FDR < 0.01 and at least a 3-fold change in binding. Blue dots represent peaks not differentially
bound between siNS/siNS and siHic5/siNS with blue smears indicating overrepresentation of
blue dots. (C) Identification of Hic-5 blocked and non-blocked GBR by statistical analysis of
differential GR binding. Three-way Venn diagram overlapping GBR in Hic-5 positive and Hic-5
depleted cells identified by MACS2+IDR method with GBR having significantly different GR
binding between these two conditions as defined by MACS2+DiffBind method. DiffBind Hic-5
blocked GR peaks are GBR found only in Hic-5 depleted cells using the MACS2+IDR method
that have a significant difference in GR binding intensity between Hic-5 depleted cells and Hic-5
positive cells using the MACS2+DiffBind method. DiffBind non-blocked GBR are GR binding
sites found in both Hic-5 positive and Hic-5 depleted cells using the MACS2+IDR method but
have no significant difference in binding intensity between Hic-5 depleted cells and Hic-5
positive cells as defined by the MACS2+DiffBind method.

82
Figure 3.1

A

B

C

83
Differential requirement for chromatin remodelers CHD9 and BRM for GR occupancy at
blocked versus non-blocked GBR
I previously showed in chapter 2 that CHD9 and BRM are required for GR occupancy at
2 blocked GBR but not at 4 non-blocked GBR (Lee and Stallcup, 2017). Therefore, after
defining genome-wide sets of blocked and non-blocked GBR, I compared the requirement of the
two sets of GBR for chromatin remodelers CHD9 and BRM. Since GR occupancy at the blocked
GBR occurs only when Hic-5 is depleted, double depletion of a chromatin remodeler and Hic-5
is required to identify GBR that are dependent on the chromatin remodeler. The set of 3102 Hic-
5 blocked GBR determined by the DiffBind analysis (from Figure 3.1C) was overlapped
simultaneously with the MACS2+IDR-called GBR (from Table 3.1) from cells depleted of CHD9
and Hic-5 (siCHD9/siHic5) and from cells depleted of BRM and Hic5 (siBRM/siHic5) (Figure
3.2A; left side). Of the 3102 Hic-5 blocked GBR, 80% (382+2103=2485) were dependent on
CHD9 for GR occupancy and 72% (124+2103=2227) were dependent on BRM (Figure 3.2A, B;
left side). The CHD9 and BRM requirements were largely overlapping, such that 68% of the Hic-
5 blocked peaks required both CHD9 and BRM for GR occupancy. In contrast, in a similar
analysis of the 2156 non-blocked GBR, only 35% (477+268=745) and 16% (70+268=338) of the
non-blocked peaks were dependent on CHD9 and BRM, respectively, for GR occupancy; only
13% of the non-blocked GBR required both CHD9 and BRM (Figure 3.2A, B; right side).
To conduct a more rigorous statistical analysis of blocked and non-blocked GBR that
were dependent on chromatin remodelers CHD9 and BRM for GR occupancy, I first used the
DiffBind method to identify CHD9-dependent GBR and BRM-dependent GBR. For each GBR
the log
2
fold change of GR binding signal between cells doubly depleted of Hic-5 and CHD9 and
cells depleted of Hic-5 alone (y-axis) was plotted against the average binding intensity across all
six experimental conditions (x-axis). In this analysis GR occupancy was significantly changed
(FDR < 0.01, no fold change cut-off) for 3022 GBR due to depletion of CHD9; and 3903 GBR
were differentially occupied due to depletion of BRM (Figure 3.3A). The resulting sets of GBR

84
Figure 3.2. Hic-5 blocked GBR require CHD9 and BRM. Genome-wide GR ChIP-seq analysis
was performed to evaluate the GR peaks dependent on CHD9 and BRM. (A) Three-way Venn
diagram overlapping the following GBR sets: DiffBind Hic-5 blocked GBR (left) or non-blocked
GBR (right) from Figure 3.1C; GBR in cells depleted of both CHD9 and Hic-5 (siCHD9/siHic5)
from Table 3.1; and GBR in cells depleted of BRM and Hic-5 (siBRM/siHic5) from Table 3.1.
Dark grey regions, GBR dependent on CHD9 and BRM; yellow regions, GBR dependent on
CHD9, not BRM; blue regions, GBR dependent on BRM, not CHD9; light grey regions, GBR not
dependent on CHD9 or BRM. (B) Pie charts showing percentage of blocked or non-blocked
GBR that are dependent on CHD9 and/or BRM. The number of GBR in each colored
compartment from A was divided by the total Hic-5 blocked GBR or non-blocked GBR to
calculate percent of the whole.

85
Figure 3.2

A

B

86
for which GR occupancy is significantly dependent on CHD9 or BRM were overlapped with each
other and with the set of DiffBind blocked GBR or DiffBind non-blocked GBR (Figure 3.3B). In
these overlaps, 43% (329+978=1307) of the 3102 peaks in the DiffBind blocked GBR required
CHD9 and 59% (852+978=1830) were dependent on BRM (Figures 3.3B, C). In contrast, 18%
(136+261=397) and 38% (558+261=819) of the 2156 non-blocked DiffBind GR peaks were
dependent on CHD9 and BRM, respectively. Thus, the requirement of CHD9 and BRM for GR
occupancy is strongly associated with the GBR that are blocked with Hic-5 and not with the non-
blocked GBR.

