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Epigenetic regulation of non CPG island gene promoters
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Epigenetic regulation of non CPG island gene promoters
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Content
EPIGENETIC REGULATION OF NON CpG ISLAND GENE PROMOTERS
by
Connie C. Cortez
_____________________________________________________________________
A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(BIOCHEMISTRY AND MOLECULAR BIOLOGY)
December 2008
Copyright 2008 Connie C. Cortez
ii
DEDICATION
This thesis is dedicated the sources of my strength and inspiration: God, for consistently
amazing me with all his creations, my parents, who have always made my education a
priority, to my sister and brother, for being wonderful role models, and of course, my
sweet husband, for his support, patience, and ability to understand me. I also could not
have done this without the love and support of my extended family and friends.
iii
ACKNOWLEDGEMENTS
There are those that have helped me get to this point in my scientific career, who without,
none of this would have been possible: Dr. Peter A. Jones, my thesis advisor, for teaching
me how to pay attention to detail, for encouraging me to think independently, for helping
me with my writing and for supporting me during those times when experiments just
wouldn’t work.
Drs. Louis Dubeau, Judd Rice, Robert Stellwagen, and Austin Mirchef, for their scientific
guidance. Drs. Gangning Liang and Tina B. Miranda, for being wonderful role models
and friends as well as helping me to grow as a scientist, and of course for their technical
assistance. Drs. Gerda Egger and Anna Van Reitschoten Coyajee for their scientific
insights and willingness to help me with my thesis work.
Past and current members of the Jones lab and other USC labs: Masanobu Abe, Ana
Aparicio, Jonathan Cheng, Yoshitomo Chihara, Jody Chuang, Sonia Escobar, Merhnaz
Fatemi, Einav Nili Gal-Yam, Shinwu Jeong, Theresa K Kelly, Tony Li, Joy Lin,
Yoshimasa Saito, Daiya Takai, Yvonne C. Tsai, Dan Weisenberger, Phillippa Oakford
Taberlay, Christine Yoo, Xiangning Qiu, Flora Han, Jennifer Sims, Shikhar Sharma,
Omar Khalid, Li Jia, and Kwangho Lee for help with experiments, in boosting moral, and
for an overall great experience here at USC. Erika Wolff was also instrumental in
completing the thesis writing as without her it would have been a mess.
Jones lab meeting participants, in particular, Drs. Gerry Coetzee, Peter Laird, and Allen
Yang for their constructive criticisms and comments.
iv
TABLE OF CONTENTS
DEDICATION ii
ACKNOWLEDGMENTS iii
LIST OF FIGURES v
ABSTRACT viii
CHAPTER 1: EPIGENETIC REGULATION OF GENE EXPRESSION AND
EPIGENETIC THERAPY 1
Table 1.1 Epigenetic drugs used in the clinic 14
CHAPTER 2: RUNX3: DNA METHYLATION ANALYSIS OF TWO
PROMOTERS 26
CHAPTER 3: LAMB3: DNA METHYLATION OF A NON CPG ISLAND
GENE PROMOTER AND ITS IMPLICATIONS IN CANCER 65
CHAPTER 4: S-ADENOSYLHOMOCYSTEINE HYDROLASE INHIBITORS
AND THEIR EFFECTS ON THE EPIGENOME 91
CHAPTER 5: SUMMARY AND CONCLUSIONS 121
REFERENCES 127
v
LIST OF FIGURES
Figure 1.1 Post-translational histone modifications on histone tails 3
Figure 2.1 Map of RUNX3 gene 27
Figure 2.2 Protein schematic of RUNX3 28
Figure 2.3 DNA methylation status of RUNX3 P1 and P2 in normal tissues 38
Figure 2.4 Bisulfite genomic sequencing of RUNX3 P1 40
Figure 2.5 Imprinting analysis of RUNX3 P1 42
Figure 2.6 Bisulfite sequencing of EBV transformed B cells at RUNX3 P1 44
Figure 2.7 Determination of monoallelic expression of RUNX3 P1 46
Figure 2.8 Bisulfite genomic sequencing of RUNX3 P1 in fractionated
populations of white blood cells 48
Figure 2.9 RUNX3 expression in normal cells 50
Figure 2.10 DNA methylation status of both RUNX3 promoters in bladder, colon
and prostate cancer cell lines 51
Figure 2.11 DNA methylation status of RUNX3 in normal bladder tissue, primary
tumors and matched adjacent normal tissues 54
Figure 2.12 RUNX3 P1 expression by real time RT-PCR and analysis of histone
H3 acetylation levels by ChIP analysis 56
Figure 2.13 Induction of RUNX3 in T24 and LD419 cells after 5-Aza-CdR
treatment 58
Figure 2.14 Possible explanations for demethylated subclones 61
Figure 3.1 Schematic of laminin-5 in the basement membrane and in detail 66
Figure 3.2 Bisulfite genomic sequencing of the LAMB3 promoter in normal
tissues 75
vi
Figure 3.3 DNA methylation analysis in sperm and testis by bisulfite genomic
sequencing of DNA at the LAMB3 and SERPINB5 promoters 77
Figure 3.4 Expression of LAMB3 in normal tissues 79
Figure 3.5 LAMB3 methylation and expression in cell lines 81
Figure 3.6 Expression and DNA methylation analysis of LAMB3 in the RT4
cell line and the UM-UC-3 cell line after 5-Aza-CdR treatment 83
Figure 3.7 LAMB3 ChIP analysis of histone H3 lysine 9/14 acetylation, lysine 4
dimethylation, and lysine 9 trimethylation in the RT4 and UM-UC-3
cell lines 85
Figure 3.8 DNA methylation status of LAMB3 methylation in bladder cancer
matched sets 87
Figure 4.1 Methyltransferase inhibitors 93
Figure 4.2 The effects of methyltransferase inhibitors on EZH2 and H3K27me3 100
Figure 4.3 The effects for DZNep on global histone methylation 102
Figure 4.4 Map of KRT7 104
Figure 4.5 KRT7 P1 and P2 expression after drug treatments 105
Figure 4.6 KRT7 P2 transcript occurs in immortalized keratinocytes and
in epithelial bladder cancer cells 107
Figure 4.7 KRT7 P1 and P2 DNA methylation status after 5-Aza-CdR or
DZNep treatment 108
Figure 4.8 KRT7 P1 and P2 ChIP analysis 110
Figure 4.9 Microarray validation 112
Figure 4.10 Scatter plots from expression microarray 113
Figure 4.11 Venn diagram showing the overlap of genes upregulated in MCF7
cells treated with 5-Aza-CdR or DZNep 114
vii
Figure 4.12 EASE analysis showing the categories of genes turned on by DZNep
or 5-Aza-CdR 115
Figure 4.13 Comparison of the heritability of gene expression upon treatment of
MCF7 cells with either 5-Aza-CdR or DZNep 117
viii
ABSTRACT
DNA methylation and post-translational modifications of histones can specify
transcriptional competency in both normal and cancer cells. However these epigenetic
processes have largely been studied in the context of genes that contain CpG islands in
their promoters. Therefore the epigenetic regulation of genes without CpG islands was
explored in order to understand how epigenetics controls the expression of the remainder
of the genome and provide insight into what aberrancies occur at these regions during
cancer. Additionally, because there is an ongoing quest to find drugs that target the
aberrancies that occur in the epigenome during cancer, methyltransferase inhibitors were
also studied to test their potential as an epigenetic therapeutic.
Two genes, RUNX3 and LAMB3, were studied because they both contain non
CpG island promoters. However, RUNX3 has two contrasting promoters: a non CpG
island promoter that was found to have a cell type specific DNA methylation pattern and
a second CpG rich promoter that was found to have a cancer specific DNA methylation
pattern in bladder cancer. LAMB3 also displayed a tissue specific methylation and
expression pattern in normal tissues. Interestingly, the non CpG island promoters of
RUNX3 and LAMB3 could be induced in bladder cancer cell lines by the inhibition of
DNA methylation with 5-Aza-2´-deoxycytidine, supporting the role DNA methylation
has in their transcriptional control, regardless of CpG density. Additionally, chromatin
immunoprecipitation performed at the non CpG island promoters of both genes
demonstrated that DNA methylation and histone modifications together can regulate
ix
RUNX3 and LAMB3. Taken together, these data demonstrate the type of control
epigenetic mechanisms have in the regulation of non CpG island promoters.
The histone methylation transferase inhibitor, 3-deazaneplanocin, was
investigated to better understand its effects on cancer cells and was shown to be able to
inhibit many types of histone methylation marks. Treatment of cancer cells with 3-
deazaneplanocin was able to induce expression from the non CpG island promoters of
LAMB3 and KRT7 as well as many genes that could not be induced by 5-Aza-2´-
deoxycytidine, suggesting a combination treatment of these two types of drugs may be
promising in cancer treatment.
1
CHAPTER 1
EPIGENETIC REGULATION OF GENE EXPRESSION AND EPIGENETIC
THERAPY
Introduction to Chromatin Structure
The organization of chromatin has been intensely studied and now the focus in the
field of epigenetics has shifted to understanding the biological relevance and function of
chromatin structure. Epigenetics, which is defined as the study of heritable changes in
gene expression that occur without a change in the DNA sequence, provides insight into
the extent by which chromatin structure exerts control on transcriptional regulation.
Interpreting the patterns of post-translational histone modifications as well as DNA
methylation and how these epigenetic mechanisms contribute to gene expression in a
normal state and in cancer are key to developing drugs that can reverse abnormalities that
occur during tumorigenesis.
Chromatin is comprised of DNA, histone proteins and non-histone proteins. The
fundamental repeating unit of chromatin is the nucleosome, which consists of an octamer
of histones with two each of the four small and highly basic histones (H3, H4, H2A, and
H2B) (Kornberg, 1974). Approximately 146 bp of DNA are wrapped twice around each
histone core providing a means for higher order packaging of DNA in the nucleus. The
histone amino terminal tails that project out of the nucleosome core are subject to many
post-translational modifications such as phosphorylation, ubiquitination, sumoylation,
2
acetylation and methylation on specific amino acid residues (Strahl and Allis, 2000;
Berger, 2002). The most highly studied modifications are the acetylation and
methylation of histones H3 and H4 (Figure 1.1).
Post-translational Histone Modifications
Acetylation by histone acetyl transferases (HATs) occurs on the lysine residues of
histone tails and is strongly correlated with active gene expression. The basic charges of
the histone tails become neutralized upon acetylation. This causes increased accessibility
for further modifications or access to the DNA for binding factors and transcriptional
machinery (Hebbes et al., 1992; Turner, 1993; Kouzarides, 2007). Unlike acetylation,
methylation of histones does not change the charge of the histone tails (Rice and Allis,
2001). Lysine residues can accept up to three methyl groups, which are added by various
histone methyltransferases (HMTs). The degree of methylation is informative for both
the state of gene activity as well as which proteins/complexes might bind and read the
message displayed by those marks (Fischle et al., 2003; Santos-Rosa and Caldas, 2005).
Methylated lysine residues may constitute either active or inactive marks. Active marks
include histone H3 lysine 4 (H3K4), lysine 36 (H3K36) and lysine 79 (H3K79) (Liang et
al., 2004; Bannister et al., 2005; Shilatifard, 2006). The methylation marks on lysines 9
and 27 on histone H3 and lysine 20 of histone H4 are associated with an inactive
chromatin state (Peters et al., 2002; Peters et al., 2003; Sims et al., 2006). Interestingly,
there is cross regulation between different marks such as the competition for lysine 9 on
3
Figure 1.1. Post-translational histone modifications on histone tails. Modifications
made on the N-terminal tails of histones are important in establishing the activity state of
chromatin. Many modifications are possible, however, only acetylation and methylation
of a subset of lysine residues are depicted here for simplification. Active acetylation or
methylation marks (green triangles or squares), can act to “loosen” chromatin to allow for
access of transcriptional machinery while also serving as docking points for nucleosome
remodeling complexes. Conversely, inactive marks such as methylation of specific
residues can cause an inactive conformation of chromatin and can recruit repressive
complexes.
SGRGKGGKGLGKGGAKRHRKVL
ARTKYTARKSTGGKAPRKQLATKAARKSAPATGGVKK
Acetylation (Active)
Methylation (Active)
Methylation (Inactive)
H4
H3
4
histone H3 between an inactive methyl mark and acetylation (Nicolas et al., 2003;
Latham and Dent, 2007). The distinct patterns of post-translational modifications make
up the "histone code" and the precise combinations determine how the chromatin is read
(Jenuwein and Allis, 2001).
Nucleosome Positioning/Occupancy
The position of nucleosomes on the DNA further adds to the complexity of
chromatin structure. Nucleosome positioning and occupancy can also play a key role in
regulating gene expression and the presence of a nucleosome at the transcription start site
is commonly seen in inactive genes (Schones et al., 2008). Studies have shown evidence
of the loss of a nucleosome directly upstream of the transcription start site upon gene
activation. This may allow greater access for binding of transcription complexes or
factors (Shivaswamy et al., 2008). It has been shown that a promoter of a gene with a
basal level of transcription can already be depleted of nucleosomes, which allows for
quick induction upon stimulation (Gal-Yam et al., 2006). Also, the reactivation of a
completely silenced gene is associated with nucleosome loss (Lin et al., 2007). These
studies demonstrate the importance of nucleosomes in gene regulation.
Mammalian DNA methylation
DNA methylation influences gene regulation in concert with histone
modifications and nucleosome positioning (Vaissiere et al., 2008). DNA methylation at
the transcriptional start sites of genes is associated with inactivity and is important in
5
imprinting, X inactivation and the silencing of retrotransposons. The 5 carbon on the
cytosine ring in DNA can be modified by the placement of a methyl group by DNA
methyltransferases (DNMTs). DNMT1 is referred to as the "maintenance" methylase due
to its preference for hemimethylated CpG sites in DNA (Pradhan et al., 1999). DNMT3a
and DNMT3b are considered to be de novo methylases because they can methylate
unmethylated DNA (Okano et al., 1999; Pradhan et al., 1999). However, all three
DNMTs have been shown to act cooperatively and the functional differences between the
methylases may to a large extent be due to the genomic regions that they act upon (Liang
et al., 2002; El-Osta, 2003).
Methylation occurs in the context of CpG dinucleotides, which are
underrepresented in the genome possibly due to evolutionary depletion (Ehrlich and
Wang, 1981). Regions of high CpG content are termed "CpG islands" and are found at
the promoters of more than 50% of genes in the genome. CpG islands are often located
at the promoter regions of housekeeping genes in an unmethylated state (Razin and
Shemer, 1995). The DNA methylation mark can act both directly and indirectly to silence
a gene by either inhibiting the binding of transcription factors or by possibly recruiting
methyl-binding domain proteins (MBDs), which further recruit histone deacetylases
(HDACs) (Wade et al., 1998).
Tissue Specific DNA Methylation
An increasing number of studies have reported evidence of tissue specific DNA
methylation contributing to tissue specific gene expression. One of the first individual
6
genes studied, SERPINB5, showed a lack of DNA methylation in normal expressing cells
at the promoter region accompanied by acetylated histones and an accessible chromatin
structure (Futscher et al., 2002). However, in normal cells not expressing SERPINB5, the
promoter regions were highly methylated and had hypoacetylated histones indicative of
an inaccessible chromatin structure. This monumental study gave way to further
investigation of tissue specific genes and how they might be epigenetically regulated.
Since then, many genome wide studies have strived to analyze CpG islands en masse to
determine which display tissue specific DNA methylation patterns. In one genome wide
study, a class of tissue specific genes containing CpG islands within their promoters were
discovered to be highly methylated in non-expressing tissues and unmethylated in tissues
in which they were expressed (Shen et al., 2007). Yet another genome wide study
expanded the number of CpG islands of genes that were identified as having tissue
specific methylation patterns (Illingworth et al., 2008) Studies such as these provide
evidence that DNA methylation aids in the maintenance of cell identity by silencing
certain tissue-specific genes with CpG island promoters. Many of these genome wide
studies aim to compare DNA methylation in different tissues across promoters with
different CpG densities and further correlate this data to expression.
A by-product of the genome wide studies has been the information obtained about
non CpG islands. It was found that non CpG island genes can show an inverse
correlation between gene expression and DNA methylation (Eckhardt et al., 2006).
During the DNA methylation profiling study of human chromosomes 6, 20, and 22,
specifically the non CpG island OSM (oncostatin M) gene was shown to have a tissue
7
specific methylation and expression pattern (Eckhardt et al., 2006). This study indicates
that DNA methylation may also be a mechanism of regulation for non CpG island
promoter genes as well. Overall, the role of DNA methylation in the tissue specific
expression of genes of CpG islands is gaining acceptance, and for non CpG island genes,
the role of DNA methylation is beginning to be understood.
Epigenetic regulation of genes with non CpG island promoters
Several studies of the epigenetic regulation of non CpG island promoter genes
have involved genes in immunological response pathways. For instance when naïve T
cells are stimulated the demethylation of one CpG site at the IL2 promoter occurs
followed by the recruitment of Oct-1 and changes in histone modifications, resulting in
the increased expression of IL2 (Bruniquel and Schwartz, 2003). Interestingly the
epigenetic changes that occur at this gene can serve as an epigenetic memory for future
events, meaning that subsequent T cell activations become quicker because the IL2
promoter is in a primed conformation for transcription (Murayama et al.,
2006).Additionally, a study of the non CpG island IL-1α gene demonstrated that active
transcription is associated with an unmethylated, nucleosome free promoter accompanied
by active chromatin marks (van Rietschoten et al., 2008).
There are also non-immunological genes without CpG islands that have been
shown to be regulated by DNA methylation. The glial fibrillary acidic protein (GFAP)
promoter contains a STAT3 binding site that when methylated inhibits STAT3 binding
and results in repression of the gene (Takizawa et al., 2001). Another study found there
8
to be two classes of non CpG island promoter genes that could be activated by different
mechanisms (Lande-Diner et al., 2007). The first class involved the non CpG island
promoters genes Rhox5 and Slpi, which are normally methylated and not expressed in
fibroblasts. However, these genes showed a 40 fold difference in induction in a
DNMT1 knock out (KO) fibroblast cell line, suggesting that DNA methylation was the
main mechanism repressing these genes in normal fibroblasts (Lande-Diner et al., 2007).
The second class of genes, also methylated in normal fibroblasts, was not found to be re-
expressed in the KO cell line. However, only upon administration of an HDAC inhibitor
was there activation of their expression. This suggests that the second class of genes are
epigenetically regulated perhaps by histone acetylation, not solely by DNA methylation.
Again, these data show that non CpG islands can be influenced by DNA methylation and
histone modifications, either together or separately, which is similar to what has been
observed with CpG rich genes.
MicroRNAs
MicroRNAs (miRs) are another mechanism used by the cell to regulate the
expression of genes involved in differentiation, cell proliferation and apoptosis (Zhang et
al., 2007). They are short RNAs 19-24 nucleotides in length that often bind to the 3'UTR
of their target mRNA to either inhibit that mRNA's translation or cause its degradation
(He and Hannon, 2004). MiR expression profiles differ depending on cell type and like
DNA methylation, they help to establish the cells identity. Currently more than 400
human miRs have been experimentally identified and are proposed to regulate more than
9
30% of all mRNAs post-transcriptionally (He and Hannon, 2004; Griffiths-Jones et al.,
2006).
Epigenetic Changes in Cancer
Epimutations in cancer can result in the activation of oncogenes, the silencing of
tumor suppressors, and ultimately in the cell's ability to proliferate uncontrollably. These
changes are often linked to the presence of altered levels of chromatin modifying
enzymes and a shift in the genome-wide distribution of DNA methylation. Changes in
histone marks work together with DNA methylation or independently to silence gene
expression depending on the region of chromatin and the type of gene. Advances in our
understanding of how these abnormalities occur will help in designing and improving
drugs to target the factors that cause these changes during tumorigenesis.
Altered activity of the histone lysine methyltransferases can contribute to the
deviant histone methylation patterns found in cancer. For example, histone lysine
methylation on histone H3K9 and H3K27 are normally present at transcriptionally
inactive or heterochromatic regions, yet they can be found at genes that are aberrantly
repressed in cancer cells (Cao et al., 2002; Nguyen et al., 2002). The methyltransferase
MLL, which methylates H3K4, is involved in translocations that lead to the inappropriate
expression of various homeotic (Hox) genes, which contributes to leukemic progression
(Krivtsov and Armstrong, 2007). Methyltransferases within complexes well known for
their suppressive activities are also up-regulated in cancer.
10
The Polycomb group (PcG) complexes are chromatin modifiers that are crucial to
development, and have been implicated in the development of cancer (Tonini et al.,
2008). These negative regulators of gene expression are very important in sustaining the
repressive state of their target genes through the cell cycle (Kingston et al., 1996). Two
of the PcG repressive complexes (PRC1 and PRC2) have both been shown to be involved
in various cancers. Enhancer of zeste homologue 2 (EZH2), a component of PRC2 with
H3K27 methyltransferase activity, is upregulated in mantle cell lymphoma, breast and
prostate cancer (Visser et al., 2001; Kleer et al., 2003; Rhodes et al., 2003). RING1, a
component of PRC1 that aids in the ubiquitylation of histone H2A lysine 119, is
upregulated in prostate cancer (van Leenders et al., 2007).
The demethylation of histones is important in transcriptional regulation. Histone
lysine methylation had been previously thought to be a very stable mark. However, the
discovery of LSD1, a demethylase of mono- and dimethylated histone H3K4, showed
that these chromatin marks are reversible (Shi et al., 2004). LSD1's mechanism of action
is through the amine oxidation of the methylated histone H3K4. Several histone lysine
demethylases have been found since LSD1, including the Jumonji C domain (JmjC)
proteins, which can specifically demethylate mono-, di- and trimethylated lysines
(Wissmann et al., 2007). Histone demethylases have been found to play a role in cancer
progression as seen with JMJD2A, JMJD2B, and JMJD2C, which are expressed at high
levels in prostate cancer (Cloos et al., 2006).
Interestingly, LSD1 has been found to associate with HDACs, therefore HDAC
inhibitors can potentially affect the function of demethylases (Lee et al., 2006). Currently,
11
histone demethylases have been identified which demethylate both active and inactive
marks thereby functioning as both co-repressors and co-activators (Agger et al., 2008).
Therapeutic inhibition of specific demethylases may be a possible direction for the
treatment of cancer, however there is still much to uncover about the precise functions
and associations of histone demethylases.
The acetylation of histones is held at equilibrium by the action of HATs and
HDACs and an imbalance of one or the other enzyme can lead to phenotypic changes in
the cell. Alterations in HAT activity have been found in cancer, stressing the importance
of strict regulation of histone acetylation. For example, a translocation involving MOZ
and p300, both with HAT activity, results in a fusion protein and is associated in
leukemogenesis (Kitabayashi et al., 2001). Errant HAT complexes such as these disturb
normal epigenetic processes through inappropriate acetylation, altering chromatin
organization and gene expression patterns. Inappropriate deacetylation can also
contribute to cancer progression. HDACs are upregulated in various types of cancer,
such as gastric, prostate, oral squamous cell, and lung (Halkidou et al., 2004; Bartling et
al., 2005; Song et al., 2005; Sakuma et al., 2006). Over-expression of HDACs can also
lead to the transcriptional inactivation of tumor suppressors, such as p53 (Kim et al.,
2001).
DNA methylation patterns are altered in the progression of cancer. Both the
hypomethylation and hypermethylation of different regions of the genome play roles in
contributing to tumorigenesis. During tumorigenesis, a genome-wide demethylation
occurs and this can promote genomic instability possibly by activating silenced
12
retrotransposons (Ehrlich, 2002). Global demethylation of repetitive sequences such as
satellite DNAs can lead to increased chromosomal rearrangements further adding to
genomic instability (Dunn, 2003). It is also possible that demethylation could lead to the
activation proto-oncogenes, such as the R-Ras activation in gastric cancer (Fruhwald and
Plass, 2002; Nishigaki et al., 2005).
Focal hypermethylation of CpG islands has been intensively studied in cancer.
The list of genes found to have increased levels of methylation in their promoter regions
accompanied by decreased expression in cancer has grown rapidly. Nearly all types of
cancers have transcriptional inactivation of tumor suppressor genes due to DNA
hypermethylation (Jones and Baylin, 2007). However, the exact mechanism responsible
for the appearance of DNA methylation in a given promoter is not fully understood.
Cancer cells can attain a growth advantage through the hypermethylation and silencing of
genes that are involved in cell cycle regulation, DNA repair, cell signaling, and
apoptosis. Additionally, the levels of DNMTs may also be important (Sigalotti et al.,
2007). Over-expression of the DNA methyltransferases 1 and 3A was found in the bone
marrow of patients with myelodysplastic syndrome (MDS) (Langer et al., 2005).
Upregulation of DNMTs has also been shown in prostate cancer cell lines and tissues
(Patra et al., 2002).
MiRs have different expression profiles in cancer (Lu et al., 2005). If a miR is
found to be downregulated in cancer then its potential function as a tumor suppressor has
been lost. Conversely, if it is upregulated the miR may be acting as an oncogene by
downregulating a tumor suppressor gene. Little is known about the regulation of miRs,
13
however it has now been shown that miRs can be regulated by DNA methylation and
histone modifications as shown with miR 127 (Saito et al., 2006). In this study miR 127
was reactivated in a cancer cell line upon treatment of both a DNA methylation and
HDAC inhibitor. This demonstrates that miRs can be potential targets for epigenetic
therapy.
Cancer and Epigenetic Therapy
Epigenetic errors in cancer, unlike genetic lesions, can be reversed relatively
easily through chemotherapeutic intervention, which makes epigenetic therapy
promising. The goal of epigenetic therapy is to target the chromatin in rapidly dividing
tumor cells and return it to a more "normal state" while only mildly disturbing the
epigenome of healthy cells. This section will focus on the epigenetic drugs that are
currently used in the clinic (Table 1) and their effects on cancer cells. It is important to
note both their benefits and shortcomings so that improvements can be made in the next
generation of epigenetic drug therapies.
The nucleoside analogues 5-azacytidine (5-Aza-CR) and 5-aza-2´-deoxycytidine
(5-Aza-CdR), known clinically as azacitadine (Vidaza) and decitabine (Dacogen),
respectively, are FDA approved demethylating agents used to treat myelodysplastic
syndrome (MDS) (Ghoshal and Bai, 2007). They differ from cytosine by a nitrogen
substitution at the 5-carbon position. During replication these drugs are incorporated into
DNA and their modified cytosine rings inhibit methylation by trapping DNMTs, thereby
14
Drug Name Cancer
DNA Methylation Inhibitors
5-azacytidine (FDA approved) MDS, AML, CML
5-aza-2’-deoxycytidine (FDA approved) AML, CML, MDS
MG98 Renal cell carcinoma
RG108 Colon cancer cell line
Procainamide Colon cancer cell line
HDAC Inhibitors
SAHA (FDA approved) CTCL, various solid tumors
PXD101 Various solid tumors
LBH589 CTCL
Depsipeptide Multiple cancer cell lines, MDS, AML
Phenylbutyrate MDS
Valproic Acid Neuroblastoma cells
MS-275 Prostate cancer cell lines, various sold tumors
and lymphoid malignancies
CI-994 Various solid tumors
Table 1.1. Epigenetic Drugs Used in the Clinic
15
depleting the cell of these enzymes and resulting in the reduced methylation of cytosines
in DNA synthesized after drug treatment (Jones and Taylor, 1980; Momparler, 2005). In
the study leading to the FDA approval of 5-Aza-CR, there was a 60% response rate of the
patients with MDS (Silverman et al., 2002). It was also shown that 5-Aza-CR prolongs
survival rate in high risk MDS patients (Itzykson et al., 2008). Encouraging results were
also obtained when MDS patients were treated with 5-Aza-CdR and there was a 70%
overall response rate (Kantarjian et al., 2007). In addition to MDS, these drugs have
proven to be useful in other hematological malignancies such as acute myeloid leukemia
(AML) and chronic myeloid leukemia (CML) (Plimack et al., 2007).
These drugs potentially act by restoring normal cellular functions by allowing
aberrantly hypermethylated tumor suppressor genes to become re-expressed, although the
relationship between therapeutic activity and DNA methylation inhibition has not been
formally proven. They have shown promising response rates in patients with MDS and
CML along with a reversal of p15 hypermethylation in bone marrow (Daskalakis et al.,
2002; Yang et al., 2006; Aribi et al., 2007). Interestingly, 5-aza-CdR can restore drug
sensitivity to cells that have become unresponsive to chemotherapy. For example,
treatment of melanoma cell lines with 5-Aza-CdR restores sensitivity to cells that are
unresponsive to chemotherapy by aiding in the re-expression of a crucial player in the
apoptotic pathway, APAF-1 (Soengas et al., 2001).
The use of these drugs raises questions regarding their potential to affect non-
cancerous cells epigenetically. However, normal cells divide at a slower rate than
malignant cells and incorporate less of these drugs into their DNA resulting in less of an
16
effect on DNA methylation. Also, long-term negative effects of DNA methylation
inhibitors in patients have not been found to date (Yang et al., 2003). Drawbacks to these
drugs are their chemical and in vivo labilities as well as their acute hematological
toxicities. A next generation DNA methylation inhibitor, such as zebularine, might
possibly overcome these problems (Marquez et al., 2005; Yoo et al., 2008).
Other small molecule inhibitors such as RG108 or MG98 are not incorporated
into DNA but instead bind to the catalytic site of DNMTs thereby causing inhibition of
DNA methylation. RG108 (N-Phthalyl-1-tryptophan) has been shown to be minimally
toxic in colon cancer cell lines and was successful at inhibiting DNMTs (Brueckner et al.,
2005; Zheng et al., 2008). The antisense oligonucleotide MG98 (2´-O-CH3-substituted
phosphorothioate oligo deoxynucleotide) targets the 3' UTR of DNMT1 (Klisovic et al.,
2008) and can cause a methylation decrease in cell lines and animal models. Phase I trials
with MG98 did show DNA demethylation in patients, however the Phase II trials had no
effect on methylation reduction in patients with renal cell carcinoma (Winquist et al.,
2006; Gronbaek et al., 2007). Although procainamide (4-Amino-N-(2-
diethylaminoethyl)benzamide hydrochloride) is FDA approved for the use of cardiac
arrhythmias, it can also reduce DNMT1's affinity for both DNA and S-adenosyl-
methionine in a colon cancer cell line causing a decrease in DNA methylation (Lee et al.,
2006). Generally, these non-nucleoside analogue inhibitors are not are not as potent as
the nucleoside analogues and therefore the need for improvement for these drugs still
exists (Chuang et al., 2005).
17
DNA methylation inhibitors are successful in affecting one epigenetic pathway
that leads to the progression of cancer. HDAC inhibitors have also been proven to be
useful in cancer treatment by allowing the re-establishment of acetylation to reactivate
silenced genes (Karagiannis and El-Osta, 2006). HDAC inhibitors are divided into 4
groups based on their structures: hydroxamic acids, cyclic peptides, short chain fatty
acids, and benzamides. There are 18 HDAC isoenzymes that have been categorized into
4 classes. The challenge is in designing HDAC isoform-specific inhibitors and
determining their potential clinical advantages over general inhibitors (Zheng et al.,
2008). The specificities of the HDAC inhibitors used in the clinic vary from one to three
classes of HDACs (Gronbaek et al., 2007). HDAC inhibitors have pleiotropic effects
including inhibition of angiogenesis, induction of apoptosis and cell cycle arrest (Stearns
et al., 2007).
The hydroxamic acid HDAC inhibitors have been successful in treating both
hematologic malignancies and solid tumors. X-ray crystallography has shown that the
catalytic site of HDACs contains a zinc atom. The hydroxamic acid moiety of these
HDAC inhibitors can fit into the catalytic site and bind to the zinc atom thereby
inhibiting the HDAC (Marks et al., 2000). Suberoylanilide hydroxamic acid (SAHA;
vorinostat), a general inhibitor, targets HDACs from Class I and Class II by binding to
the active site of the enzyme (Finnin et al., 1999). SAHA can be administered orally, is
minimally toxic and has been FDA approved for the treatment of cutaneous T-cell
lymphoma (CTCL). The overall response rate in a recent CTCL Phase IIb trial was 30%
and those that did not respond still benefited from relief of pruritus early in the trial
18
(Olsen et al., 2007). SAHA also is in phase II trials to treat solid tumors (Yoo and Jones,
2006; Gronbaek et al., 2007; Xu et al., 2007). A recent use of SAHA in women with a
recurrence of ovarian cancer showed a progression-free survival over 6 months (Modesitt
et al., 2008). Other hydroxamic acids, PXD101 ((E)-N-hydroxy-3-[3-
(phenylsulfamoyl)phenyl]prop-2-enamide) and LBH589 ((E)-N-hydroxy-3-[4-[[2-(2-
methyl-1H-indol-3-yl)ethylamino]methyl]phenyl]pro p-2-enamide), have also been
evaluated in clinical trials. PXD101 treatment of patients with advanced refractory solid
tumors was shown to cause an increase in acetylation in their peripheral blood
mononuclear cells, stabilize their disease, and was well tolerated (Steele et al., 2008).
LBH589 is best known for its role in hyperacetylation of histones H3, H4 and the protein
Hsp90 (George et al., 2005). LBH589 has shown clinical activity in cutaneous T-cell
lymphoma (CTCL) and soon will be studied in chronic myeloid leukemia, and multiple
myeloma (Glaser, 2007).
Depsipeptide (FK228), an example of a cyclic peptide HDAC inhibitor, is more
specific in that it exerts its effect on three of the Class I HDACs (Gronbaek et al., 2007).
Depsipeptide has been recently shown to cause a decrease in methylation of DNA while
increasing acetylation in lung, pancreatic, and colon cancer cell lines, however the
mechanism is not well understood (Wu et al., 2008). Depsipeptide has been shown to
inhibit growth of human prostate cancer cells (Lai, 2008). Concerns have arisen with this
drug's potential cardiac toxicity (Shah et al., 2006), however none was observed in a
phase I clinical trial to treat patients with MDS or AML (Klimek et al., 2008).
19
Short chain fatty acids such as butyrate and valproic acid (2-Propylpentanoic
acid) have the longest history of being used as HDAC inhibitors (Sigalotti et al., 2007).
Valproic acid (VPA), originally used to treat epilepsy, has been used for the last decade
as an anti-cancer drug since it can inhibit proliferation and induces differentiation in
human neuroblastoma cells (Cinatl et al., 2002). VPA is well tolerated, has low toxicity
in adults, and is relatively stable (Blaheta et al., 2005). Phenylbutyrate is in Phase I trials
for MDS and was shown to be safe for treatment of solid tumors (Gore et al., 2002;
Camacho et al., 2007). The shortcoming of these HDAC inhibitors is that a high
concentration of drug is required for efficacy resulting in limited use in the clinic
(Johnstone, 2002).