Hic-5 blocked GBR are preferentially associated with Hic-5 blocked GR target genes
After defining the sets of Hic-5 blocked and non-blocked GBR, we determined the
distribution of blocked and non-blocked GBR near the block, ind, and mod genes. From the
ChIP-seq data, I first examined the GBR that are closest to representative genes from the block,
ind, and mod gene classes. As I previously observed with ChIP-qPCR experiments on 2
representative block genes (Chodankar et al., 2014; Lee and Stallcup, 2017), the current ChIP-
seq data showed weak or undetectable GR occupancy at GBR nearest to the transcription start
sites (TSS) of the representative block genes ANO2 and RP1L1 in cells containing Hic-5 and
robust GR occupancy in cells depleted of Hic-5, such that MACS2+IDR only called the peak in
cells depleted of Hic-5 (Figure 3.4A), as indicated by presence or absence of solid bars under
the peaks. This phenotype is consistent with their classification as Hic-5 blocked GBR. After
double depletion of CHD9 and Hic-5 or BRM and Hic-5, GR occupancy was reduced to a level
that was not identified by the peak caller. In contrast, GBR closest to representative ind genes
MSX2 and IGFBP1 and representative mod genes SCNN1A and SPINK13 had called GR peaks
in Hic-5 containing cells as well as in Hic-5 depleted cells (Figures 3.4B, C), consistent with their
classification as non-blocked GBR. GR occupancy at the GBR closest to these representative
ind and mod genes was reduced by depletion of CHD9 or BRM, but peaks were still called.

87
Figure 3.3. Differential GR peak analysis shows Hic-5 blocked GBR require CHD9 and
BRM. Hic-5 blocked and non-blocked GBR were evaluated genome-wide with DiffBind to
identify GBR with significantly different GR occupancy in Hic-5 depleted cells containing or
depleted of CHD9 or BRM. (A) MA plot analysis to identify GR peaks with statistically altered
level of GR binding between cells depleted of Hic-5 only (siHic5/siNS) and cells doubly depleted
of CHD9 and Hic-5 (left side) or doubly depleted of BRM and Hic-5 (right side). For each GBR
the log
2
fold-change in GR binding between siCHD9/siHic5 and siHic5/siNS samples or between
siBRM/Hic5 and siHic5/siNS samples (Y-axis) is plotted against the log
2
of the average GR
binding intensity from all conditions tested (cpm). Each red dot represents a differential GR peak
with significantly altered GR binding (FDR < 0.01) between the doubly depleted and Hic-5
depleted samples. Blue dots represent peaks not differentially bound between the indicated
conditions with blue smears demonstrating overrepresentation of blue dots. (B) Three way Venn
diagrams overlapping the following GBR sets: DiffBind Hic-5 blocked GBR (left side) or non-
blocked GBR (right side); the set of GBR that had significantly reduced GR occupancy when
CHD9 and Hic-5 were depleted compared with depletion of Hic-5 alone; and the set of GBR that
had significantly reduced GR occupancy when BRM and Hic-5 were depleted compared with
depletion of Hic-5 alone. Yellow regions, GBR dependent on CHD9, not BRM; blue regions,
GBR dependent on BRM, not CHD9; dark gray regions, GBR dependent on both CHD9 and
BRM; light grey regions, GBR not dependent on CHD9 or BRM. (C) Pie charts showing
percentage of blocked and non-blocked GBR that are dependent on CHD9 and/or BRM. The
number of GBR in each colored compartment from B was divided by the total Hic-5 blocked
GBR or non-blocked GBR to calculate percent of the whole.

88
Figure 3.3

A

B

C

89
Figure 3.4. ChIP-seq GR occupancy near representative genes for each class. Integrative
Genomics Viewer displaying the ChIP-seq track of GR occupancy near block genes ANO2 and
RP1L1 (A), ind genes MSX2 and IGFBP1 (B), and mod genes SCNN1A and SPINK13 (C).

90
Figure 3.4

A

91
Figure 3.4 continued

B

92
Figure 3.4 continued

C

93
To assess genome-wide whether Hic-5 blocked GBR are preferentially enriched near the
block genes compared with ind and mod genes, we determined the proportion of Hic-5 blocked
GBR and non-blocked GBR within a specified distance of the TSS for all block, ind, and mod
genes. The sets of block, mod, and ind genes used in this analysis were taken from my
previously analyzed RNA-seq data (Lee and Stallcup, 2017). Within windows extending 10, 50,
or 100 kb in either direction from the TSS of the 534 block genes, the percentages of GBR that
were classified as Hic-5 blocked GBR were 67%, 60%, and 58%, respectively. In contrast, the
corresponding percentages for the 105 ind genes were 39%, 37%, and 42%, and for the 364
mod genes the percentages were 44%, 47%, and 49% (Figure 3.5). For each of these windows
the percentages were significantly different (chi-square test) between block and ind and
between block and mod gene sets. However, when the window was extended to 1 Mb from the
TSS, the proportion of Hic-5 blocked to non-blocked GBR was similar across all gene classes
(no significant differences), presumably due to the inclusion of many additional GBR that are not
regulating the target genes. These data indicate that Hic-5 blocked GBR are preferentially
associated with and enriched near the block gene class and not with ind and mod gene classes.

Chromatin structure at Hic-5 blocked GBR is less accessible than at non-blocked GBR in
cells containing Hic-5
Since chromatin accessibility affects binding of GR and other transcription factors (Lee
and Stallcup, 2017; Morris et al., 2014), I assessed the chromatin accessibility at GBR
associated with the three gene classes. Previous experiments using formaldehyde-assisted
isolation of regulatory elements (FAIRE) followed by qPCR showed that chromatin accessibility
at the GBR closest to the TSS of 3 representative block genes dramatically increased upon Hic-
5 depletion and dex treatment (Chodankar et al., 2014; Lee and Stallcup, 2017). However,
FAIRE-qPCR was not sensitive enough to detect changes in chromatin structure due to Hic-5
depletion in cells not treated with dex. Therefore, I performed Assay for Transposase-

94
Figure 3.5. Hic-5 blocked GBR are preferentially enriched near the block genes. Proportion
of Hic-5 blocked GBR and non-blocked GBR within 10 kb, 50 kb, 100 kb and 1 Mb of TSS for all
genes in each of the three gene classes. Blue bars represent the proportion of Hic-5 blocked
GBR; orange bars represent the proportion of non-blocked GBR; the total number of GBR is
indicated on each bar. c
2
test was performed to compare the ratio of blocked and non-blocked
GBR near the ind genes and mod genes with the ratio of blocked and non-blocked GBR near
the block genes. (* = p < 0.001, ns = not significant)