MS-275 (N-(2-aminophenyl)4-[N-(pyridine-3-yl-methoxycarbonyl)aminomethyl]
benzamide) and CI-994 (N-(2-aminophenyl)-4-acetylaminobenzamide) are two of the
most well known synthetic HDAC inhibitors of the benzamide group. MS-275 can
induce p21 expression and increase acetylation in prostate cancer cell lines and inhibit
tumor growth in mouse xenograft models (Camphausen et al., 2004; Qian et al., 2007).
In Phase I trials patients with a variety of solid tumors and lymphoid malignancies
showed increased levels of acetylation in peripheral blood mononuclear cells and the
drugs were well tolerated (Kummar et al., 2007). CI-994 has undergone Phase I trails
and can be used alone or in combination with other chemotherapeutic drugs to treat solid
tumors in patients (Prakash et al., 2001; Pauer et al., 2004; Zheng et al., 2008). Neither
of these drugs is as potent as the other classes of HDAC inhibitors and seems to have the
greatest effect when used in a combinatorial treatment (Kouraklis and Theocharis, 2006).
20
The future directions for the development of epigenetic drugs will rely on the elucidation
of their mechanisms and the downstream effects of treatment.
Many clinical trials are now studying the combination of either two epigenetic
drugs or a non-epigenetic chemotherapeutic and an epigenetic drug in an effort to
increase response rates and maximize the efficacy of these drugs. Since HDAC inhibitors
work primarily to increase acetylation, they may have a limited effect on genes that have
been silenced by DNA methylation. However, HDAC inhibitors and DNA methylation
inhibitors in combination can work synergistically to cause the re-expression of such
genes. A study on colon cancer cell lines showed genes that were only expressed when
the HDAC inhibitor and 5-Aza-CdR were coupled (Cameron et al., 1999). It was also
found that DNA methyltransferase inhibitors could enhance the anti-tumor effects of
depsipeptide in leukemic cells with the AML/ETO fusion protein (Klisovic et al., 2003).
Also, phenylbutyrate and 5-Aza-CdR have synergistic effects on reducing lung tumor
formation in mice by more than 50% than with 5-Aza-CdR alone (Belinsky et al., 2003).
A similar study in xenograft hepatoma models only showed a decrease in tumor
formation when treated with both SAHA and 5-Aza-CdR (Venturelli et al., 2007). DNA
methylation inhibitors and HDAC inhibitors are now used together in the clinic after
garnering encouraging results in vitro.
For example, in humans a phase I trial involving MDS and AML patients that
were treated with both sodium phenylbutyrate and 5-Aza-CR showed reduced promoter
methylation and increased global histone acetylation. The results from this trial suggest
an increased response rate and it is hypothesized that in a phase II trial, using a longer
21
exposure and lower dose of 5-Aza-CR and HDAC inhibitor could further increase the
response rate (Gore et al., 2006).
Often cells undergoing treatment with one epigenetic drug can have increased
sensitivity to an additional drug. Pretreatment with an HDAC inhibitor can greatly
increase cytotoxicity in various cell lines when followed by subsequent treatment of a
chemotherapeutic drug (Kim et al., 2003). This particular study suggests that pre-
treatment causes the chromatin structure to become more open therefore increasing the
efficiency of the drug to follow. Likewise, cisplatin resistant cells from head and neck
cancer cell lines can be reprogrammed to become responsive after treatment with
phenylbutyrate (Burkitt and Ljungman, 2008). A phase I study in patients with solid
tumors showed that CI-994 can be safely administered with paclitaxel and carboplatin
and can cause a partial or complete response (Pauer et al., 2004). The increased
sensitivity to other drugs after use of an epigenetic drug is encouraging since drug
resistance does present a challenge in effective cancer treatment.
Additionally, it is important to target other enzymes that can disturb the
epigenetic balance during carcinogenesis such as histone methyltransferases. Reagents
that inhibit S-adenosylhomocysteine (AdoHcy) hydrolase lead to an increase in AdoHcy
levels in the cell which inhibits methyltransferases, including histone methyltransferases
(Chiang and Cantoni, 1979; Cools and De Clercq, 1990). While AdoHcy hydrolase
inhibitors such as 3-deazaneplanocin (DZNep) have been used as anti-viral compounds,
how they may be effective in cancer is in need of exploration (Huggins et al., 1999).
22
The Future of Epigenetic Therapy
As the field of epigenetics advances, a better understanding is developing of the
precise mechanisms by which DNA methylation and post-translational histone
modifications play central roles in gene regulation. The therapeutics designed thus far
have had encouraging results in counteracting the epimutations that occur during
tumorigenesis. With the FDA approval of 5-Aza-CR, 5-Aza-CdR and SAHA, the use of
epigenetic drugs has gained momentum and has proven useful in hematological
malignancies and some solid tumors. Additionally, the combinatorial use of DNA
methylation inhibitors and HDAC inhibitors in the clinic is gaining traction due to their
synergistic effects in re-establishing the expression of tumor suppressor genes.
However, much work remains in designing drugs that will be more stable, less toxic and
more specific in their enzyme inhibition. Expanding the use of these drugs to treat more
types of solid tumors should also be possible. Broadening combinatorial drug therapies
to include different permutations of the DNA methylation inhibitors, HDAC inhibitors
and non-epigenetic chemotherapies will also be key in better cancer treatment.
Fortunately, advancements in technology will help to further elucidate the understanding
of epigenetic mechanisms. As a result, drugs that can better target chromatin modifiers
that improperly function during carcinogenesis will be developed. Future epigenetic
drugs can also be designed to target histone methyltransferases, histone demethylases or
other chromatin modifiers not yet discovered. The rising interest in epigenetics research
should therefore lead to improved cancer treatment.
23
Overview of Thesis Research
DNA methylation and the post-translational modifications that occur on histones
are key regulators of gene expression. Many epigenetic studies focus on genes that
contain CpG islands at their promoters and role these epigenetic processes play in their
transcriptional control. Although these studies are important, not much emphasis is
directed towards the epigenetic regulation of genes that have a lower density of CpG sites
in their promoter. We hypothesize that non CpG island promoter genes are also
epigenetically regulated and that these genes may also impact cancer progression. The
study of genes without CpG islands compliments what is known about how epigenetics
controls the expression of the genome and provides insight into what aberrancies occur at
these regions during cancer and is therefore explored in this thesis. Additionally, because
there is an ongoing quest to find drugs that target the aberrancies that occur in the
epigenome during cancer, methyltransferase inhibitors were also studied to test their
potential as an epigenetic therapeutic.
In Chapter 2, the first gene studied was RUNX3, which has two promoters. The
RUNX3 non CpG island promoter was found to have a cell type specific DNA
methylation pattern while the second CpG rich RUNX3 promoter was found to have a
cancer specific DNA methylation pattern in bladder cancer. Both promoter's transcripts
could be induced in a bladder cancer cell line by the inhibition of DNA methylation with
5-Aza-CdR, supporting the role DNA methylation has in the transcriptional control of
both RUNX3 promoters, regardless of CpG density. Additionally, the presence or lack of
histone acetylation found at the promoter regions was indicative of the gene's
24
transcriptional state, therefore demonstrating that DNA methylation and histone
modifications together can regulate RUNX3 at either promoter.
To further elucidate the control epigenetics might have on a non CpG island
promoter, the LAMB3 gene was studied in Chapter 3. This gene was also shown to
display a tissue specific methylation and expression pattern in normal tissues. Further
examination in cancer cell lines showed an inverse correlation between DNA methylation
and expression. Interestingly, LAMB3 becomes hypomethylated in bladder cancer, which
in other studies has been shown to lead to increased cell motility, therefore possibly
providing a causative role in cancer. This non CpG island gene can also be induced upon
administration of 5-Aza-CdR. The active histone marks can be found at the LAMB3
promoter in the cells in which LAMB3 is expressed. Taken together, these data
demonstrate the type of control epigenetic mechanisms have in the regulation of non CpG
island promoters.
Chapter 4 describes the epigenetic effects that occur when AdoHcy hydrolase
inhibitors are administered to cancer cells. The goal of epigenetic therapy is to utilize
drugs that can reverse the epigenetic abnormalities that occur during carcinogenesis. The
use of methyltransferase inhibitors is meant to counteract the over-active histone
methyltransferases that cause the aberrant transcriptional repression of genes in cancer.
The drug DZNep was compared to other methyltransferase inhibitors, to better
comprehend its mechanism and specificity on histone methyl marks. It was found to
work in a similar fashion to the other drugs, and to have the ability to affect multiple
histone methyltransferases. Additionally, DZNep, but not 5-Aza-CdR, was able to
25
activate a downstream non CpG island promoter of the KRT7 gene. This difference in
regulation of epigenetic marks between the two drugs was again observed in the
microarray results. This study found that DZNep was able to epigenetically reactivate a
different cohort of genes than those of 5-Aza-CdR, suggesting that there are two
mechanisms involved in silencing genes in cancer. Taken together, this data provides
greater detail of the types of genes affected by methyltransferase inhibitors, and supports
their use in epigenetic therapy.
Studies such as these, which are extended over non CpG island genes, have the
capacity to influence the way we study the epigenome. This is because such studies are
inclusive and expand the targets of genes that can be studied to better understand normal
development and tissue specific expression. Additionally, by considering what occurs at
both CpG rich and non CpG rich regions studies can be more complete in terms of what
may be all the factors which become aberrantly regulated during disease. Studies of
epigenetic therapeutic targets also have the capacity to improve because they are not
limited to specific regions. As knowledge is increased on how epigenetics affects the
genome in its entirety, the more complete our knowledge becomes and that is the key to
successful treatment of disease.
26
CHAPTER 2
RUNX3: METHYLATION ANALYSIS OF TWO PROMOTERS
INTRODUCTION
The RUNX3 transcription factor is located on human chromosome 1p36.1. It is
the most ancient member of the highly conserved DNA binding RUNT domain family of
transcription factors (RUNX1, RUNX2, RUNX3) (Bangsow et al., 2001). All RUNX
proteins possess the ability to heterodimerize with their partner subunit CBF beta,
enhancing their DNA binding affinity. The RUNX proteins are key regulators of lineage-
specific gene expression in major developmental pathways (Levanon and Groner, 2004).
While RUNX1 is essential for hematopoiesis (Speck et al., 1999), and RUNX2 is
required for osteogenesis (Komori et al., 1997), gene knock out studies have shown that
RUNX3 is important in neurogenesis, thymopoesis and in the regulation of cell growth
and apoptosis of gastric epithelium (Inoue et al., 2002; Levanon et al., 2002; Li et al.,
2002; Taniuchi et al., 2002; Woolf et al., 2003).
RUNX3 as well as its other family members (RUNX1 and RUNX2) are expressed
from two promoters and can act to either activate or repress the transcription of their
target genes. RUNX3 promoter 1 (P1) is a non CpG island promoter and is separated by
35 kilobases (kb) from promoter 2 (P2), which lies within a 3000 basepair (bp) CpG
island (Figure 2.1). P1 gives rise to a protein that is 14 amino acids longer and differs
by19 amino acids than the P2 protein (Figure 2.2). Recently it has been shown that these
27
Figure 2.1. Map of human RUNX3 gene. RUNX3 is expressed from two promoters
separated by 35kb. Upper light blue boxes are exons specific for promoter 1 (P1) and
lower blue boxes are exons specific for promoter 2 (P2). The very highly conserved Runt
domain is shown as a purple box. The two promoters have been expanded for detail. P1
does not contain a CpG island as illustrated by the sparse amount of CpG sites (lower
black tick marks). However, P2 is located within a 3000 bp CpG island and is CpG rich.
The three CpG sites analyzed in each promoter by methylation sensitive single nucleotide
primer extension (Ms-SNuPE) are indicated by the black small arrows underneath the
tick marks.
P1 P1
P2 P2
Runt Domain
35 kb
Ms-SNuPE primers
200 bp
Ms-SNuPE primers
Map of RUNX3 (P1 and P2 promoters)
28
Figure 2.2. Protein Schematic of RUNX3. The two gene promoters (Figure 2.1) give
rise to two different transcripts, which ultimately encode the two RUNX3 protein
isoforms. The P1 protein differs from the P2 protein at the N terminus by 19 amino acids
(orange box). The P2 protein contains 5 amino acids specific to this protein (purple box).
The remainder of the two proteins are homologous (blue boxes).
P1
P2
19 a.a.
5 a.a.
29
19 amino acids located at the N-terminal region of RUNX3 code for an additional
activation domain that may be important in CD8
+
T cell function (Chung et al., 2007). In
mouse, Runx3 P2 is ubiquitously expressed whereas P1 expression is limited to a few
lineages/physiological states (activated T cells), which shows that there is differential
Runx3 promoter usage. These data also suggest that P1 is expressed in a tissue specific
manner (Rini and Calabi, 2001).
Studies of RUNX3 mainly involve the transcript from RUNX3 P2. It has been
shown that this transcript has tumor suppressor activity associated with the TGF-β
signaling pathway in gastric epithelium (Bae and Choi, 2004), indicating that the P2
transcript is important in regulating growth and proliferation. An increasing number of
studies have reported hypermethylation of P2 and loss of expression in a variety of
cancers (Kang et al., 2004; Kim et al., 2004; Ito et al., 2005; Jiang et al., 2008).
There is increasing evidence that DNA methylation plays a regulatory role in
tissue specific gene expression. Genome wide methylation studies have revealed that
DNA methylation plays a role in tissue specificity (Eckhardt et al., 2006). Tissue specific
methylation can be found in areas where there is less than the observed CpG/expected
CpG ratio of .65 needed to qualify as a CpG island (Takai and Jones, 2002; Weber et al.,
2007). The RUNX3 P2 region is a typical region to study since DNA methylation studies
mainly focus on CpG islands. The methylation status of RUNX3 P1 has not been studied,
which is not unexpected since the significance of DNA methylation in non CpG dense
promoters is not well studied.
30
To gain a better understanding of how epigenetics may regulate RUNX3 P1 and
P2, we analyzed the DNA methylation status of both promoters as well as their
expression in normal and cancer cells. We showed RUNX3 P1 to have tissue specific
methylation and expression. In contrast, RUNX P2 was found to be have a cancer
specific methylation pattern as seen in DNA from cancer cell lines as well as DNA from
primary tumor tissues and matched adjacent normal tissues. Both promoters could be
induced by treatment with 5-azacytidine (5-Aza-CdR), which supports the hypothesis that
methylation can influence the expression of genes with non CpG dense promoters.
31
MATERIALS AND METHODS
Cell lines. Cell lines obtained from the American Type Culture Collection (ATCC)
(Rockville, MD) were: bladder cancer cell lines J82, HT9, HT-1376, TCCSUP, T24,
SCABER, and UM-UC-3; prostate cancer cell lines DUC-145, PC3 and LNCAP; colon
cancer cell lines SW480, SW837, HT29, HCT116, HT15, SW48 and LoVo; and the T-
cell lymphoma cell line Jurkat. These cell lines were cultured as recommended by the
ATCC. Our laboratory generated the bladder cancer cell lines LD71, LD137, LD583,
LD600, LD605, LD627, LD630, LD660, LD679, LD692, and the fibroblast cell lines
LD98 and LD419. These bladder cancer cell lines were cultured in DMEM (Mediatech
Inc., Herndon, VA) supplemented with 10% FCS except for LD660 and LD692 which
were cultured in keratinocyte media (Sigma, St. Louis, MO). The fibroblast cell lines
were cultured in McCoy’s 5A supplemented with 20% FCS. The cell line 623 melanoma
was kindly provided by Dr. Jeff Weber (University of Southern California, Los Angeles,
CA) and was maintained in RPMI 1640 medium with 10% fetal calf serum. The B
lymphocyte cell line obtained from the Coriell Cell Repositories was established by
Epstein-Barr Virus transformation of peripheral blood mononuclear cells and was grown
in RPMI 1640 with 2mM L-glutamine and 15% FCS. All cell lines were maintained in a
humidified incubator at 37ºC in 5% carbon dioxide.
Tissue samples. Matched sets of bladder tumor tissue and adjacent normal tissue were
obtained from patients from the Los Angeles County/University of Southern California
32
Medical Center and the University of Southern California/Norris Comprehensive Cancer
Center (Los Angeles, CA), following institutional guidelines. Mucosal tissue was
dissected from surrounding muscle and adipose tissue. DNA was extracted from these
tissues.
Imprinting anaylsis. DNA (digested with RSA I) from buccal swabs was obtained from
healthy human individuals to screen for heterozygotes of the A/T single nucleotide
polymorphism (SNP) rs 6672420. This SNP is specific for the RUNX3 P1 coding region.
PCR amplification surrounding the SNP was done using the following primer set for
genomic DNA: sense 5´- CATGGCATCGAACAGCATCTTC- 3´ and antisense 5´-
CACCAACTCCCCACCCAAA-3´. PCR amplification surrounding the SNP in cDNA
was done using the 5´and 3´RT primers described below. PCR products were then used
in single nucleotide primer extension (SNuPE) reactions with either the radioactive
nucleotide dATP [α-32P] or dTTP [α-32P] (Perkin Elmer, Waltham, Massachusetts).
Subcloning of B lymphoblastoid cells. A double layer of Difco Nobel agar was plated
on each 60-mm bacteriological grade dish. Maintaining a 45ºC temperature, the bottom
layer solution was made by mixing one part 2.5% agar solution to three parts medium
(20% FCS). This bottom layer solution (5ml) was added to each dish and allowed to set
for at least 1hr. The second layer consisted of 1ml of the “bottom layer” solution, .5ml
media (20%FCS) and the cell suspensions, which ranged from 1x10
5
to 1x10
3
cells/1.5
ml. This was added on top of the bottom layer and was allowed to set at least one hour
33
before it was placed into 37ºC incubator. Exactly 24 hrs later, 2 ml of regular medium
was carefully added on top of the second layer and the medium was changed once per
week. Two weeks later colonies were isolated using a sterile Pasteur pipette and a
rubber-bulb aspirator. Cells were placed into 35cm
2
dishes and were allowed to grow to
densities large enough for DNA and RNA isolation.
Isolation of human lymphocytes from whole blood. Blood (40 ml) was drawn (in
accordance with the IRB protocol) from a healthy donor into a BD vacutainer (BD,
Franklin Lakes, NJ) containing sodium heparin to prevent coagulation. Blood was
layered on top of Ficoll-Paque PLUS (GE healthcare, Piscataway, NJ) in 15 ml centrifuge
tubes. Tubes were spun in a clinical centrifuge (440g) for 30 min. The lymphocytes
which are found at the interface between the plasma and the Ficoll-Paque PLUS were
recovered and subjected to short washing steps with Hank’s balanced salt solution
(HBSS) and phosphate buffered solution (PBS) (Mediatech Inc., Herndon, VA) to
remove any platelets. CD8
+
and CD4
+
lymphocytes were further purified using
MicroBeads (Miltenyi, Auburn, CA) conjugated to monoclonal anti-human CD8
+
or
CD4
+
antibodies. Also recovered separately and washed were the remaining
granulocytes, which were in the layer immediately above the erythrocytes. DNA and
RNA were both isolated from these sub-populations and fractions.
DNA isolation. DNA from healthy human tissues was either obtained through BioChain
(Hayward, CA) or was prepared in our lab from healthy anonymous donors. DNA was
34
extracted from cells by digestion in lysis buffer (400 mM NaCl, 100 mM Tris-HCl (pH
8.5), 5 mM EDTA, 0.2% SDS, 20 µg/ml RNase A and 500 µg/ml Proteinase K) for 16
hours at 55ºC (Jiang et al., 2008). DNA was phenol/chloroform extracted, ethanol
precipitated and dissolved in TE buffer.
DNA methylation analysis. Genomic DNA (4µg) was digested with EcoR I (New
England Biolabs, Ipswich, MA) and then bisulfite converted as described previously
(Laird et al., 1991; Wu et al., 1995). After PCR amplification of bisulfite-converted DNA
using bisulfite specific primers. Methylation levels were measured by methylation-
specific polymerase chain reaction (Ms-SNuPE) (Frommer et al., 1992). Primer
sequences used for bisulfite-PCR are: for RUNX3 P1 region, sense 5´-
AGAGGTAGTTATAAGATTTTTTAAAAG-3´ and antisense 5´-
AACTTACCCTTAACAAAACCC-3´; for RUNX P2 region, sense 5´-
GGGGTTGTAGAAGTTATAGGT-3´ and antisense 5-
CCAATACCACAACCCAAAAC-3´. The bisulfite PCR reaction conditions consisted of
the following: 95ºC for 3 min, 40 cycles of 95ºC for 1 min, annealing temperature for 1
min, 72ºC for 1 min, followed by 72 ºC for 10 min. The annealing temperature for
RUNX3 P1 was 51ºC and for RUNX3 P2 it was 58ºC. Ms-SNuPE bisulfite primers were:
for RUNX3 P1, 5´-GTTATAAGATTTTTTAAAAGGT-3´, 5´-
TTTACTTAATGAGTTAAGGT-3´ and 5´-ATAAGGTTAGGTTTTGT with the
annealing temperatures of 47ºC, 47ºC and 46ºC, respectively; for RUNX P2 region, 5´-
GGGGTTGTAGAGTTATAGGTT-3´, 5´-TAGTAAGAGTTGGGGGAAGTT-3´ and 5´-
35
TTAGTGGGGAGGAGGAG-3´ with the annealing temperatures 56ºC, 53ºC and 57ºC,
respectively. Cloning of bisulfite genomic DNA was done using the TOPO TA cloning
kit (Invitrogen, Carlsbad, CA) and sequencing was done by the USC DNA Core Facility.
Reverse transcription-PCR. Total RNA was isolated using Trizol reagent (Invitrogen,
Carlsbad, CA) according to the manufacturer’s instructions. Moloney murine leukemia
virus reverse transcriptase (MMLV) (Invitrogen, Carlsbad, CA), random hexamers
(Promega, Madison, WI), and 4µg of RNA were used for first-strand cDNA synthesis.
The cDNA from normal tissues was purchased from Clontech (Mountain View, CA) The
RT-PCR primer sequences were: RUNX3 P1, sense 5´-
CATGGCATCGAACAGCATCTTC- 3´ and antisense 5´-
GTCATTGCCTGCCATCACAG-3´ and for RUNX3 P2 sense 5´-
GCTGTTATGCGTATTCCCGTAG - 3´ and antisense 5´-
AAGTGGCTTGTGGTGCTGAG -3´. The RT-PCR conditions were 95ºC for 5 min, 32
cycles (19 cycles for GAPDH) of 95ºC for 1 min, annealing for 1 min, 72ºC for 1 min,
and lastly 72 ºC for 10 min. Annealing temperatures for RUNX P1, P2 and GAPDH were
61ºC, 58ºC and 58ºC, respectively. PCR products were resolved on 2% agarose gels
which were then often blotted onto a GeneScreen Plus (Perkin Elmer, Waltham, MA )
membrane overnight in .4N NaOH denaturing solution. Probes specific for either the P1,
P2 or GAPDH amplicon were radioactively end labeled and incubated with the
membrane followed by a series of washes that removed excess probe and background
signal.
36
5-Aza-CdR treatment. Cells were plated at 1x10
6
cells per 10cm plate and were treated
the next day with 3µM 5-Aza-CdR (Sigma, St. Louis, MO). The medium was changed
24 h after treatment, and cells were harvested 3 and 8 days after drug treatment.
5´ RACE. Total RNA was extracted from cell lines as described above, and the 5´ ends
of mRNA were determined by using the RLM-RACE Kit (Ambion, Austin, TX)
according to the manufacturer’s instructions. 5´ RACE reaction products were cloned
using the TOPO TA Cloning Kit (Invitrogen, Carlsbad, CA) and the USC DNA Core
Facility carried out the DNA sequencing.
ChIP assays. ChIP assays were performed as described previously (Gonzalgo and Jones,
1997). Sonicated chromatin was incubated with the following antibodies: 10 µl of anti-
Histone H3 (1µg/µl) (Abcam), anti-acetylated Histone H3 (1µg/µl) (Upstate), or 1µl
rabbit IgG (10µg/µl) (Upstate) used as a nonspecific antibody control.
37
RESULTS
DNA methylation analysis of both RUNX3 promoters in normal tissues by Ms-
SNuPE.
In order to determine if DNA methylation could play a role in the regulation of
RUNX3, the methylation statuses of both RUNX3 P1 and P2 were examined. DNA from
a panel of normal tissues was analyzed by methylation-sensitive single nucleotide primer
extension (Ms-SNuPE) at three CpG sites located at each RUNX3 promoter. Figure 2.3
shows the DNA methylation levels of the two promoters in normal tissues. The DNA
methylation levels of P2, the promoter within a CpG island, were less than 15% in the
tissues analyzed and there was no methylation found in the DNA isolated from sperm. It
has been previously demonstrated that the RUNX3 P2 transcript is necessary for normal
cellular processes such as apoptosis and cell differentiation (Ito, 2004), therefore, the low
levels of DNA methylation found were as anticipated for RUNX3 P2.
In contrast, RUNX3 P1 was found to exhibit high levels of DNA methylation in
most normal tissues. DNA methylation levels were above 65% in DNA extracted from
sperm, brain, kidney, and liver, while DNA from lung and white blood cells was found to
have lower methylation levels (42% and 41% respectively). The low levels of DNA
methylation observed in both promoters in white blood cells were expected because
RUNX3 is a known key regulator in hematopoietic cells (Woolf et al., 2003). This tissue
specific methylation pattern of P1 was intriguing because it had not been observed
38
Figure 2.3. DNA methylation status of RUNX3 P1 and P2 in normal tissues. DNA
methylation levels of RUNX3 P1, shown in gray bars, and P2, shown in black bars, were
analyzed by Ms-SNuPE in normal tissues. The average DNA methylation (%) at 3 CpG
sites in each promoter is shown.
0
20
40
60
80
100
Sperm Brain Kidney Liver Lung WBC
% Methylation
P1 P2
39
previously. This result suggests that the RUNX3 P1 transcript may be regulated by DNA
methylation in a tissue specific manner. Taken together, these data show that RUNX3 P1
and P2 can both have differential DNA methylation patterns. Due to a lack of
information on P1, its tissue specific methylation pattern was further investigated.
Bisulfite genomic sequencing of RUNX3 P1 in normal tissues.
The DNA methylation levels of 3 CpG sites within RUNX3 P1 were found to be
high in most of the normal tissues examined by Ms-SNuPE analysis, except for lung and
white blood cells. Although Ms-SNuPE analysis provides a good indication of the DNA
methylation status at specific CpG sites, bisulfite sequencing offers a DNA strand-by-
strand view and further elucidates the DNA methylation patterns found at gene promoters
(Frommer et al., 1992; Clark et al., 1994). In order to confirm the DNA methylation
analysis results obtained by Ms-SNuPE, and to expand the panel of tissues analyzed,
DNA isolated from sperm, testis, brain, lung, placenta and white blood cells was bisulfite
sequenced at RUNX3 P1 (Figure 2.4). The bisulfite genomic sequencing, which queried
13 CpG sites within a span of 240 base pairs (bp), confirmed the methylation results that
were observed by Ms-SNuPE and provided further detail in regards to the extent of
methylation in individual tissues.
Sperm, testis, and placenta were highly methylated with most DNA strands being
completely methylated. A few DNA strands containing a mixed pattern of DNA
40
Figure 2.4. Bisulfite genomic sequencing of RUNX3 P1. A. A schematic
representation of RUNX3 P1 is shown. Lower black tick marks represent individual CpG
sites. The three black arrowheads indicate the three CpG sites analyzed by Ms-SNuPE.
B. The black or white circles represent methylated or unmethylated CpG sites,
respectively.
Brain
Lung
Sperm
Testis
Placenta
20 bp
White
Blood
cells
A.
B.
41
methylation were also observed, mainly in placental DNA. DNA from brain, lung and
white blood cells contained many strands that were also highly methylated, however it
was noted that many completely unmethylated strands were also present. The P1 DNA
methylation patterns in these three tissues, as well as the presence of complete
methylation of this region in sperm, were reminiscent of the typical methylation patterns
seen across an imprinted gene (Lees-Murdock and Walsh, 2008). Since RUNX3
expression is crucial for proper hematopoietic processes, we next explored the possibility
that the transcript generated from RUNX3 P1 was imprinted.
Imprinting analysis of RUNX3 P1
An A/T single nucleotide polymorphism (SNP), located within the coding region
of RUNX3 P1, was used to distinguish between two alleles in determining the imprinting
status of RUNX3 P1 (Figure 2.5a). Using single nucleotide primer extension (SNuPE),
we first identified heterozygotes or homozygotes for the SNP using human genomic
DNA (Figure 2.5b). To determine if RUNX3 P1 was imprinted, we again utilized SNuPE
to check white blood cell cDNA from two individuals, one an A/A homozygote and the
other an A/T heterozygote (Figure 2.5c). The homozygote, because they contain two A
alleles, could only show expression from an A containing allele as indicated by the
radioactivity being present only in the lane where dATP[∝32P] but not where
dTTP[∝32P] was incorporated into the PCR product. In contrast, it was found that the
heterozygote could express the RUNX3 P1 transcript from both alleles as indicated by the
42
Figure 2.5. Imprinting analysis of RUNX3 P1. A. This schematic diagram shows the
SNP (A/T), located in the coding region of RUNX3 P1, which was used to determine if
RUNX3 P1 was an imprinted gene. Lower black tick marks represent individual CpG
sites. The black arrow signifies the transcription start site and blue boxes are exons. B.
Genomic DNA was first analyzed by SNuPE to distinguish heterozygotes from
homozygotes. C. RNA was extracted from white blood cells and was reverse transcribed.
cDNA was then analyzed by SNuPE to test for the presence of expressed RUNX3 P1
transcripts from either allele in the heterozygote. The homozygote was used as a control.
Heterozygote Homozygote
Genomic DNA cDNA
A T A T A T A T
Heterozygote Homozygote
100 bp
A
T
A.
B.
C.
43
presence of the radioactive mark in both lanes. The data from the heterozygote
demonstrated that the transcript generated from RUNX3 P1 was not imprinted. However,
there was still the possibility that there may be random monoallelic expression as has
been seen with a number of genes involved in the immune system, such as the mouse
immunoglobulin light chain locus (Mostoslavsky et al., 1998), interleukin-2 gene
(Hollander et al., 1998), and the human interleukin-1 alpha (IL1A) gene (van Rietschoten
et al., 2006).
Determination of monoallelic expression in B cells.
To determine if RUNX3 P1 was monoallelically expressed, we subcloned
GM14611, an Epstein bar virus (EBV) transformed B lymphocyte cell line originally
isolated from a healthy individual. EBV transformed B cells were readily attainable
through the Coriell Repository and were the closest to normal blood cells that could be
used for these experiments. First, we confirmed that GM14611 was heterozygous for the
A/T SNP found in the coding region specific to RUNX3 P1. Then the DNA from these
parent B cells was bisulfite sequenced (Figure 2.6). The results showed a few DNA
strands with no methylated CpG sites, a few strands with all but two CpG sites
methylated and two strands that were methylated on a few CpG sites on one side of the
strand. Such a pattern suggests a monoallelically-expressed gene because there were
both highly methylated and completely unmethylated strands.
B cells were plated in soft agar at 1x10
5
cells per plate for subcloning. After two
weeks, individual colonies were isolated and placed onto 60mm dishes. Subclones were
44
Figure 2.6. Bisulfite sequencing of EBV transformed B cells at RUNX3 P1.
GM14611, a lymphoblastoid cell line, is available through the Coriell Repository. These
cells have been immortalized by transfection with the Epstein bar virus (EBV). DNA
from these EBV transformed B cells was bisulfite sequenced at RUNX3 P1.
20 bp
EBV transformed
B cells
45
allowed to grow to densities large enough for DNA and RNA isolation (Figure 2.7a).
RNA was extracted from the parent B cells as well as the subclones and was reverse
transcribed. Similar to the imprinting study, the SNP (A/T) specific to the coding region
of the RUNX3 P1 transcript was used to analyze cDNA for the presence of RUNX3 P1
transcripts from either allele. A SNuPE was performed and the heterozygote parent B
cells and subclones both showed expression from both alleles indicating this was not a
monoallelically expressed gene (Figure 2.7b). DNA methylation analysis at 3 CpG sites
at RUNX3 P1 by Ms-SNuPE was performed on DNA from parent cells and subclones.
The results showed that the parent B cell has an average DNA methylation level of 50%,
similar to the bisulfite sequencing results (Figure 2.7c). Interestingly, the subclones had
very low levels of DNA methylation suggesting that the growth conditions of the
subcloning procedure led to the loss of DNA methylation. The data does not disprove
that there may be monoallelic expression in the parent B cell, since the subcloning
experiments cannot confirm that RUNX3 P1 is monoallelically expressed.
These results led us to reconsider the significance of the methylation patterns
found at RUNX3 P1. We have shown that RUNX3 P1 is methylated in sperm (Figure 2.4)
and since it is known that tissue specific genes can also be methylated in sperm (Shemer
et al., 1990; Antequera, 2003; Suzuki and Bird, 2008) we decided to further investigate
the tissue specific methylation pattern of RUNX3 P1 in white blood cells because of the
important role RUNX3 has in that tissue.
46
Figure 2.7. Determination of monoallelic expression of RUNX3 P1. A. EBV
transformed B cells were plated in soft agar at 1x10
5
cells per plate for subcloning. After
two weeks, individual colonies were isolated and placed onto 35cm
2
dishes. Subclones
(A3, D1, C2, C4, C5) were allowed to grow to densities large enough for DNA and RNA
isolation. B. RNA was extracted from the parent B cells as well as the subclones and was
reverse transcribed. Using the same SNP as in the previous imprinting study, cDNA was
analyzed by SNuPE to test for the presence of expressed RUNX3 P1 transcripts from
either allele in the heterozygote parent B cells and subclones. C. DNA methylation
analysis by Ms-SNuPE was performed on DNA from parent cells and subclones. The
average DNA methylation (%) at 3 CpG sites in P1 is shown.
47
Bisulfite sequencing of RUNX3 P1 in fractionated populations of white blood cells.
Considering the important role RUNX3 has in blood cell development (Egawa et
al., 2007) subsequent experiments focused on the DNA methylation status of RUNX3 P1
in fractionated white blood cells. Whole human blood from a healthy individual was
fractionated by a Ficoll-Paque PLUS density gradient allowing isolation of granulocytes
in one fraction, and lymphocytes in another fraction. The lymphocytes were then further
purified into CD8
+
or CD4
+
T lymphocytes. The DNA was extracted from each of these
cell fractions, treated with bisulfite, and the methylation of each CpG site was determined
by sequencing at RUNX3 P1 (Figure 2.8).
The granulocytes had a mixed pattern of methylation, as previously seen in the
unfractionated white blood cells (Figure 2.4). However, the bisulfite sequenced DNA
from purified CD8
+
and CD4
+
T lymphocytes was almost completely devoid of
methylation. This striking result of complete depletion in methylation across P1 was not
observed in any other tissue or cell type analyzed, indicating the importance of an
unmethylated RUNX3 P1 in these T lymphocytes where the role of RUNX3 is crucial to
their function (Chung et al., 2007). These data show that RUNX3 P1 has a lineage-
specific methylation pattern in CD8
+
and CD4
+
T lymphocytes. It would be interesting to
test the DNA methylation of the pre-cursor cells of T lymphocyte. If they were found to
be methylated it would then be interesting to determine if treatment with 5-Aza-CdR
and/or HDAC inhibitors could differentiate the precursor cells into T cells by changing
the epigenetic landscape and expression pattern to resemble that of a T cell. Such
experiments would also better define the functional role of the P1 transcript in T cells.