95
Figure 3.5

96
Accessible Chromatin using sequencing (ATAC-seq) to assess global chromatin accessibility in
cells treated with dex or equivalent volume of vehicle ethanol, both in the presence and absence
of Hic-5 (Table 3.2). In cells containing Hic-5 and treated with ethanol or dex, my analysis
revealed 80,006 and 73,785 open chromatin regions, respectively. In cells depleted of Hic-5 and
treated with ethanol or dex, I obtained 98,693 and 92,338 open chromatin regions, respectively.
The ATAC-seq peaks were overlapped with the genome-wide sets of Hic-5 blocked and non-
blocked GBR, as determined from the DiffBind ChIP-seq analyses (from Figure 3.1C), to assess
chromatin accessibility at the GBRs. In cells containing Hic-5 (treated with either dex or ethanol)
the percentage of Hic-5 blocked GBR with open chromatin (defined by ATAC-seq peaks called
by MACS2+IDR) was significantly lower than the percentage of non-blocked GBR with open
chromatin (Figure 3.6A). In cells depleted of Hic-5, the difference in the percentage of
accessible blocked versus non-blocked GBR was less than in cells containing Hic-5 and was
significant only in cells treated with dex.
I also examined the chromatin accessibility of the GBRs that lie within 100 kb of the TSS
of the genes in each of the three gene classes. The average RPM from ATAC-seq data,
centered at the GBR summits were graphed to create an average chromatin accessibility profile
of GBR for each of the four conditions tested by ATAC-seq: cells containing Hic-5 with or
without dex (siNS etoh and siNS dex) and cells depleted of Hic-5 with or without dex (siHic-5
etoh and siHic-5 dex) (Figure 3.6B). Chromatin accessibility of blocked GBR associated with all
three gene classes did not change between etoh and dex treatment in cells containing Hic-5,
but in cells depleted of Hic-5 dex caused a robust increase in accessibility of the blocked GBR.
In contrast, for the non-blocked GBR associated with all three gene classes, dex increased
chromatin accessibility in the presence or absence of Hic-5. In general the chromatin
accessibility of blocked GBR was less than the accessibility of non-blocked GBR; this was true
in all four conditions tested (± Hic-5, ± dex) and for GBR near all three gene classes (block, ind,
and mod). Of particular note was that the accessibility of blocked GBR in ethanol-treated cells

97
Table 3.2. Total number of open chromatin regions from ATAC-seq analysis. Genome-
wide ATAC-seq analysis was performed to evaluate changes in chromatin structure caused by
Hic-5 depletion in cells treated with dex or vehicle ethanol (etoh).

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Figure 3.6. Chromatin at Hic-5 blocked GBR is less accessible than at non-blocked GBR.
Genome-wide ATAC-seq analysis was performed to evaluate changes in chromatin structure
caused by Hic-5 depletion in cells treated with dex or vehicle ethanol (etoh). (A) Percentage of
GBR with open chromatin in Hic-5 blocked GBR and non-blocked GBR for each condition
described in A. c
2
significance test was performed to compare the proportion of Hic-5 blocked
GBR with open chromatin and the proportion of non-blocked GBR with open chromatin for each
condition. (* = p < 0.001, ns = not significant). (B) Average chromatin accessibility profile for all
GBR near block, mod and ind genes. GBR within 100 kb of the TSS for all genes in each class
were evaluated. Chromatin accessibility in reads per million mapped reads (RPM) per 10
nucleotides was averaged for all GBR in the indicated GBR sets within a window extending 1 kb
in either direction from the GBR peak summits in cells treated as follows: siNS etoh (purple),
siNS dex (red), siHic-5 etoh (green), and siHic-5 dex (blue).

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Figure 3.6

A

B

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was higher when Hic-5 was depleted than when Hic-5 was present; this was true for blocked
GBR near all three gene classes. In contrast, the accessibility of the non-blocked GBR in
ethanol treated cells was lower in Hic-5 depleted cells than in cells containing Hic-5. These data
indicate that Hic-5 selectively interferes with the chromatin accessibility of blocked versus non-
blocked GBR, both before dex treatment and after dex is added.

ETS1 binding motif is enriched near blocked GBR and near GBRs associated with block
genes
To explore further the characteristics of Hic-5 blocked versus non-blocked GBR, motif
analyses were performed on different sets of GBR identified in this study. GBR within 100 kb of
the TSS of all genes in each of the three gene classes were examined using de novo motif
analysis with HOMER to identify enriched motifs in a 1-kb window centered on the GBR
summits. Motifs were ranked by p-value, and the top three motifs for each gene class are shown
(Figure 3.7A). A score indicating the degree of match between the de novo motif identified and
the consensus sequence for the matched transcription factor is also shown, along with the
percentage of GBR that contain this motif in the 1-kb window around the GBR peak. Beyond the
top three motifs identified, the remaining motifs were found at less than 10% of the GBRs in
each set examined. As expected, the most significant and most prevalent motif in all three gene
classes matched closely to the GC response element (GRE), which is the consensus binding
sequence for GR. The next two top-ranked motifs were AP-1 and ETS1 for GBR near block
genes, AP-1 and PROX1 for GBR near mod genes, and androgen receptor half-site and
ZNF519 for ind genes. The ETS1 motif was present near 37% of the GBR associated with block
genes, but it was not significantly enriched near GBR associated with mod and ind genes. When
the distribution of the GRE and ETS1 motifs were examined relative to the center of a 1-kb
window centered on the GBR peaks associated with the block genes, the GRE motif (as
expected) was highly enriched at the center of the GBR peaks associated with block genes