48
Figure 2.8. Bisulfite genomic sequencing of RUNX3 P1 in fractionated populations
of white blood cells. Whole blood was fractionated by a Ficoll-Paque PLUS density
gradient to separate lymphocytes from granulocytes. To obtain a pure population of T
cells, lymphocytes were further separated into CD8
+
and CD4
+
cells by antibody coupled
magnetic beads. A. Granulocytes and the B. isolated T cells, as indicated, were then
bisulfite genomic sequenced at RUNX3 P1. The black or white circles represent
methylated or unmethylated CpG sites, respectively. Known estimated population
percentages of each cell type are indicated in parentheses.
Lymphocytes (33% of all WBC)
NK Cells (~1% of lymphocytes)
T cells (~ 90% of lymphocytes)
B cells (~5% of lymphocytes)
Granulocytes (67%of all WBC)
T cell CD8
+
T cell CD4
+
20 bp
20 bp
B.
A.
49
Expression of RUNX3 P1 and P2 in normal tissues
To understand the relationship between the DNA methylation levels observed
through our experiments and the expression of RUNX3, reverse transcriptase PCR (RT-
PCR) was performed on cDNA from brain, kidney, liver, lung, prostate, colon as well as
resting and activated mononuclear cells, which included both monocytes and
lymphocytes (Figure 2.9). RUNX3 P1 had little to no expression in the tissues analyzed
except for the activated mononuclear cells. Many of these tissues, except for the
hematopoietic cells, had high levels of methylation, which further supports the role of
DNA methylation in the tissue-specific regulation of RUNX3 P1. The expression seen in
the activated mononuclear cells, particularly the transcript originating from P1, is
indicative of the important role that RUNX3 has in hematopoietic cells. Previously
observed low levels of methylation and high expression in all tissues except for the brain
also suggest a role for methylation in the regulation in RUNX3 P2.
Methylation status of both RUNX3 promoters in bladder, colon and prostate cancer
cell lines
Hypermethylation of RUNX3 P2 has been implicated in many cancers including
but not limited to lung and gastric cancer (Li et al., 2002; Sato et al., 2006). For that
reason it was interesting to examine the methylation status of both RUNX3 promoters in
several bladder, colon and prostate cancer cell lines (Figure 2.10). DNA methylation of
RUNX3 P1 and P2 in these cell lines was analyzed by Ms-SNuPE and the methylation
levels of the three CpG sites analyzed were averaged.
50
Figure 2.9. RUNX3 expression in normal cells. cDNA from normal human tissues and
blood fractions was purchased from Clontech. mRNA expression of transcripts from
both RUNX3 P1 and P2 was determined by RT-PCR in each tissue. Ubiquitously
expressed GAPDH served as a control for the input cDNA. Mononuclear cells consist of
both monocytes as well as lymphocytes.
Brain
Liver
Lung
Kidney
Colon
Prostate
Mononuclear
Mononuclear
activated
Negative
Control
P1
GAPDH
P2
51
Figure 2.10. DNA methylation status of both RUNX3 promoters in bladder, colon
and prostate cancer cell lines. RUNX3 P1 (gray bars) and P2 (black bars) methylation
levels in various cell lines were analyzed by Ms-SNuPE. The average DNA methylation
(%) at 3 CpG sites in each promoter is shown. The first 2 cell lines (blue bar) are normal
fibroblasts, followed by 17 bladder cancer cell lines (red bar), 3 prostate cancer cell lines
(black bar), and 7 colon cancer cell lines (orange bar).
0
20
40
60
80
100
LD98
LD419
T24
5637 (HT9)
HT-1376
J82
SCABER
TCCSUP
UM-UC-3
LD137
LD583
LD600
LD605
LD627
LD630
LD660
LD679
LD692
LD71
DUC-145
PC3
LNCAP
LoVo
SW837
HT116
SW480
HT29
HT15
SW48
Cell Lines
% Methylation
P1
P2
52
The normal fibroblast cell lines generated in our lab, LD419 and LD98 served as
our controls. These two control cell lines showed high levels of methylation at RUNX3
P1, in agreement with our previous data showing that normal tissues exhibit generally
high levels of methylation at this promoter (Figure 2.3). Most bladder cancer cell lines
exhibited high levels of methylation within RUNX3 P1 except for HT-1376, which lost
methylation (16%). It was also observed that although the DNA methylation levels in the
majority of the bladder cancer cell lines were high, there were still some cell lines that
became hypomethylated such as TCCSUP, LD600, LD605, or LD627.This suggests the
possibility that at RUNX3 P1 the level of DNA methylation may decrease during
carcinogenesis. Similar results were obtained for the prostate and colon cancer cell lines
in that RUNX3 P1 exhibited higher levels of DNA methylation with only SW48 showing
a loss of methylation (42%).
In contrast, very low levels of DNA methylation were observed at RUNX3 P2 in
normal fibroblasts. RUNX3 P2 became hypermethylated in 16/17 of the bladder cancer
cell lines with 13 of these cell lines showing methylation levels above 50%. RUNX3 P2
methylation levels in the prostate and colon cancer cell lines were found to be high but
not as dramatically as that observed for the bladder cancer cell lines. The loss of
methylation at RUNX3 P1 and the gain of methylation at RUNX3 P2 led us to further
investigate the expression status of RUNX3 in primary tumors.
53
DNA methylation status of RUNX3 in normal bladder tissue, primary tumors and
their adjacent normal tissues.
To determine whether the methylation levels found in cell lines were similar to
primary tumors, RUNX3 P1 DNA methylation levels were analyzed in normal tissues and
both P1 and P2 DNA methylation levels were analyzed in primary tumors as well as their
adjacent normal tissues. Using Ms-SNuPE, DNA methylation at RUNX3 P1 from 10
normal bladder samples was found to be consistently around 50% methylated (Figure
2.11a). The methylation status was then analyzed in matched sets of DNAs from primary
tumors and their adjacent normal tissues. RUNX3 P1 still exhibited high levels of
methylation in the adjacent normal tissues, but there were adjacent normal tissues that did
contain less than 40% methylation (Figure 2.11b). However the primary tumors often
had higher RUNX3 P1 DNA methylation levels in comparison to all the adjacent normal
tissues.
RUNX3 P2 was not methylated in the adjacent normal tissues, however in many
cases, a dramatic hypermethylation was observed at this promoter between the adjacent
normal and tumor tissues (Figure 2.11c). This demonstrates that in bladder cancer,
RUNX3 P2 becomes hypermethylated possibly leading to the transcriptional silencing of
its transcript.
Chromatin immunoprecipitation (ChIP) analysis of RUNX3 P1 in expressing and
non-expressing cells.
Our data has suggested a role for methylation in the regulation of the CpG
54
Figure 2.11. DNA methylation status of RUNX3 in normal bladder tissue, primary
tumors and matched adjacent normal tissues. DNA methylation of RUNX3 promoters
was analyzed by Ms-SNuPE. A. RUNX3 P1 methylation was analyzed in normal bladder
tissue or B. matched sets of primary tumors and matched adjacent normal tissues as
indicated. C. Similarly, RUNX3 P2 DNA methylation was analyzed in the matched sets
of primary tumors and matched adjacent normal tissues as indicated. The average DNA
methylation (%) at 3 CpG sites is shown.
RUNX3 P2: Bladder Matched Sets
0
20
40
60
80
100
Bladder
2607
Bladder
2705
Bladder
2915
Bladder
3028
Bladder
1999
Bladder
3047
Bladder
2813
Bladder
2852
Bladder
2939
Bladder
2758
% Methylation
Adjacent Normal
Tumor
RUNX3 P1: Bladder Matched Sets
0
20
40
60
80
100
Bladder
2607
Bladder
2705
Bladder
2915
Bladder
3028
Bladder
1999
Bladder
3047
Bladder
2813
Bladder
2852
Bladder
2939
Bladder
2758
% Methylation
Adjacent Normal
Tumor
RUNX3 P1: Normal Bladder
0
20
40
60
80
100
4896 NN 4920 NN 4921 NN 4947 NN 4950 NN 4959 NN 4967 NN 4968 NN 5000 NN 5162 NN
% Methylation
A.
B.
C.
55
poor RUNX3 P1. It is known that DNA methylation and histone modifications together
regulate transcription. To better understand if the acetylation of histones may
additionally influence the regulation of this promoter, ChIP analysis was performed on
the RUNX3 P1-expressing 623 melanoma cell line as well as the non-expressing
immortalized keratinocyte cell line HaCaT and bladder cancer cell line UM-UC-3 (Figure
2.12).
In region 1 (R1), located 600bp from the transcriptional start site, there was a 50%
higher level of histone H3 acetylation than at region 2 (R2). However, the most
acetylation was observed upstream of the transcription start site at region 3 (R3).
Acetylated histone H3 is associated with transcriptional activation (Kouzarides, 2007),
therefore this result suggests that the presence of histone H3 acetylation may additionally
aid in the regulation of the RUNX3 P1 transcript. In the HaCaT or UM-UC-3 cell lines,
low levels of histone H3 acetylation were detected at all regions. This was expected
because these cell lines do not express the RUNX3 P1 transcript. Together these data
suggest that histone acetylation may influence the regulation of RUNX3 P1.
RUNX3 expression can be induced and methylation levels are decreased in normal
fibroblast and bladder cancer cell lines upon 5-Aza-CdR treatment.
Next, it was interesting to determine whether the deoxyribonucleoside analogue
5-aza-2'-deoxycytidine (5-Aza-CdR) could induce the expression of RUNX3 from either
promoter. The T24 human bladder cancer cell line and the LD419 human fibroblast cell
56
Figure 2.12. RUNX3 P1 expression by real time RT-PCR and analysis of histone H3
acetylation levels by ChIP analysis. A. Real time RT-PCR specific to the P1 transcript
was performed on cDNA from the 623 melanoma cell line, the immortalized keratinocyte
HaCaT cell line or the bladder cancer cell line UM-UC-3. Expression was normalized to
GAPDH. B. ChIP was performed on the 623 melanoma, HaCaT or UM-UC-3 cell lines
as indicated, with anti-histone H3 or anti-acetylated histone H3 K9/K14 antibodies. Real
time PCR analysis was performed with immunoprecipitated DNA at the 3 RUNX3 P1
regions (R1, R2 and R3) shown. The data are presented as anti-acetylated histone H3
relative to histone H3 levels. Results from a single assay are shown.
RUNX3 P1: HaCaT
0
0.3
0.6
0.9
1.2
1.5
1.8
R1 R2 R3
Acetyl H3/ H3
RUNX3 P1: UM-UC-3
0
0.3
0.6
0.9
1.2
1.5
1.8
R1 R2 R3
Acetyl H3/ H3
RUNX3 P1: 623 melanoma
0
0.3
0.6
0.9
1.2
1.5
1.8
R1 R2 R3
Acetyl H3/H3
200 bp
R2 R1 R3
0
0.005
0.01
0.015
623mel HaCaT UMUC3
Cell Line
Expression/GAPDH
A.
B.
57
line were treated for 24 hours with 5-Aza-CdR (3µM) and harvested on day 3 or day 8
after initial drug treatment (Figure 2.13a). RUNX3 was not expressed from P1 in either
cell line before treatment, however upon administration of the DNA methylation
inhibitor, expression could be detected in both cell lines. Additionally, expression from
RUNX3 P2 was observed in the normal fibroblast cell line but not in the T24 bladder
cancer cells prior to treatment. However upon treatment with 5-Aza-CdR, induction of
RUNX3 from the P2 promoter occurred at both day 3 and day 8 in the T24 cell line.
Many studies have shown an association between the DNA methylation at RUNX3 P2
and its loss of expression (Jiang, 2008; Sato, 2006; Levanon, 2003), however their
analysis was never extended to RUNX3 P1. Therefore, DNA methylation was analyzed
by Ms-SNuPE at RUNX3 P1 in T24 and LD419 cell lines treated with 3 µM 5-Aza-CdR.
A reduction in methylation levels was found in both cell lines at this region in
comparison to the untreated cells (Fig. 2.13b). The observed reduction in DNA
methylation correlates with the ability of these cell lines to express RUNX3 from P1 when
treated with 5-Aza-CdR, which is particularly intriguing since RUNX3 P1 is not located
within a CpG island.
58
Figure 2.13. Induction of RUNX3 in T24 and LD419 cells after 5-Aza-CdR
treatment. A. RT-PCR was performed on cDNA from the bladder cancer cell line T24
or the fibroblast cell line LD419 before and after treatment with 5-Aza-CdR (3µM) at day
3 or day 8. B. RUNX3 P1 DNA methylation was analyzed by Ms-SNuPE in the T24 or
LD419 cell lines before and after treatment with 5-Aza-CdR (3µM) at day 8. The
average DNA methylation (%) at 3 CpG sites is shown.
Untreated
5-Aza-CdR at day 3
5-Aza-CdR at day 8
Untreated
5-Aza-CdR at day 3
5-Aza-CdR at day 8
T24 LD419
RUNX3-P1
RUNX3-P2
GAPDH
Induction of RUNX3 by 5-Aza-CdR
A.
B.
RUNX3 P1 DNA Methylation
0
20
40
60
80
100
T24 LD419
% DNA Methylation
Untreated
5-Aza-CdR
59
DISCUSSION
In this study, the DNA methylation status of RUNX3 at both P1 and P2 was
investigated in order to better understand how DNA methylation regulates both a CpG
rich promoter and a non CpG dense promoter in both normal tissues and cancer. We
showed RUNX3 P1, a non CpG dense promoter, to have a tissue specific DNA
methylation pattern and RUNX3 P2, a CpG dense promoter, to have a cancer specific
methylation pattern. Hypermethylation of RUNX3 P2 and silencing of this transcript
have been shown to occur in many cancers (Kang et al., 2004; Kim et al., 2004; Ito et al.,
2005; Jiang et al., 2008; Kim et al., 2008), therefore our RUNX3 P2 results were
consistent with the findings in literature. However, our studies are the first to report
tissue specific DNA methylation at the non CpG dense RUNX3 P1.
Previous reports (Bangsow et al., 2001) as well as our own data have shown that
the RUNX3 P1 transcript is not expressed in many tissues. However, the DNA
methylation status could not be predicted at RUNX3 P1 because there are cases in which
inactive genes are found unmethylated, such as the dopamine receptor gene DRD3, which
also does not contain a CpG island (Shen et al., 2006). Results from bisulfite genomic
sequencing at RUNX3 P1 demonstrated how heavily methylated DNA from sperm, testis
and placenta were at this region. In addition, a mixture of methylated and unmethylated
DNA strands were found in brain and lung tissue, which do not express the P1 transcript.
Since the P1 transcript is expressed in white blood cells and has displayed low levels of
DNA methylation it is possible that these unmethylated DNA strands originated from
60
blood cells that were contaminating the normal tissue. In addition, the cDNA and DNA
were obtained from two different companies, which may have different protocols for
preparing tissue prior to isolating nucleic acids. If contamination was a factor, then the
DNA methylation levels observed in brain and lung might have been higher.
The RUNX3 P1 methylation patterns found in white blood cells together with the
methylation found in sperm was intriguing because imprinted genes display a 50:50
methylated/unmethylated DNA methylation pattern, and can be methylated in sperm
(Lees-Murdock and Walsh, 2008). Since the RUNX3 P1 transcript is known to be
expressed in white blood cells, we pursued the possibility that this transcript was
imprinted. However, our studies concluded that RUNX3 P1 was not imprinted since we
observed expression from both alleles.
Since imprinting was not a mechanism used by RUNX3 P1 to control its
expression, it was still possible that the methylation pattern was indicative of monoallelic
expression. However our subcloning experiments, which were used to determine if
RUNX3 P1 was monoallelically expressed, were inconclusive. It was interesting that the
DNA methylation levels in the subclones decreased dramatically. This could indicate
that either one of two scenarios may have occurred (Figure 2.14). In the first scenario,
the parent B cells may have contained one allele that was methylated and one allele that
was unmethylated, and the only cells that survived were those that underwent a
demethylation process leading to the two unmethylated alleles (Figure 2.14a).
Alternatively, if the parent B cells were a mixed population of cells containing either two
methylated alleles or two unmethylated alleles, then after subcloning these results may
61
Figure 2.14. Possible explanations for demethylated subclones. EBV transformed B
cells were subcloned to determine if there was monoallelic expression from RUNX3 P1,
however after subcloning, only demethylated alleles remained indicating possibly one of
the two scenarios depicted above. A Parent B cells contain one allele that was methylated
and one allele that was unmethylated, and surviving cells were those that underwent a
demethylation process leading to the two unmethylated alleles. B. Parent B cells were a
mixed population of cells containing either two methylated alleles or two unmethylated
alleles. After subcloning these only the cells with two unmethylated alleles survived.
Conditions of subcloning
cause demethylation.
Only parent cells with
unmethylated alleles
survives subcloning
conditions.
Subclone
Allele 1
Allele 2
Subclone
Allele 1
Allele 2
Subclone
Allele 1
Allele 2
Parent B cell
Allele 1
Allele 2
Allele 1
Allele 2
Parent B cell
Parent B cell
Allele 1
Allele 2
A. B.
Parent B cell
Allele 1
Allele 2
Subclone
Allele 1
Allele 2
62
have meant that only the cells with two unmethylated alleles survived for unknown
reasons (Figure 12.14b). Either situation indicates that RUNX3 P1 is important for the
survival and growth of these B cells and that the expression of RUNX3 P1 was needed
from both alleles. The parent B cells however might still be controlled by monoallelic
expression because they did display a 50:50 unmethylated/methylated DNA methylation
pattern. However, the reason why B cells might be monoallelically methylated and
expressed or have a mixed population to begin with is unknown, but might be attributed
to the tight regulation that is needed at RUNX3 P1.
We now understand that many of the unmethylated DNA strands we observed in
the white blood cells were attributed to the unmethylated state of CD4
+
and CD8
+
T cells.
The bisulfite sequencing of fractionated whole blood gave us much insight into the
methylation pattern that could be seen in a certain lineage of cells. Although many
RUNX3 studies do not report on the contribution of expression from P1, a recent study
recognized that the P1 transcript is selectively expressed in mature mouse CD8
+
T cells
(Chung et al., 2007). Additionally, similar to our expression analysis where we
demonstrated P1 to be expressed in activated mononuclear cells, this study showed that
the P1 transcript becomes upregulated in activated mouse splenocytes. This study helped
corroborate a functional link between expression from P1 and the importance of an
unmethylated RUNX3 P1 in T cells.
RUNX3 has been shown to be hypermethylated at P2 and its expression is
downregulated in many cancers studies (Li et al., 2004; Jiang et al., 2008). However
there are no reports on the methylation status of both RUNX3 promoters. To gain a better
63
understanding of RUNX3 methylation status in cancer, RUNX3 P1 and P2 DNA
methylation levels were analyzed in cancer cell lines, primary tumors and matched
normal adjacent tissues. DNA methylation levels at RUNX3 P2 were low in fibroblasts
and were hypermethylated in the bladder, prostate and colon cancer cell lines. RUNX3
P2 also showed a dramatic hypermethylation in the primary tumors as compared to the
matched normal adjacent tissues. Therefore the DNA methylation of RUNX3 P2 is
cancer specific. In contrast, RUNX3 P1 DNA methylation levels were high in fibroblasts
as well as many of the cancer cell lines, primary tumors and the matched adjacent normal
tissues. Because a global demethylation occurs during carcinogenesis (Ehrlich, 2002), it
was interesting to note that a few cell lines did exhibit a loss of methylation and in
matched Bladder 2813 set (Figure 2.11), the adjacent normal and tumor were both less
than 40% methylated. This suggests that genes with non CpG dense promoters may be
affected by global demethylation.
Epigenetic regulation of CpG poor regions is not traditionally studied, however
our data suggests that DNA methylation regulates RUNX3 P1. ChIP analysis of histone
H3 acetylation at K9/K14 suggested that histone H3 acetylation additionally aids in the
regulation of the RUNX3 P1 transcript by allowing increased accessibility to the DNA
for binding factors and transcriptional machinery (Rice and Allis, 2001; Kouzarides,
2007). The nucleosome dip that is often seen at the transcription start site was not as
dramatic as previously noted in literature (Gal-Yam et al., 2006). This is because
although the R2 primers were designed to be as close to the transcription start site as
possible, they were still about 300 bps away.
64
Next we found that treating the T24 bladder cancer and the LD419 human
fibroblast cell lines with 5-Aza-CdR could induce the expression of RUNX3 P1 and P2.
This further supports the idea that methylation plays a role in the regulation in a non CpG
rich and CpG rich promoter. However, when induction of a gene is observed, it should
be taken into account that the induction may be an indirect result.
Overall we have shown that methylation may play a role in both RUNX3 P1 and
P2, albeit in different scenarios. Furthermore our data shows that 5-Aza-CdR can induce
the expression of a methylated non CpG dense promoter in addition to a CpG island
promoter. The DNA methylation pattern of RUNX3 P2 is cancer specific. This promoter
seems to remain unmethylated and expressed in most tissues and it is during
carcinogenesis that it becomes methylated leading to its repression. In contrast, the
methylation of RUNX3 P1 is displayed in tissues where its expression is not found in
order to silence this transcript in tissues where it is not needed. However, in blood cells,
it is known that the RUNX3 P1 transcript is crucial to T cell function and is therefore
unmethylated and expressed. These results are similar to what has been found with the
maspin gene, which it is preferentially expressed and unmethylated in epithelial cells but
not expressed and methylated in other cell types (Futscher et al., 2002). These studies
suggest that methylation can control tissue specific expression. However, RUNX3 P1
shows that this tissue specific methylation and expression can occur in a non-CpG dense
promoter.
65
CHAPTER 3
LAMB3: TISSUE SPECIFIC DNA METHYLATION OF A NON CpG ISLAND
GENE PROMOTER AND ITS IMPLICATIONS IN CANCER
INTRODUCTION
Laminins are large basement membrane glycoproteins and are essential
components of the extracellular matrix. They contribute to the structural framework of
the basement membrane and are believed to have a role in cell differentiation,
proliferation, adhesion and migration (Scheele et al., 2007). All laminins contain one of
each of the alpha (α), beta (β) and gamma (γ) chains assembled in a cross shaped
structure (Figure 3.1). There are five types of alpha chains, three types of beta chains, as
well as three types of gamma chains, which have been found to produce twelve different
heterotrimers (Katayama and Sekiguchi, 2004). Each laminin variant’s function is
distinct to its environment and has a specific biological activity (Miyazaki, 2006).
Laminin-5, newly named in the literature as laminin 332 because it is comprised
specifically of the α3, β3 and γ2 chains, was first discovered in keratinocytes. Laminin-
5, along with the integrin α6β4, facilitates the attachment of epidermal cells to the
basement membrane through hemidesmosomes (Figure 3.1). In addition, epidermolysis
bullosa is a severe skin blistering disease that that occurs when there are mutations in the
66
Figure 3.1. Schematic of laminin-5 in the basement membrane and in detail.
Laminin-5 has a cruciform structure that is comprised of the alpha (α) 3, beta (β) 3, and
gamma (γ) 2 polypeptide chains (inset), which are the products of the genes LAMA3,
LAMB3, and LAMC2. Each chain contains different domains that allow the heterotrimer
to interact with various extracellular molecules. The simplified schematic of
keratinocytes (pink) shows the secretion (arrows) of laminin-5 into the basement
membrane (left) followed by its binding to the α6 β4 integrins (right) to form stable
hemidesmosomes. These junctions allow for the stable anchorage of keratinocytes to
their underlying connective tissue which is made up of other filaments such as collagen.
67
LAMA3, LAMB3 or LAMC2 genes, which code for laminin-5. This study focuses on the
LAMB3 gene, which has a non CpG island promoter.
Investigation of the control of LAMB3, a gene with a non CpG island promoter,
has shown that DNA methylation can play a role in its expression. Studies have shown
that the aberrant methylation of the LAMB3 promoter can cause its silencing in lung,
prostate, and bladder cancer (Sathyanarayana et al., 2003a; Sathyanarayana et al., 2003b;
Sathyanarayana et al., 2004). The loss of LAMB3 may disrupt the integrity of the
basement membrane allowing for the degradation of the extracellular matrix and
eventually metastasis. However, there are also studies that show cancer and possibly
metastasis can be caused by an over-expression of laminin-5. For example, an increase in
the levels of laminin-5 is observed in the basal lamina of cervical cancers (Skyldberg et
al., 1999). Similarly, cells from squamous cell carcinoma of the tongue and colorectal
carcinoma strongly coexpress laminin-5 β3 and γ2 chains at the cancer-stromal interface
and invasive front (Akimoto et al., 2004). Also, the LAMB3 transcript was shown to be
upregulated in an expression analysis study in transformed human cell lines and
xenograft tumors (Stull et al., 2005). However, the overexpression studies do not explore
how epigenetics may influence the upregulation of the genes that code for the laminin-5
chains.
To further investigate how DNA methylation and chromatin structure might
influence the expression of the non CpG island LAMB3 gene promoter, normal tissues,
cancer cell lines, as well as bladder cancer matched sets were analyzed. These studies
indicated that epigenetics does play a role in the regulation of LAMB3. LAMB3 was
68
found to have a tissue specific methylation pattern, in which it was heavily methylated in
most normal tissues including sperm, but not in keratinocytes where it was highly
expressed. Additionally, the DNA methylation and expression analysis in cell lines
showed an inverse correlation. DNA methylation levels decreased at the LAMB3
promoter and there was an induction of the LAMB3 transcript in a bladder cancer cell line
upon administration of 5-Aza-CdR, further suggesting a role of DNA methylation in the
expression of LAMB3. Chromatin immunoprecipitation (ChIP) analysis demonstrated
that the chromatin marks associated with active genes were present in LAMB3 expressing
cells, while the marks associated with inactive genes were present in the non expressing
cells showing how chromatin may aid in the regulation of LAMB3. Contrary to the
results of Sathyanarayana et. al., 2003, we found DNA hypomethylation in human
primary bladder tumors in comparison to their paired adjacent normal tissues as well as
normal bladder tissue. This last finding is interesting in that LAMB3 may contribute to
metastasis due to the DNA hypomethylation at its promoter leading to its aberrant re-
expression.
69
MATERIALS AND METHODS
Cell lines. Cell lines obtained from the American Type Culture Collection (ATCC)
(Rockville, MD) were: bladder cancer cell lines UM-UC-3, TCCSUP, SCABER, RT4
and HT-1376. These cell lines were cultured and maintained as recommended by the
ATCC. Our laboratory generated the bladder cancer cell lines LD611, LD605, LD71
LD137 and the normal fibroblast cell line LD419. These bladder cancer cell lines were
cultured in DMEM (Mediatech Inc. Herndon, VA) supplemented with 10% fetal calf
serum (FCS). The fibroblast cell line was cultured in McCoy’s 5A supplemented with
20% FCS. The cell line 623 melanoma was kindly provided by Dr. Jeff Weber
(University of Southern California, Los Angeles, CA) and was maintained in RPMI 1640
medium with 10% FCS. The immortalized keratinocyte HaCaT cell line was obtained as
described previously (Boukamp et al., 1988) and was grown in modified MEM
supplemented with 10% FCS. Normal human epithelial keratinocytes (NHEK) were
purchased from Lonza (Walkersville, MD) as well as Promocell (Heidelberg, Germany)
and were maintained in the recommended media supplied by the company. All cell lines
were maintained in a humidified incubator at 37°C in 5% carbon dioxide.
DNA isolation. DNA from healthy human tissues was either obtained through BioChain
(Hayward, CA) or was prepared in our lab from healthy anonymous donors. DNA was
extracted from cells by digestion in lysis buffer ( 400mM NaCl, 100mM Tris-HCl (pH
8.5), 5mM EDTA, .2% SDS, 20µg/ml RNase A and 500 µg/ml Proteinase K) for 16
70
hours at 55ºC. DNA was phenol/chloroform extracted, ethanol precipitated and dissolved
in TE buffer.
5-Aza-CdR treatment. UM-UC-3 cells were plated at 1x10
5
cells/100-mm dish and
treated the next day with 3uM 5-Aza-CdR (Sigma Chemical Co., St. Louis, MO). The
medium was changed 24 hour after treatment, and RNA and DNA was extracted at day 3
and 8 from exponentially growing cultures.
Tissue collection and DNA isolation. DNA from healthy un-diseased tissue was
purchased from BioChain Institute Inc. (Hayward, California). DNA from oral
keratinocytes was obtained from healthy volunteers in our lab with informed consent and
IRB approval. DNA from bladder matched sets of adjacent normal and tumor specimens
was obtained from bladder cancer patients and the 2 normal urothelial controls were from
individuals without bladder cancer at University of Southern California/Norris
Comprehensive Cancer Center (Los Angeles, CA). DNA was isolated using standard
procedures by treatment with proteinase K and phenol extraction.
Real-time RT-PCR. Total RNA from the cell lines was reverse transcribed using 2 ug
of RNA and random hexamers, deoxynucleotide triphosphate (Boehringer Mannheim,
Mannheim, Germany), and Superscript II reverse transcriptase (Life Technologies, Inc.)
in 50 µl reaction. The mixture was placed at room temperature for 10 min., 42°C for 45
min, and 90°C for 3 min and then rapidly cooled to 0°C. Human Multiple Tissue cDNA
71
was purchased from BD Bioscences Clontech. Primers and probe for LAMB3: sense
primer, GCTTTCAGGCGATCTGGAGA; antisense primer,
GGGTGATCCCCAGAAAGGA; and probe,
AGAACGGCAGAACACACAGCAAGGAAAG. Primers and probe for GAPDH:
sense primer, TGAAGGTCGGAGTCAACGG; antisense primer,
AGAGTTAAAAGCAGCCCTGGTG; and probe, TTTGGTCGTATTGGGCGCCTGG.
All PCR reactions were carried out under the same conditions: 95 °C for 15 s and 59 °C
for 1 min for 45 cycles. With each set of PCR, titrations of known amounts of DNA were
included as a standard for quantitation.
ChIP assays. ChIP analyses were performed as described previously (Nguyen et al.,
2001). Antibodies used were: 10 mg of either anti-Histone H3 (Abcam), anti-acetylated
Histone H3 (Upstate), anti-dimethyl K4 (Abcam), or trimethyl K9 Histone H3 (Abcam)
and 1 mg of rabbit IgG (Upstate) as nonspecific antibody control.
Real-time PCR analysis of immunoprecipitated DNA. Chromatin
Immunoprecipitation Assay (ChIP) assay was performed in RT4 and UM-UC-3 cell lines.
The detailed method has been previously described (Nguyen et al., 2001; Liang et al.,
2004). Ten µl of anti-acetylated H3-K9/14, or 10 µl of anti-dimethylated H3-K4
(Upstate Biotechnology), or 10 µl of anti-trimethylated H3-K9 which has been described
(Tamaru et al., 2003), or 10 µl normal mouse IgG as negative control (Santa Cruz
Biotechnology, Inc.) was used. Quantitative PCR was performed with a DNA Engine
72
Opticon System (MJ Research, Inc.) using AmpliTaq Gold DNA polymerase (Applied
Biosystems) with either 5 µl of immunoprecipitated DNA, 5 µl of no antibody sample
(NAC), or 1 µl of input sample (1%). Fluorescently labeled TaqMan probes were
synthesized by Biosearch Technologies. Region 1: sense primer,
CTCTGGGACGGACCGACC; antisense primer, TCACACCCATCTGTCGTCACTG;
and probe, CCACTTGCCCAGTCCCGTCCTG. Region 2: sense primer,
TAAAAACCTGGAGCCGGGA; antisense primer, CCGTTCTTTCTCCAGATCGC;
and probe, AGACCCCCACATTCAAGAGGAGCTTTCA. Region 3: sense primer,
AACGGCAGAACACACAGCAA; antisense primer, CCTCCTCCCGCATCCG; and
probe, CTGTCTTTACCTGCCTGGTGCGGG. All PCR reactions were carried out
under the same conditions: 95 °C for 15 s and 59 °C for 1 min for 45 cycles. With each
set of PCR, titrations of known amounts of DNA were included as a standard for
quantitation. DNA from the ChIP samples immunoprecipitated with anti-acetylated H3-
K9/14, anti-dimethylated H3-K4, anti-trimethylated H3-K9, and the ChIP samples
immunoprecipitated with non-specific antibody (NAC) were included in each PCR set.
The fraction of immunopreciptated DNA was calculated as: (amount of IP sample with
antibody - amount of NAC)/(amount of Input DNA (1%) - amount of NAC).
Quantitation of DNA Methylation by Ms-SNuPE: We have developed a rapid
quantivative method called Ms-SNuPE for assessing methylation differences at specific
CpG sites (Gonzalgo and Jones, 1997). Briefly, Genomic DNA was first treated with
sodium bisulfile to convert unmethylated cytosine to uracil while leaving 5-methycytosin
73
unchanged. Amplification of the desired target sequence was performed using PCR
primers (sense primer, GTATTGTGGA GTTTATTATA AGAATTTTGG; antisense
primer, CAAACCCCAA ACATCCAAAA ATACAA) specific for bisulfite-converted
DNA. The PCR products were then isolated and used as a template for methylation
analysis at three CpG sites by three specific Ms-SNuPE primers (primer 1,
TATTATAAGAATTTTGGGA; primer 2, GATTAATTTATTTGTTTAGT TT; primer
3, GATAGATGGGTGTGA). PCR conditions were 95°C for 2 min. and 40 cycles for
95°C for 1 min., 56°C for 1 min., and 72 °C for 1 min., followed by 72°C for 10 min.
Ms-SNuPE conditions were one cycle primer extension of 95°C for 1 min., 45°C for 1
min., and 72°C for 1 min. The final products were then resolved on 15 % polyacryamide
gel. Results were quantitated with a Molecular Dynamics PhosphorImager. The
percentage methylation of each sample is the average of the three CpG sites examined by
Ms-SNuPE. Cloning of bisulfite genomic DNA was done using the TOPO TA cloning
kit (Invitrogen, Carlsbad, CA) and sequencing was done by the USC DNA Core Facility.
74
RESULTS
LAMB3 DNA methylation status in normal human tissues
Since the DNA methylation status in normal human tissues of LAMB3 is
unknown, we analyzed DNA methylation of the LAMB3 promoter in a panel of normal
tissues using bisulfite genomic sequencing. There were a total of 13 CpG sites within the
340 bp promoter region that was analyzed. This promoter, and specifically this 340bp
region is not considered a CpG island because although the GC content is .59, the
observed CpG/expected CpG ratio is only .39, which is lower that the observed/expected
ratio of .65 needed in order to qualify as a CpG island (Takai and Jones, 2002; Takai and
Jones, 2003; Hackenberg et al., 2006). Our study found a tissue specific methylation
pattern of the non CpG dense LAMB3 promoter. The DNA methylation analyzed by
bisulfite genomic sequencing at this region revealed high levels of methylation in most
tissues with the exceptions of placenta and both oral and skin keratinocytes (Figure 3.2).