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Figure 3.7. ETS1 motif is enriched at Hic-5 blocked GBR and GBR near the block genes.
(A) ETS1 motif is enriched in GBR near block genes but not in GBR near mod and ind genes.
De novo motif analysis was performed using HOMER; the top 3 ranked motifs are shown with
their p-value, score for concordance of the de novo motif with the identified match, and
prevalence near the GBR set examined. Motif analysis was performed in a 1-kb window
centered on the GBR peak for all GBR within 100 kb of the TSS of the block, mod and ind
genes. Motifs below the dotted lines are for the highest scoring member of the ETS family
(shown for comparison to the block genes), which were not found as one of the top three motifs
for mod and ind genes and indicated as possible false positives by HOMER. (B) GR and ETS1
motif distribution in a 1-kb window centered on the peak of all GBR near the block genes.
Orange, GRE motif; blue, ETS1 motif. (C) ETS1 motif is enriched at Hic-5 blocked GBR but not
at non-blocked GBR. Motif analysis was performed, as in A, for all Hic-5 blocked and non-
blocked GBR. The top 5 motifs are shown. ETS motif below the dotted line is shown for
comparison, but was not one of the top 5 motifs and was indicated as a possible false positive
by HOMER. (D) GR and ETS1 motif distribution near Hic-5 blocked GBR, presented as in B.

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Figure 3.7

A

B

103
Figure 3.7 continued

C

D

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(Figure 3.7B). The ETS1 motif had a broader distribution within the 1-kb window but was also
enriched near the center of the 1-kb window surrounding the GBR associated with the block
genes. When de novo motif analysis was also conducted for all of the 3102 DiffBind Hic-5
blocked GBR and for all of the 2156 DiffBind non-blocked GBR (from Figure 3.1C), the ETS1
motif was significantly associated with the blocked GBR and was found near the majority of the
blocked GBR (Figure 3.7C). In contrast, a sequence resembling the ETS protein family binding
motif was found near less than 3% of the non-blocked GBR and was not significantly enriched.
As observed with the ETS1 motifs near the block genes (Figure 3.7B), the ETS1 motif near the
3102 blocked GBR was broadly distributed within the 1-kb window around the center of the
GBR but was enriched near the center of the GBR (Figure 3.7D).
De novo motif analysis was also performed for the sets of Hic-5 blocked and non-
blocked GBR that became newly accessible (by called ATAC-seq peaks) upon depletion of Hic-
5 (Figure 3.8, the shaded sector of the Venn diagrams). A de novo motif closely resembling an
ETS1 consensus binding site was significantly enriched within a 1-kb window centered on the
peaks of the blocked GBR that became newly accessible upon Hic-5 depletion in cells treated
with dex or vehicle ethanol (Figure 3.8A,B). However, motifs resembling ETS1 binding sites
were not significantly enriched near non-blocked GBR that became newly accessible upon Hic-5
depletion (Figure 3.8C,D). Furthermore, the percentage of blocked GBR that became newly
accessible upon Hic-5 depletion (15% in ethanol-treated cells and 22% in dex-treated cells),
was almost twice that for the non-blocked GBR (8% in ethanol-treated cells and 13% in dex-
treated cells). Thus, the ETS1 motif was found to be significantly associated with block genes
and with blocked GBR, but not with non-blocked GBR or with ind or mod genes.

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Figure 3.8. ETS1 motif is enriched at Hic-5 blocked GBR that become newly chromatin-
accessible when Hic-5 is depleted. Hic-5 blocked GBR (A-B) or non-blocked GBR (C-D) were
overlapped with two other sets: open chromatin regions identified by ATAC-seq in Hic-5 positive
cells; and open chromatin regions in Hic-5 depleted cells. This was done for ATAC-seq data
from cells treated with vehicle ethanol (etoh, A and C) or cells treated with dex (C and D). The
shaded region of each three-way Venn diagram indicates the GBR that become newly
chromatin accessible upon Hic-5 depletion. De novo motif analysis was performed on the GBR
in each shaded region, using HOMER. The top 3 ranked motifs are shown with their p-value,
score for concordance of the de novo motif with the consensus sequence of the identified
match, and prevalence near the GBR in the set examined. Motif analysis was performed in a 1-
kb window centered on the GBR peak for all GBR belonging to the set examined. Motifs below
the dotted lines in C and D (shown for comparison to A and B) are for the highest scoring
member of the ETS family, which were not one of the top three motifs and were indicated as
possible false positives in C and D by HOMER.

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Figure 3.8

A

B

C

D

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3.4 Discussion
My results demonstrate that Hic-5 has a major influence on binding site selection by GR.
The number of GBR detected by a simple peak calling analysis nearly quadrupled upon Hic-5
depletion (Table 3.1), while a DiffBind analysis of sites with significantly enhanced GR binding
upon Hic-5 depletion found a doubling of the number of GBR when Hic-5 was depleted (Figure
3.1B,C). By integrating this information on Hic-5 effects on GR binding at specific GBR with an
analysis of chromatin remodeler requirements, chromatin conformation analysis by ATAC-seq,
and previously obtained RNA-seq data from chapter 2 (Lee and Stallcup, 2017), I defined
specific characteristics that distinguish the Hic-5 blocked GBR from the non-blocked GBR.

Hic-5 blocked GBR are preferentially associated with block genes, compared with ind
and mod genes
I examined the GBR located within various distances of the TSS of all block, ind, and
mod genes to determine the relative prevalence of Hic-5 blocked and non-blocked GBR near
genes in each of the three gene classes. There was an enrichment of Hic-5 blocked GBR near
the block genes compared to the ind and mod genes. 67% of the GBR located within 10 kb of
any block gene TSS were Hic-5 blocked GBR, compared to 39% and 44% for ind and mod
genes, respectively, and the ratio of blocked to non-blocked GBR near the block genes was
significantly different from the ratios for the ind and mod genes (Figure 3.5). Similar results were
found when the distance from the TSS was increased to 50 kb or 100 kb. In contrast, when the
distance from the TSS was increased to 1 Mb, the ratios of blocked to non-blocked GBR were
indistinguishable between the three gene classes, indicating that the blocked GBR appear to be
preferentially concentrated near the block genes. The enrichment of Hic-5 blocked GBR, and
hence, GBR that require CHD9 and BRM for GR occupancy, distinguishes the GBR near the
block genes from the GBR near ind and mod genes. In spite of the correlative nature of the data

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linking blocked GBR with the block gene class, the preferential co-localization of Hic-5 blocked
GBR with genes that are blocked by Hic-5 from responding transcriptionally to dex suggests the
logical conclusion that the blocked GBR are responsible for mediating the dex regulation of the
block class of genes, while the non-blocked GBR are the regulatory sites responsible for dex
regulation of the ind and mod genes.