We found only the epithelial cells such as the placental lining, oral and skin keratinocytes
to be unmethylated. Because LAMB3 is important to the function of keratinocytes, these
results suggest that DNA methylation plays a role in tissue specific expression
The tissue specific DNA methylation pattern found at LAMB3 was similar to the
DNA methylation pattern found at the non CpG dense promoter of RUNX3 (Chapter 2).
Since RUNX3 P1 is methylated in DNA from sperm and testis, we examined if LAMB3
was methylated in these tissues as well by bisulfite sequencing DNA from sperm and
75
Figure 3.2. Bisulfite genomic sequencing of the LAMB3 promoter in normal tissues.
The DNA methylation status of the LAMB3 promoter in normal tissues was analyzed by
bisulfite sequencing. A schematic of the LAMB3 promoter is shown above the bisulfite
genomic sequencing results. Lower black tick marks represent individual CpG sites and
the black arrow represents the transcription start site. The black or white circles represent
methylated or unmethylated CpG sites, respectively. Expression status (+ or -) of
LAMB3 in these tissues is indicated.
Placenta
Brain
Lung
Kidney
WBC
Skin
Keratinocytes
Oral
Keratinocytes
50 bp
Expression
76
testis (Figure 3.3a). In addition we bisulfite sequenced the promoter of SERPINB5
because it too has been found to have a cell-type specific expression and methylation
pattern, being unmethylated at its promoter and expressed only in epithelial tissues
(Figure 3.3b) (Futscher et al., 2002). Both sperm and testis showed high levels of DNA
methylation with most DNA strands being completely methylated at the LAMB3
promoter, while the SERPINB5 gene was unmethylated in sperm and testis, with most
strands being completely unmethylated. Interestingly, these results show that two genes
that have tissue specific methylation can both be differentially methylated in sperm. The
SERPINB5 promoter meets the criteria of a CpG island using the older standard (a 200bp
sequence of DNA with a GC content of .50 and an observed CpG/expected CpG ratio
over 0.6) (Gardiner-Garden and Frommer, 1987; Futscher et al., 2002) while the LAMB3
promoter does not. This suggests that the finding of DNA methylation in sperm at the
LAMB3 and RUNX3 P1 promoter may either be a characteristic of non CpG dense
promoter genes or indicates a difference in the regulation of gene expression throughout
development. The methylation in sperm DNA and subsequent loss of methylation in
somatic tissues suggests that a demethylation occurs through differentiation to allow
expression of LAMB3, a process previously reviewed (Weiss and Cedar, 1997). This is in
contrast to the de novo methylation that occurs to silence SERPINB5 in any non-epithelia
cell type.
77
Figure 3.3. DNA methylation analysis in sperm and testis by bisulfite genomic
sequencing of DNA at the LAMB3 and SERPINB5 promoters. DNA methylation at
the LAMB3 and SERPINB5 promoters was analyzed by bisulfite sequencing in sperm and
testis. A schematic of either the A. LAMB3 promoter or B. SERPINB5 promoter is shown
above the bisulfite genomic sequencing results. Lower black tick marks represent
individual CpG sites and the black arrow represents the transcription start site. The black
or white circles represent methylated or unmethylated CpG sites, respectively.
Testis
Sperm
50kb
50kb
Sperm
Testis
A.
B.
78
LAMB3 expression in normal cells
DNA methylation has been known to play an important role in the regulation of
gene expression, especially in promoters with CpG islands, however whether DNA
methylation influences the expression of genes with non-CpG island promoters is not
known. LAMB3 is known to be very important to the function of keratinocytes and other
types of epithelial cells. The expression of LAMB3 was analyzed by real time RT-PCR in
normal tissues to better understand how the DNA methylation results we obtained
through bisulfite sequencing may be associated with gene expression. LAMB3 was found
to have little to no expression in testis, placenta, brain, lung, kidney, and white blood
cells in comparison to primary skin keratinocytes, which had a 50 fold higher level of
expression (Figure 3.4). This data suggests that DNA methylation plays a role in the
regulation of LAMB3 in normal tissues since the tissues that were highly methylated did
not express LAMB3 whereas the unmethylated keratinocytes highly expressed LAMB3.
LAMB3 promoter methylation status and expression in cell lines.
We next tested various cell lines to understand how the DNA methylation of the
LAMB3 promoter is associated with expression. This study included the fibroblast cell
line LD419, the bladder cancer cell lines LD611, LD605, HT 1376, SCABER, RT4,
LD71, LD137, UM-UC-3, and TCCSUP, as well as the immortalized keratinocyte
HaCaT cell line and the melanoma cell line 623 mel. Both DNA and RNA were isolated
from the same cells for each cell line and DNA methylation analyzed by Ms-SNuPE at
three CpG sites located in the LAMB3 promoter (Figure 3.5a). High levels of DNA
79
Figure 3.4. Expression of LAMB3 in normal tissues. cDNA from testis, placenta,
brain, lung and kidney was purchased from Clontech. mRNA from white blood cells
(WBC) and from primary keratinocytes was isolated in our lab and was reverse
transcribed into cDNA. LAMB3 expression was analyzed by real time RT-PCR.
Expression was normalized to the average GAPDH and β-actin values.
Testis Placenta Brain Lung Kidney WBC Skin
Keratinocytes
0.00
0.01
0.02
0.03
0.04
1.38
1.39
1.40
Ratio of LAMB3/GAPDH
80
methylation were found at the LAMB3 promoter in LD419, LD137, UMUC3, TCCSUP
and 623 mel cell lines, while little DNA methylation was found (less that 10%
methylation) in the remaining cell lines (Figure 3.5b). To determine if the observed DNA
methylation levels correlated with gene expression and could play a role in the regulation
of LAMB3, LAMB3 expression in these cell lines was analyzed (Figure 3.5c).
Real time RT-PCR showed that LAMB3 was expressed from the bladder cancer
cell lines that were unmethylated, as well as the HaCaT cell line. LAMB3 expression was
not detected or was very low in LD419 fibroblasts, the 623 mel cell line as well as the
bladder cancer cell lines that had DNA methylation at the LAMB3 promoter. These
results further support the role of DNA methylation in the regulation of the non CpG
dense promoter LAMB3 gene.
Decreased DNA methylation of the LAMB3 promoter and expression of LAMB3
upon 5-Aza-CdR treatment in the bladder cancer cell line UMUC3
The DNA methylation status of LAMB3 corresponded to its expression status in
cell lines, however whether DNA methylation might be directly involved in controlling
the non CpG island LAMB3 promoter was still in need of determination. To further
investigate the role DNA methylation plays in the expression of LAMB3, UM-UC-3 cells,
which have a methylated promoter, were treated for 24 hours with 5-Aza-CdR, a DNA
methyltransferase inhibitor, and DNA methylation as well as LAMB3 expression was
assessed. DNA from UM-UC-3 cells showed high levels of methylation at the LAMB3
promoter as well as no expression of LAMB3 prior to treatment with 5-Aza-CdR as
81
Figure 3.5. LAMB3 methylation and expression in cell lines.. DNA and mRNA were
isolated from the fibroblast cell line LD419, the bladder cancer cell lines LD11, LD615,
HT 137 SCABER, RT4, LD137, UMUC3 and TCCSUP, the immortalized keratinocyte
cell line HaCaT, and the melanoma cell line 623 mel. A. LAMB3 promoter map
indicating the three CpG sites (small lower arrows) analyzed by Ms-SNuPE. Lower black
tick marks represent individual CpG sites and upper black arrow represents the
transcription start site. B. The average DNA methylation (%) at 3 CpG sites in the
LAMB3 promoter as measured by Ms-SNuPE is shown. C. mRNA from cell lines was
reverse transcribed into cDNA. LAMB3 expression was analyzed by real time RT-PCR.
Expression was normalized to GAPDH.
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
LD419
LD611
LD605
HT 1376
SCABER
RT4
LD71
LD137
UMUC3
TCCSUP
HaCaT
623 mel
LAMB3/GAPDH
0
10
20
30
40
50
60
70
80
90
100
LD419
LD611
LD605
HT 1376
SCABER
RT4
LD71
LD137
UMUC3
TCCSUP
HaCaT
623 mel
% DNA methylation
A.
B.
50 bp
C.
82
determined by bisulfite sequencing and real time RT-PCR, respectively. However, after
5-Aza-CdR treatment, UM-UC-3 cells harvested on day 3 and day 8 showed expression
from LAMB3. In addition, the DNA methylation levels at the LAMB3 promoter were
decreased from 94% in untreated UM-UC-3 to 75% on day 3 and 67% on day 8. We also
used the RT4 bladder cancer cell line as a positive control, as it had a fully unmethylated
LAMB3 promoter and showed expression of LAMB3 (Figure 3.6). The increase in
LAMB3 expression and a decrease in methylation of the LAMB3 promoter after 5-Aza-
CdR treatment suggests that DNA methylation is directly involved in silencing the
expression of LAMB3.
Chromatin immunoprecipitation analysis at the LAMB3 promoter
Post-translational histone modifications cause changes in chromatin structure,
which can result in an increased accessibility of the DNA to transcriptional activators or
repressors (Rice and Allis, 2001). It is known that DNA methylation and histone
modifications can specify transcriptional competency and cooperatively establish long-
term, cell-type specificity (Bird, 2001; Rice and Allis, 2001). The methylation of histone
H3 lysine 4 (H3K4) and acetylation of histone H3 lysine 9 (H3K9) is important for
transcriptional activation (Nguyen et al., 2001; Liang et al., 2004) while the methylation
83
Figure 3.6. Expression and DNA methylation analysis of LAMB3 in the RT4 cell
line and the UM-UC-3 cell line after 5-Aza-CdR treatment. A. LAMB3 expression
was measured by real time RT-PCR in the bladder cancer cell line RT4 as well as
untreated and 5-Aza-CdR (3uM) treated UM-UC-3 (day 3 or day 8). Expression was
normalized to GAPDH. B. DNA methylation of LAMB3 was analyzed by bisulfite
sequencing in the RT4 cell line and the UM-UC-3 cell line before and after treatment
with 5-Aza-CdR (3uM) at day 3 or day 8. The black or white circles represent methylated
or unmethylated CpG sites, respectively.
50 bp
RT4 UM-UC-3
UM-UC-3 (D3)
+ 5-Aza-CdR
UM-UC-3 (D8)
+ 5-Aza-CdR
0
5
10
15
20
25
30
35
RT4 UM-UC-3 UM-UC-3
(D3)
UM-UC-3
(D8)
Expression of LAMB3/ GAPDH
A.
B.
84
of histone H3K9 is associated with transcriptional repressive heterochromatin formation
(Iizuka and Smith, 2003). Recent studies have demonstrated that the trimethylation of
histone H3K9 is critical for the establishment of cytosine methylation and
heterochromatinization (Stancheva, 2005). It might be expected that the accompanying
histone marks would reflect the inactive transcriptional state and would further prohibit
access for transcription factors to the LAMB3 promoter. Chromatin accessibility was
analyzed around the transcriptional start site of LAMB3 by chromatin
immunoprecipitation (ChIP) analysis (Figure 3.7). ChIP analysis was performed on both
the unmethylated and expressing RT4 bladder cancer cell line as well as the methylated
and expressing UM-UC-3 bladder cancer cell line. Both acetylation of histone H3 and
di-methylation of H3K4 was observed in the bladder cancer cell line RT4. In contrast the
bladder cancer cell line UM-UC-3 showed extremely low levels of acetylated histone H3
and methylated H3-K4 in region 2 and 3 with a high level of the inactive chromatin mark,
trimethylated H3-K9 in region 2 (Fig. 3.7).
As we expected, our results clearly showed a link between DNA methylation and
chromatin reconfiguration. Our previous study and others have showed that methylated
H3-K4 and acetylated H3 are both highly localized to transcriptional start sites of
transcriptionally active genes (Liang et al., 2004; Schneider et al., 2004). These results
also confirmed that the region we studied was the true promoter region of LAMB3.
85
Figure 3.7 LAMB3 ChIP analysis of histone H3 lysine 9/14 acetylation, lysine 4
dimethylation, and lysine 9 trimethylation in the RT4 and UM-UC-3 cell lines. A.
LAMB3 promoter map displaying the location of the regions analyzed by ChIP. B. ChIP
was performed on the RT4 and UM-UC-3 cell lines as indicated, with anti-acetylated
histone H3 K9/14, anti-dimethyl histone H3 K4 and anti-trimethyl histone H3 K9
antibodies. Real time PCR analysis was performed with immunoprecipitated DNA at the
3 LAMB3 regions (R1,R2 and R3) shown. The experiment was done in duplicate and the
average was plotted relative to input. Error bars represent the range.
Acetylated H3-K9/14
0
0.5
1
1.5
2
2.5
3
R1 R2 R3
% (IP/Input)
Dimethylated H3-K4
0
2
4
6
8
R1 R2 R3
%(IP/Input)
Trimethylated H3-K9
0
0.2
0.4
0.6
0.8
R1 R2 R3
% (IP/Input)
UMUC3
RT4
A.
R2
R3
R1
B.
86
Hypomethylation of LAMB3 in bladder cancer
The promoter of LAMB3 gene has been reported to be hypermethylated in
prostate, lung, breast, and bladder cancers and unmethylated in normal tissues
(Sathyanarayana et al., 2003a; Sathyanarayana et al., 2003b; Sathyanarayana et al.,
2003c; Sathyanarayana et al., 2004). We further analyzed the DNA methylation status in
the promoter region of LAMB3, which had been previously reported to be
hypermethylated in bladder cancer (Sathyanarayana et al., 2004), in 2 normal urothelial
controls and 21 matched sets of primary tumors and matched adjacent normal bladder
mucosa by the quantitative Ms-SNuPE assay (Figure 3.8a). The promoter region was
highly methylated from about 60 to 65% in the 2 normal controls. The bladder matched
sets revealed hypomethylation in the primary tumors in12 of the 21 pairs. To confirm our
results, set #34 was bisulfite genomic sequencing along with a normal urothelial control
(NB1). We were able to confirm that the tumors did lose methylation at the LAMB3
promoter in comparison to the methylated adjacent normal and normal urothelium
(Figure 3.8b). These results indicate that LAMB3 might play a role during tumorigenesis
in bladder cancer by loss of methylation, not by gain of methylation as previously
indicated by another group (Sathyanarayana et al., 2004). This is interesting because that
would suggest two mechanisms by which methylation may play a role in the
carcinogenesis in the same organ, such as the bladder.
87
Figure 3.8. DNA methylation status of LAMB3 methylation in bladder cancer
matched sets. A. DNA methylation of the LAMB3 promoter was analyzed by Ms-
SNuPE in normal bladder tissues as well as 21 sets of primary bladder tumors and
matched adjacent normal tissues, as indicated. DNA from metastasized bladder tumor
cells of 2 matched sets was also analyzed for methylation. The average DNA
methylation (% ) at 3 CpG sites is shown. B. DNA from a normal bladder (NB1) and
from matched set #34 was bisulfite sequenced. Tumor and adjacent normal are indicated
as T and N, respectively. The LAMB3 promoter map is shown above the bisulfite
genomic sequencing results. Lower black tick marks in map represent individual CpG
sites. The arrows indicate the 3 CpG sites analyzed by Ms-SNuPE. Black and white
circles represent methylated or unmethylated CpG sites, respectively.
R2
NB1
Matched
Set #34
N T
0
10
20
30
40
50
60
70
80
90
100
NB1
NB2
#1
#5
#16
#28
#30
#34
#36
#79
#305
#38
#652
#833
#1001
#3629
#3743
#4221
#4421
#4556
#4566
#4606
#4707
% DNA methylation
Adjacent Normal
Tumor
A.
B.
Normal Bladder
88
DISCUSSION
DNA methylation is thought to be essential to development and in establishing
tissue specific expression patterns in differentiated cells. It is well documented that DNA
methylation in gene promoters with CpG islands can lead to gene silencing (Jones and
Baylin, 2007). However, the role of DNA methylation in non-CpG island promoters is
not well understood and it is possible that these promoters may be also involved in the
development and establishment of tissue specific expression patterns. Our results
regarding DNA methylation in the LAMB3 promoter was similar to the tissue specific
methylation data shown with the SERPINB5 gene (Futscher et al., 2002), except that
LAMB3 promoter was heavily methylated in sperm and testis. The DNA methylation
found in sperm and testis at the LAMB3 promoter indicates an occurrence of
demethylation through development resulting in the undermethylation of LAMB3 in its
cell type of expression (Yeivin and Razin, 1993; Weiss and Cedar, 1997). Also, the
similar tissue specific methylation patterns observed in the LAMB3 and SERPINB5
correlated with their expression in normal cells. The limited expression we observed in
normal tissues reflected where the LAMB3 promoter was found to be highly methylated.
This demonstrated that not only can a correlation between tissue specific methylation and
expression be found in a CpG island promoter such as SERPIN5, it be present in a non
CpG island promoter gene, such as LAMB3.
When the association between DNA methylation and expression of LAMB3 was
further investigated in cell lines, again an inverse correlation was observed. This
prompted us to employ the DNA methylation inhibitor 5-Aza-CdR to test if expression of
89
LAMB3 can occur if DNA methylation is removed. Upon treatment with 5-Aza-CdR,
expression was induced from the UM-UC-3 bladder cancer cell line that had been
previously highly methylated and displayed no expression. These results suggested that
the removal of DNA methylation from a methylated non CpG island promoter can cause
expression of that gene.