Chromatin remodelers CHD9 and BRM are preferentially required for Hic-5 blocked GBR
versus non-blocked GBR
The previous chapter showed that for the 2 GBR that are the closest to 2 selected block
genes, Hic-5 blocks GR binding, and after Hic-5 depletion CHD9 and BRM are required for
robust GR binding (Lee and Stallcup, 2017). In contrast Hic-5 did not block binding to the most
proximal GBR to 2 ind genes and 2 mod genes, and CHD9 and BRM were not required for GR
binding to those GBR (Lee and Stallcup, 2017). In the current chapter I found the same pattern
genome-wide. By a peak calling analysis 84% of Hic-5 blocked GBR required CHD9 or BRM,
and 68% required both of them for GR binding; on the other hand, only 38% of non-blocked
GBR required CHD9 or BRM and only 13% required both (Figure 3.2B). When the same
question was examined by a statistical method (DiffBind) to assess significant changes in GR
binding, 70% of blocked Hic-5 GBR required CHD9 or BRM (32% required both), while only
44% of non-blocked GBR required either CHD9 or BRM (12% required both). Previous studies
have shown that chromatin remodeling enzymes work in a coordinated fashion with GR to
remodel the chromatin and allow GR to bind (Engel and Yamamoto, 2011; Lee and Stallcup,
2017; Nagaich et al., 2004; Swinstead et al., 2016; Voss and Hager, 2014). Thus, a
distinguishing feature of Hic-5 blocked GBR genome-wide is their requirement for CHD9 and
BRM. In chapter 2, I previously showed that Hic-5 actually prevents GR binding to CHD9 and
BRM but not to other chromatin remodelers such as BRG1 (Lee and Stallcup, 2017). Since it
was previously demonstrated that many if not most DNase I hypersensitive sites require more

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than one chromatin remodeler to maintain their chromatin accessibility and different
hypersensitive sites require different combinations of chromatin remodelers (Morris et al., 2014),
my findings of differential requirements for CHD9 and BRM explain mechanistically why Hic-5
blocks only a specific subset of GBR. The large overlap of the CHD9 and BRM requirement
among Hic-5 blocked GBR suggests that they may act cooperatively to remodel the GBR, with
each chromatin remodeler contributing specific remodeling actions.
The results support my hypothesis that gene-specific actions of Hic-5 depend on the
gene-specific chromatin remodeler requirements, with block genes requiring CHD9 and BRM for
GR binding to the GBR, but not ind or mod genes. Site-specific action of the chromatin
remodeling enzymes has also been observed in other studies (Engel and Yamamoto, 2011;
Morris et al., 2014). The site-specific chromatin remodeler requirements support the idea that
CHD9 and BRM regulate specific pathways of glucocorticoid function (Lee and Stallcup, 2017).

Effects of Hic-5 on chromatin conformation at Hic-5 blocked and non-blocked GBR
Chromatin structure plays an important role in the binding of GR and other transcription
factors to DNA (Burd and Archer, 2013; John et al., 2011; Love et al., 2017). Genome-wide
studies of GR binding and chromatin accessibility, as measured by DNAse I hypersensitivity,
have shown that GR binding mainly occurs at chromatin accessible sites that exist prior to
hormone treatment (John et al., 2011). However, upon GR activation the preexisting accessible
GR binding sites can undergo additional chromatin remodeling (Burd et al., 2012; Chodankar et
al., 2014). Additionally, studies using the MMTV promoter as a model system showed that GR
was able to bind relatively inaccessible chromatin by triggering localized chromatin remodeling
with the assistance of ATP-dependent chromatin remodelers (Fletcher et al., 2002).
In the current study, I used ATAC-seq to show that the number of accessible chromatin
sites genome-wide (identified as called ATAC-seq peaks) increased when Hic-5 was depleted
(Table 3.2) indicating a global effect of Hic-5 on many chromatin sites, not just at GBR. Thus,

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Hic-5 may restrict the binding site selection of other transcription factors in addition to GR. To
specifically focus on GBR, ATAC-seq and GR ChIP-seq data were overlapped, revealing that
the fraction of Hic-5 blocked GBR that are chromatin accessible is lower than the fraction of
chromatin accessible non-blocked GBR (Figure 3.6A). Chromatin accessibility around Hic-5
blocked GBR was increased by dex only in cells depleted of Hic-5, while chromatin accessibility
at non-blocked GBR was increased by dex in cells containing or depleted of Hic-5 (Figure 3.6B).
This difference in blocked GBR and non-blocked GBR was observed for GBR near all three
gene classes. In addition, Hic-5 depletion also slightly enhanced ATAC-seq signals at Hic-5
blocked GBR before dex treatment; in contrast, the ATAC-seq signal for non-blocked GBR
decreased slightly in cells depleted of Hic-5 and not treated with dex (Figure 3.6B). Again, this
pattern was observed for GBR near all three gene classes. My results indicate a major effect of
Hic-5 on chromatin remodeling of blocked GBR in response to dex and a minor effect on the
basal (pre-dex) chromatin structure of blocked GBR, suggesting that both of these mechanisms
contribute to the overall effect of Hic-5 on the response of blocked GBR to dex. The differential
requirement for CHD9 and BRM to allow GR binding at blocked versus non-blocked GBR
provides a likely molecular mechanism for the post-dex effect of Hic-5, with Hic-5 preventing GR
from recruiting CHD9 and BRM to open the chromatin conformation and hence allow more
robust GR binding. The specific mechanism for the pre-dex effect presumably depends on some
other characteristic of blocked versus non-blocked GBR, such as less accessible chromatin
conformation (Figure 3.6B) or different occupancy by other transcription factors at blocked
versus non-blocked GBR, as suggested by motif analysis (Figure 3.7). This issue is explored in
the next section below.

ETS family binding motifs are prevalent near blocked GBR
GR binds directly to DNA as a homodimer, with a consensus 15-bp motif composed of
two pseudopalindromic 6 base pair half-sites separated by a 3 base pair spacer (Strähle et al.,

111
1987). These GRE were the most significant and most prevalent motif for the GBR in all three
gene classes, and for blocked and non-blocked GBR genome-wide (Figure 3.7A,C). The
specific nucleotide sequence of the GR binding site can affect GR conformation and function
(Love et al., 2017; Meijsing et al., 2009; Telorac et al., 2016), but the de novo motif analysis did
not find any obvious sequence differences for GREs associated with different gene classes or
for blocked versus non-blocked GBR. GR can also bind indirectly to specific DNA sites by
binding to other transcription factors that are directly binding to DNA (De Bosscher et al., 2003;
Herrlich, 2001; Langlais et al., 2012). A commonly found motif associated with GBR is the AP-1
motif. AP-1 has been shown to maintain an accessible chromatin conformation that facilitates
GR access to the GBR (Biddie et al., 2011). Indeed, the AP-1 motif was significantly associated
with a high percentage of blocked and non-blocked GBR. In comparing motifs associated with
blocked versus non-blocked GBR, the only motif my analysis found with a strong differential
enrichment was an ETS family binding motif. This motif was significantly enriched and highly
prevalent near blocked but not non-blocked GBR (Figure 3.7C) and was also significantly
enriched and highly prevalent near GBR associated with block genes but not GBR associated
with ind and mod genes (Figure 3.7A). The best match for the ETS family motif identified was
with the ETS1 family member, but the motifs for other ETS family members are also similar to
the ETS1 motif. ETS1 has previously been implicated in tumor progression (Dittmer, 2015),
immunity (Garrett-Sinha, 2013), and angiogenesis (Dejana et al., 2007). ETS1 interacts with
various other transcription factors including RUNX2 (Wai et al., 2006), STAT5 (Rameil et al.,
2000), and AP-1 (Thomas et al., 1997). However, an interaction between ETS1 and GR or Hic-5
has not yet been reported. In future experiments it will be interesting to examine what role ETS1
or another ETS family member plays in Hic-5 dependent blocking of GR binding and chromatin
remodeling at blocked GBR. Possibilities are recruitment of GR or Hic-5, or establishing
requirements for CHD9 and BRM as chromatin remodelers.

112
The results presented here strongly suggest that chromatin remodeler requirements,
chromatin structure and the DNA sequence contribute to the gene-specific actions of Hic-5 on
the binding of GR and remodeling of chromatin at GBR. The GBR near the block genes, but not
the mod or ind genes, required CHD9 and BRM for GR occupancy and dex-induced chromatin
remodeling; blocked GBR were less chromatin accessible than the non-blocked GBR; and DNA
motif analysis revealed an ETS family motif enriched at blocked GBR and GBR near the block
genes but not at non-blocked GBR or GBR near the ind and mod genes. Hic-5 has been shown
to block occupancy of other transcription factors, such as the estrogen receptor a, in a site-
specific manner and prevent hormone-induced expression of a subset of estrogen receptor
target genes (Chodankar et al., 2015). Hic-5 also serves as a coregulator for a number of other
transcription factors (Aghajanova et al., 2009; Drori et al., 2005; Leach et al., 2014; Li et al.,
2011; Shibanuma et al., 2004; Wang et al., 2008; Yang et al., 2000). Furthermore, several other
coregulators promote hormonal regulation of some GR target genes while blocking regulation of
another subset of GC regulated genes in A549 cells (Wu et al., 2014). Thus, the mechanism I
document for the gene-specific actions of GR and Hic-5 - involving chromatin environment,
differential requirements for chromatin remodelers, binding of nearby proteins, and DNA
sequence - is likely to be more broadly applicable. Although my study focuses on GR and Hic-
5, binding site selection and gene-specific actions of coregulators are important components of
transcriptional regulation for all transcription factors. My study elucidates important aspects of
the mechanisms responsible for the gene-specific requirements for coregulators that control
transcription.

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Chapter 4
Conclusion

The overall goal of my dissertation project was to understand the gene-specific actions
of coregulators in mediating transcription factor binding and activity. I explored the role of a
coregulator not just as a protein that regulates the rate of gene transcription by being recruited
to the genome by DNA binding transcription factors but also as a protein that determines the
binding of a transcription factor and thus influences gene expression. Hic-5 is particularly
interesting because it serves as a coregulator in the conventional sense for the mod genes and
has a novel role as a coregulator that blocks transcription factor binding at the block genes. By
comparing and contrasting between the different gene classes in this study, I was able to
elucidate a unique mechanism of action by Hic-5 in blocking the chromatin remodelers CHD9
and BRM required for GR binding at the block genes. Furthermore, I was also able to identify
gene-specific DNA sequence motifs specifying potential binding sites for other transcription
factors and gene-specific chromatin conformation requirements at the GBR of the block gene
class.
The results of my study suggest that differential gene-specific actions of Hic-5 are
determined by the differential gene-specific chromatin remodeler actions. I identified CHD9 and
BRM as chromatin remodelers required for GR binding to and expression of the block genes but
not the ind and mod genes. However, my project did not directly determine whether or not Hic-5
blocks the recruitment of CHD9 and BRM to the specific GBR of the block genes. The PLA
experiments indicate an increased interaction between GR and CHD9 and BRM once Hic-5 is
depleted and hence strongly suggest that CHD9 and BRM may be recruited to the GBR along
with GR after Hic-5 is depleted in dex treated cells. However it is still possible that CHD9 and
BRM may be present but inactive and inhibited from interacting with GR at or near the GBR in
the presence of Hic-5. Therefore it would be interesting to examine whether Hic-5 blocks the

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recruitment of the remodelers specifically to the Hic-5 blocked sites or Hic-5 somehow inhibits
the enzymatic activity of the chromatin remodeling enzymes. Additionally by examining Hic-5
occupancy along with CHD9 and BRM occupancy we may be able to understand more deeply
coregulator recruitment dynamics and protein interactions that result in the formation of
coregulator complexes. The dynamic GR-GBR interaction model suggests that GR recruitment
and the recruitment of coregulators that allow GR to bind is a fluid process with specific
coregulators being recruited to the GBR at specific stages to influence GR binding and
transcription. Therefore, the spatial (recruitment of coregulators to specific GBR) as well as the
temporal (the timing of the coregulator recruitment) occupancy of coregulators may further
elucidate the mechanism by which coregulator proteins can assist in transcription factor binding.
To add another layer, overlapping CHD9, BRM, and Hic-5 occupancy with open chromatin
regions from ATAC-seq can provide insight into the dynamics of chromatin remodeling
enzymatic activity and coregulator recruitment at the GBR.
In order to determine the chromatin remodeler required for the subset of GC-regulated
genes my project focused on the core ATPase subunit of the chromatin remodeler complex.
However, most chromatin remodelers form large multi-subunit complexes. The non-catalytic
accessory subunits are composed of interaction domains that may facilitate the binding of the
complex to transcription factors and coregulator proteins, regulate the enzymatic activity of the
complex, or target the complex to the DNA or nucleosomes (Hargreaves and Crabtree, 2011).
For example, subunits of the SWI/SNF chromatin remodeler complex contain several proteins
that are composed of bromodomains (PBRM1, BRD7 subunits), chromodomains (BAF155,
BAF170 subunits), and PHD finger protein (BAF45 subunit) that allow the chromatin remodeler
complex to bind to histone modifications (Kadoch and Crabtree, 2015). Therefore chromatin
remodeler subunits may contribute to the specificity of chromatin remodeler complexes. It would
be interesting to explore which subunit(s) of the CHD9 and BRM chromatin remodeling complex
are responsible for the interaction between the complex and GR or Hic-5.

115

In addition to chromatin remodeler requirement, my dissertation project explored the
chromatin accessibility at the GBR of the block genes. Chromatin accessibility with ATAC-seq
showed that although the major chromatin remodeling at the Hic-5 blocked sites occurs after
dex treatment, Hic-5 has an effect on chromatin remodeling before GR activation by dex.
Although CHD9 and BRM are chromatin remodelers required for GC-induced transcription of
the block genes, it is possible that Hic-5 may be blocking other ATP-dependent chromatin
remodelers or histone modification proteins that alter the chromatin accessibility at the Hic-5
blocked GBR before GC activation. Therefore, determining the Hic-5 regulated chromatin
remodeler proteins that act at the GBR before dex treatment would be very interesting. One
possibility is that Hic-5 may be inhibiting ATP-dependent chromatin remodelers, either the same
remodelers (CHD9 and BRM) that are recruited again at the GBR after GC activation or different
chromatin remodeler enzymes, at the block genes before dex treatment. Recent study shows
some GRE possess ATP-dependent chromatin remodeler, BRG1, that contributes to chromatin
accessibility before GR activation, and chromatin accessibility is further increased by
recruitment of more BRG1 after hormone treatment (Johnson et al., 2018). Hic-5 may also be
inhibiting a histone modifying protein before hormone treatment that converts the blocked GBR
from a closed chromatin conformation to a more poised state. Some researchers have
characterized a histone code such that certain histone modifications can recruit proteins that
recognize the modified histones and alter chromatin structure to actively promote or repress
transcription (Strahl and Allis, 2000). Hic-5 does not possess histone acetyltransferase or
methyltransferase domains and does not seem to modify histones directly. However, Hic-5 has
been shown to interact with coregulators with histone modification capabilities, including
glucocorticoid receptor-interacting protein 1 (GRIP1), E1A-associated protein p300 (EP300),
and CREB-binding protein (CBP), at glucocorticoid responsive promoters (Heitzer and
DeFranco, 2006). Therefore, determining whether or not Hic-5 inhibits histone modifying

116
proteins or ATP-dependent chromatin remodeler enzymes before hormone treatment may help
to distinguish the GBR of block genes from the ind and mod genes.
Recent advances in molecular biology technology focusing on higher order of chromatin
organization illustrate that in addition to local chromatin structure, genome-wide chromatin
interactions play a critical role in linking response elements to gene promoters. Chromatin can
be organized into topologically associated domain (TAD) structures, ranging from 100kb up to
1Mb in size, where specific looping interactions can occur (Dixon et al., 2012). Majority of the
enhancer-promoter interaction occur within the TAD boundaries defined by CCCTC-binding
factor (CTCF) (Phillips and Corces, 2009). The development of chromosome conformation
capture experiments, such as 3C, 4C, and Hi-C, have contributed to our understanding of TADs
and chromatin loops. Since most GRE are located at enhancers, chromosome conformation
capture experiments may be useful in better defining the GBR associated with each of the three
gene classes, and, hence, provide additional distinguishing characteristics for each gene class.
Furthermore, a recent study showed that BRG1 chromatin remodeler ATPase was enriched at
TAD boundaries and regulated TAD structures (Barutcu et al., 2016). Hence, CHD9 or BRM and
also Hic-5 may also be involved in regulating TAD structures.
In addition to chromatin conformation and chromatin remodeler requirements, I also
investigated the DNA sequence requirements at GR binding sites. Various studies have shown
that the DNA sequence at the GBR may influence whether GR will activate or repress
transcription (Hudson et al., 2013; Surjit et al., 2011). Furthermore, the specific composition of
the GR binding sequence has also been shown to allosterically regulate the structural
conformation of GR and the magnitude of transcriptional activation of GC-regulated genes
(Meijsing et al., 2009). Even sequences flanking the core GR binding site can modulate GR
transcriptional activity (Schöne et al., 2016). My analysis of GR binding sites indicated that the
canonical GRE with a consensus 15-bp motif composed of two pseudopalindromic 6 base pair
half sites separated by a 3 base pair spacer was the most prevalent motif in all three gene

117
classes and at Hic-5 blocked and non-blocked GBR. However, a unique DNA binding sequence
was not found at the Hic-5 blocked GBR or the GBR near the block genes compared to the ind
or mod genes.
Analysis of the DNA sequence revealed an enrichment of ETS1 transcription factor motif
at the Hic-5 blocked GBR and GBR near the block genes. Previous research has shown that
ETS1 interacts with other transcription factors including RUNX2, STAT5, and AP-1 (Rameil et
al., 2000; Thomas et al., 1997; Wai et al., 2006). However, there are no previous studies
showing ETS1 interacting with GR or Hic-5. Although motif enrichment for a specific
transcription factor most likely indicates transcription factor binding, ETS1 occupancy by ChIP-
seq with ETS1 antibody should be used to verify the presence of ETS1 at the GBR of Hic-5
blocked genes. Increased ETS1 expression and transcriptional activity has been shown to
promote aggressive and castrate-resistant phenotype in prostate cancer cells (Smith et al.,
2012). Interestingly, enhanced GR activity has also been implicated in progression of castrate-
resistant prostate cancer (Arora et al., 2013). Hic-5 has also been linked to prostate cancer and
tumorigenesis (Li et al., 2011). Further investigation to determine whether ETS1 can recruit GR
or Hic-5 to the GBR or establish an accessible chromatin with CHD9 or BRM for GR binding
may lead to some interesting clinical applications especially in prostate cancer.
By investigating a unique mechanism of coregulator action by Hic-5 regulating GR
binding site selection, my hope was to contribute to our overall understanding of gene
transcriptional regulation. Studying how Hic-5 prevents the binding of GR to the DNA for a
subset of GC-regulated genes, I uncovered a novel mechanism of action by a coregulator
protein. Although the focus of my dissertation was on the block gene class for Hic-5, the results
of my study may be applied broadly to other coregulators. Blocked expression for a subset of
GC-regulated genes also occurs with other coregulators such as cell cycle and apoptosis
regulator 1 (CCAR1), cell cycle and apoptosis regulator 2 (CCAR2), calcium-binding and coiled-
coil domain 1 (CALCOCO1), and zinc finger protein 282 (ZNF282) (Wu et al., 2014).

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Additionally, chromatin remodeler and chromatin conformation requirements along with distinct
DNA sequence requirements can probably be determined in other subsets of GC-regulated
genes including the ind and mod genes. The multiple biological roles of coregulator proteins and
the coordinated mechanisms of action between the coregulators are yet to be fully understood.
However, with emerging scientific technologies and emphasis on genome-wide experiments,
the future is bright in not only expanding our understanding of the diverse roles of coregulators,
but also honing in on therapeutic pathways involving coregulator proteins.

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Abstract (if available)

Abstract The steroid hormone-activated glucocorticoid receptor (GR) regulates cellular stress pathways by binding to genomic regulatory elements of target genes and recruiting coregulator proteins to remodel chromatin and regulate transcription complex assembly. The coregulator hydrogen peroxide-inducible clone 5 (Hic-5) is required for glucocorticoid regulation of some genes but not others and blocks the regulation of a third gene set by inhibiting GR binding. How Hic-5 exerts these gene-specific effects and specifically how it blocks GR binding to the GR binding regions (GBR) of some genes but not others is unclear. Here I report that site-specific blocking of GR binding is due to gene-specific requirements for ATP-dependent chromatin remodeling enzymes. By depletion of 11 different chromatin remodelers, we found that ATPases chromodomain helicase DNA-binding protein 9 (CHD9) and Brahma homologue (BRM, a product of the SMARCA2 gene) are required for GC-regulated expression of the blocked genes but not for other GC-regulated genes. Hic-5 selectively inhibits GR interaction with CHD9 and BRM, thereby blocking chromatin remodeling and robust GR binding at GR-binding sites associated with blocked genes. Furthermore, I identify specific differences in chromatin conformation, chromatin remodeler requirements, and local DNA sequence motifs that distinguish GR binding regions (GBR) at Hic-5 blocked genes from GBR at other GC-regulated genes. Genome-wide assessment of GR occupancy with ChIP-seq shows that blocked GBR generally require CHD9 and BRM for GR occupancy in contrast to GBR that are not blocked by Hic-5 and that Hic-5 blocked GBR are enriched near Hic-5 blocked GR target genes but not near GR target genes that are not blocked by Hic-5. Hic-5 blocked GBR are in a closed conformation prior to Hic-5 depletion, and require Hic-5 depletion and glucocorticoid treatment to create an open conformation necessary for GR occupancy. Additionally, ETS1 transcription factor binding motif was enriched near blocked GBR and blocked genes but not near non-blocked GBR or non-blocked GR target genes. Thus, Hic-5 regulates GR binding site selection by a novel mechanism, exploiting gene-specific requirements to selectively influence DNA occupancy and gene regulation by a transcription factor.

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Asset Metadata

Creator Lee, Brian Hae Kang (author)

Core Title The role of Hic-5 in glucocorticoid receptor binding to chromatin

School Keck School of Medicine

Degree Doctor of Philosophy

Degree Program Cancer Biology and Genomics

Publication Date 03/14/2018

Defense Date 02/15/2018

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

Tag BRM,CHD9,chromatin,chromatin remodeling enzyme,coregulator,glucocorticoid receptor,Hic-5,OAI-PMH Harvest,steroid hormone,TGFB1I1,Transcription

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Frenkel, Baruch (committee chair), Bajpai, Ruchi (committee member), Stallcup, Michael (committee member)

Creator Email brianhkl@gmail.com,leebh@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c40-485747

Unique identifier UC11267035

Identifier etd-LeeBrianHa-6110.pdf (filename),usctheses-c40-485747 (legacy record id)

Legacy Identifier etd-LeeBrianHa-6110.pdf

Dmrecord 485747

Document Type Dissertation

Rights Lee, Brian Hae Kang

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