Because the data suggests that DNA methylation can play a role in the expression
from the LAMB3 promoter, we next sought to investigate if the histone modifications
reflective of the activity of LAMB3 were present. The unmethylated RT4 cell line, which
expresses LAMB3 did contain histone H3 acetylation as well as the dimethylated histone
H3-K4 histone modifications which are associated with transcriptional activity.
Conversely, those active marks were not present in the UM-UC-3 cell line, but rather the
trimethylated histone H3-K9 post-translational modification was present, which is
indicative of gene inactivity.
Several studies from the same research group have reported hypermethylation in
the promoter of LAMB3 in various cancers including bladder cancer (Sathyanarayana et
al., 2003a; Sathyanarayana et al., 2003b; Sathyanarayana et al., 2003c; Sathyanarayana et
al., 2004). Our study clearly showed high levels of DNA methylation in the bladder
cancer free normal tissues as well as the adjacent normal tissues from the matched sets,
which was opposite of the low levels of DNA methylation we observed in many of the
bladder primary tumors. This discrepancy can be explained by the realization that
Laminin 5 (encoded by LAMA3, LAMB3, and LAMC2 respectively) is a protein that has
many different functions, two of which are cell adhesion and cell motility (Aumailley and
90
Rousselle, 1999). Therefore the loss of LAMB3 in some tissues may cause a loss of the
cell’s adhesion to the basement membrane contributing to the cancer phenotype while the
overexpression of LAMB3 can alternatively lead to increased cell motility. An induction
of laminin-5 occurs naturally during wound healing (Gil et al., 1994). However,
unregulated overexpression has been observed in various invasive epithelial tumors and
its role as a potential oncogene correlating with tumor invasion has been reviewed
(Katayama and Sekiguchi, 2004). Our results support that it is the latter situation that
occurs. Furthermore, hypomethylation of LAMB3 has also been shown be a diagnostic
marker of bladder cancer and can be detected not only in tumor tissues but patient urine
sediments (Friedrich et al., 2004).
The compilation of data from our study strongly suggests that epigenetics does
influence the expression of the non CpG island promoter gene LAMB3. We have shown
in normal cells, DNA methylation can play a role in the tissue specific expression of
LAMB3. Our data also suggests that as cancer progresses and a genome-wide
hypomethylation occurs, it is possible that these non CpG islands are affected and can
contribute to the progression and metastasis of cancer by acting as oncogenes. Our study
warrants further investigation of DNA methylation of other non CpG island promoter
genes for a better understanding of these types of genes in normal cellular processes as
well as in tumorigenesis.
91
CHAPTER 4
EFFECTS OF S-ADENOSYLHOMOCYSTEINE HYDROLASE INHIBITORS
ON THE EPIGENOME
INTRODUCTION
Epigenetic mechanisms regulate heritable genetic expression patterns and are
necessary for the proper development and function of mammals. DNA methylation,
covalent and noncovalent modifications of histones are epigenetic processes that work
together to control gene expression and stabilize the genome. Errors in any of these
processes can contribute to tumorigenesis and result in cancer (Jones and Baylin, 2007).
New therapeutic approaches have been developed with the goal of reversing the
epigenetic marks that lead to gene silencing (Yoo and Jones, 2006). Three such drugs
have been FDA approved for the treatment of cancer. 5-Aza-2´-deoxycytidine (5-Aza-
CdR) and 5-azacytidine (5-Aza-CR), inhibitors of DNA methylation, have been approved
for the treatment of myelodysplastic syndromes. Suberoylanilide hydroxamic acid
(SAHA), a general deacetylase inhibitor, has been approved for the use in treatment of
cutaneous T-cell lymphomas. These three drugs have been shown to have synergistic
effects when used together (Cameron et al., 1999; Egger et al., 2007).
Two separate epigenetic pathways have been shown to be dysregulated in cancer.
The first is the well known silencing of genes by aberrant DNA methylation (Miranda
92
and Jones, 2007) and the second is the silencing of genes mediated by the Polycomb
repressor complex 2 (PRC2), which is often independent of DNA methylation (Gal-Yam
et al., 2008; Kondo et al., 2008b). PRC2 plays an important role during development and
the methyltransferase component, enhancer of zeste homolog 2 (EZH2), is responsible
for the trimethylation of histone H3 at lysine 27 (H3K27me3), which is a repressive
chromatin mark (Cao and Zhang, 2004). Overexpression of EZH2 has been associated
with a number of cancers including melanoma, lymphoma, breast, and prostate cancers
(Visser et al., 2001; Varambally et al., 2002; Bracken et al., 2003; Yu et al., 2007). Since
the polycomb system and DNA methylation can work through different pathways, a
combination treatment using an inhibitor to the PRC2 complex as well as an inhibitor of
DNA methylation could target the different sets of genes silenced by the two
mechanisms. This would allow for a simultaneous re-activation of genes by epigenetic
therapy.
Recently, 3-deazaneplanocin A (DZNep) was discovered to selectively inhibit
H3K27me3 and H4K20me3 (trimethylation of histone H4 at lysine 20) as well as to
induce apoptosis in cancer cells (Tan et al., 2007). This makes DZNep a possible
candidate as an epigenetic therapeutic for the treatment of cancer. However, DZNep is a
known S-adenosylhomocysteine (AdoHcy) hydrolase inhibitor, which leads to the
indirect inhibition of S-adenosyl-methionine (AdoMet) dependent reactions including
those carried out by many methyltransferases (Figure 4.1a) (Borchardt et al., 1984;
Chiang, 1998). Therefore it is surprising that DZNep’s effects on cancer cells were found
to be relatively specific to EZH2.
93
Figure 4.1. Methyltransferase inhibitors. A. A schematic of AdoMet (S-
adenosylmethionine) metabolism and methyltransferase inhibition is shown.
AdoMet, the universal methyl donor for methylation reactions, is metabolized to AdoHcy
(adenosylhomocysteine) after interaction with methyltransferases. AdoHcy is then
converted into adenosine and homocysteine by AdoHcy hydrolase. DZNep and Adox
inhibit AdoHcy hydrolase causing an increase in AdoHcy levels. AdoHcy in turn inhibits
the methyltransferases. Sinefungin can directly inhibit methyltransferases. B. Chemical
structures of 5-Aza-CdR (5-Aza-2´-deoxycytidine), DZNep (3-deazaneplanocin A),
Sinefungin (Adenosylornithine) and Adox (adenosine-dialdehyde) are shown. The
enzymes that are inhibited by each drug are indicated below the structures in parenthesis.
AdoMet AdoHcy Hcy + Adenosine
Methyltransferase AdoHcy
Hydrolase
Adox
DZNep
Sinefungin
A.
B.
Sinefungin
(Methyltransferases)
5-Aza-CdR
(DNA
Methyltransferases)
Adox
(SAH Hydrolase)
DZNep
(SAH Hydrolase)
94
To better understand how DZNep affects cancer cells and to test its potential as an
epigenetic therapeutic, the mechanism by which DZNep inhibits H3K27me3 was further
explored by comparing its affect on cancer cells to other methyltransferase inhibitors. It
was found that the decrease in H3K27me3 was not specific to DZNep and that DZNep
affects multiple histone methyltransferases. It had been shown previously that DZNep
could activate genes without a loss in methylation (Tan et al., 2007). Further
investigation of the KRT7 gene revealed that only the transcript generated from the KRT7
promoter with a CpG island could be activated after a decrease in DNA methylation by 5-
Aza-CdR treatment. KRT7 was also found to have another downstream transcript
without a CpG island that could only be activated by treatment with DZNep, suggesting
that there are two mechanisms involved in silencing this gene in cancer. This data was
reflected in the microarray results in that DZNep was found to epigenetically reactivate a
different cohort of genes than those of 5-Aza-CdR. Taken together, this data provides
greater detail of the types of genes affected by methyltransferase inhibitors, and supports
their use in epigenetic therapy.
95
MATERIALS AND METHODS
Cells and drug treatments. MCF7 and T24 were purchased from the American Type
Culture Collection and cultured according to the ATCC recommendations. The
immortalized keratinocyte HaCaT cell line was obtained as described previously
(Boukamp et al., 1988) and was grown in modified MEM supplemented with 10% FCS.
For drug treatments, cells were seeded the day before the drugs were added. Cells were
seeded at 700,000 cells per 10 cm dish. Cells were then treated with 1, 5 or 10 µM
DZNep (Glazer, 1986), 10-100 µM Adox (Sigma), 50-150 µM Sinefungin (Sigma), and
1-3 µM 5-Aza-CdR (Sigma) for 72 h.
Western blots. Cells were harvested by treatment with trypsin and resuspended in RIPA
buffer. The resuspended cells were lysed by 2 cycles of sonication for 15 sec. Equal
amounts of protein (20-50 µg) were separated on SDS–polyacrylamide gels and
transferred to PVDF membranes. The blots were probed with antibodies against EZH2,
SUZ12, PCNA, histone H3 and the following histone modifications: H3K27me3
(Upstate), H3K9me2, H3K79me3, H4K20me3, H3K9me3, H3K4me3, H3K9me1,
H3K36me3, H3R2me2.
Rapid amplification of cDNA ends (5´-RACE). Total RNA was extracted using
TRIzol (Invitrogen) from MCF7 cells treated with either 5 µM DZNep or 1 µM 5-Aza-
CdR for 72 h. The 5´ends of mRNA were determined using the RLM-RACE Kit
96
(Ambion) according to the manufacturer’s instructions. The 5´to 3´ sequences of gene-
specific primers were: KRT7 P1 outer: 5´-TGCAGTGCCTCAAGCTGA-3´; KRT7 P1
inner: 5´-GGAGGCAAACTTGTTGTTGAGGGT -3´; KRT7 P2 outer: 5´-
ATTGAGGGTCCTGAGGAAGTTGAT-3´; KRT7 P2 inner: 5´-
CTTCAGCACCACAAACTCATTCTCAG-3´. Universal outer and inner primers were
provided with the kit. The 5´-RACE reaction products were cloned using the TOPO-TA
cloning kit (Invitrogen) and were sequenced.
Quantitative real-time PCR. Total RNA was isolated from cell lines using TRIzol
(Invitrogen) or the RNAeasy Mini Kit (Qiagen). Reverse transcription using MMLV-RT
(Invitrogen) was done according to the manufacturer’s instructions. Quantitative real-
time PCR was performed using TaqMan probes (Applied Biosystems). Expression of
KRT7 was normalized to GAPDH. Primers and probe for KRT7 P1: sense primer, 5´-
AGCAGATCAAGACCCTCAA-3´, and antisense primer 5´-
GGCCTCAAAGATGTCTGG-3´; KRT7 P2: sense primer, 5´-
GGGAGTCCAGGGAAGGAGTA-3´, and antisense primer 5´-
AGGTCAAGAGGGTGCACAGA-3´, probe, 5´-CTGCTCAGGGAGTTCCGA-3´. PCR
conditions were as follows: 95°C for 10 minutes, followed by 40 cycles of 95°C for 15
seconds and 60°C for 1 minute.
Quantitation of DNA methylation by pyrosequencing. Pyrosequencing analysis was
performed on bisulfite-converted DNA, prepared as described previously (Frommer et
97
al., 1992; Yang et al., 2006). Briefly, the promoter regions of interest were first
amplified by PCR using biotinylated anti-sense primers. Next, 10µl of the PCR product
was used for individual sequencing. Strepavidin-Sepharose beads (Amersham
Biosciences) and a Vacuum Prep Tool (Biotage AB) were used to purify the single-
stranded biotinylated PCR products according to manufacturer’s recommendation. The
primers used for amplification of promoter regions were: KRT7 P1, sense 5´-
TTGTTTGGATTGAAAGTTTGGGTTT-3´, antisense 5´-Biotin-
ACTAAACTTCCACAAATAAAA-3´; KRT7 P2 sense 5´-
GTAGGGAAGGTGTGGGGTTA-3´; antisense 5´-Biotin-
CCATACCCTCAAAATTACACTCCCT-3´.
The appropriate sequencing primer was annealed to the purified PCR product.
Pyrosequencing reactions were performed in a 96-well plate format using the PSQ 96HS
system (Biotage). The sequencing primers were KRT7 P1 S1:5´-TTGTTTG
GATTGAAAGTTTGGGTTT-3´; and KRT7 P2, S1: 5´-
GTTATTATTTAGTTTTTGTTGTTAGGATTAA-3´. Raw data were analyzed using the
allele quantitation algorithm using the provided software.
Chromatin Immunoprecipitation. ChIP analyses were done as described previously
(Liang et al., 2004) except that 25 µg of DNA was used for each chromatin
immunoprecipitation. Antibodies used are as follows: anti-histone H3 (Abcam), anti-
histone H3K27me3 (Upstate), anti-acetyl histone H3 (Abcam), anti-histone H3K9me2
(Abcam), anti-histone H3K4me3 (Abcam), and anti-IgG (Upstate) as a negative control.
98
Real time real time PCR was used to quantify each histone mark found within the KRT7
P1 and KRT7 P2 region. Sequences for primer and probes are as follows: KRT7 P1:
sense primer, 5´-AAGCCTTCCCTCACTGAGTCC-3´ and antisense primer 5´-
TTCAGTCCAAGCAGGGATGG-3´, probe, 5´- -3; KRT7 P2: sense primer, 5´-
GTACAGGGAGGGTCCCTGTGT-3´ and antisense primer 5´-
AGCCAGGTCAAGAGGGTGC-3´, probe, 5´-
CAGCTGCTCAGGGAGTTCCGATCTG-3´. PCR conditions were as follows: 95°C for
10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute.
Microarray Analysis. Cells were treated with either 5 µM DZNep or 1 µM 5-Aza-CdR
for 72 h. Total RNA was isolated from MCF7 cells with the RNAeasy Mini Kit. To look
at global gene expression, RNA was hybridized to the human 6 v2 Expression BeadChip
(Illumina) and data analysis was performed using Illumina microbead studio software.
99
RESULTS
DZNep inhibits global histone methylation through an indirect mechanism
To decipher the mechanism by which DZNep inhibits EZH2, its effects on EZH2
was compared to that of a DNA methylation inhibitor (5-Aza-CdR), another AdoHcy
hydrolase inhibitor (adenosine-dialdehyde), and a global methyltransferase inhibitor
(Sinefungin) (Figure 4.1a and 4.1b) (Vedel et al., 1978; Bartel and Borchardt, 1984).
MCF7 breast cancer and T24 bladder cancer cell lines were treated for 72h with each
compound and harvested for Western blot analysis (Figure 4.2). 5-Aza-CdR had no
effect on EZH2 protein levels or on the global amount of H3K27me3. Sinefungin, a
global and direct inhibitor of methyltransferases, caused a decrease in EZH2 and a
decrease in H3K27me3 levels in T24 cells. This suggested that the direct inhibition of
EZH2 caused this protein to be degraded. However, the same decrease in EZH2 protein
levels was not observed in MCF7 cells upon treatment with sinefungin. The differences
observed between the two cell lines may have been due to the differences in sinefungin’s
metabolism or in its cellular uptake (Xu et al., 2007). Next, the effect of AdoHcy
hydrolase inhibitor adenosine-dialdehyde (Adox) on EZH2 levels and H3K27me3 were
examined. Adox, like DZNep, was found to cause a decrease of EZH2 protein levels as
well as a global decrease in H3K27me3. This implied that the degradation of EZH2 and
subsequent decrease in H3K27me3 levels were not specific to DZNep. Other studies
have shown that AdoHcy inhibits methyltransferases upon treatment of cells with Adox
100
Figure 4.2. The effects of methyltransferase inhibitors on EZH2 and H3K27me3.
The MCF7 breast cancer and T24 bladder cancer cell lines were treated with either 5-
Aza-CdR (1µM), DZNep (5 or 10µM), Sinefungin (150µM) or Adox (50µM) for 72 h
and then harvested. EZH2 and H3K27me3 levels were measured by western blot
analysis using whole cell extracts. Histone H3 and PCNA are loading controls.
(Westerns performed by Christine B. Yoo)
EZH2
Untreated
1µM 5-Aza-CdR
10µM DZNep
150µM Sinefungin
50µM Adox
MCF-7 T24
PCNA
H3K27me3
Histone H3
Untreated
1µM 5-Aza-CdR
5µM DZNep
150µM Sinefungin
50µM Adox
101
(Bartel and Borchardt, 1984). Therefore, the inhibition of EZH2 observed in cells treated
with DZNep was most likely due to an increase in AdoHcy levels, which then directly
inhibited EZH2. Taken together, these data infer that DZNep can act indirectly to inhibit
EZH2 and subsequently cause a decrease in H3K27me3, similar to other compounds.
Next, it was investigated whether the effect of DZNep was selective for
H3K27me3 or if it also affected other histone methylation marks. MCF7 cells were
treated for 72h with either 1µM or 5µM DZNep and protein extracts were analyzed by
western blot analysis for EZH2 and several other histone marks (Figure 4.3). Treatment
with 1 µM DZNep had no effect on EZH2 levels whereas 5 µM DZNep caused a slight
reduction. It was discovered that in addition to H3K27me3 and H4K20me3, DZNep also
caused a global decrease in most histone modifications that were examined with the
exceptions of H3K9me3 and H3K36me3. It should be noted that these experiments were
repeated three times biologically and although the degree of inhibition varied from
experiment to experiment the trend still remained the same. These data demonstrated that
DZNep acts as a global inhibitor of histone methylation and is not selective to
H3K27me3 and H4K20me3 as was previously reported (Tan et al., 2007).
Even though this data has clearly demonstrated that DZNep acts globally and can
inhibit histone marks associated with both active and repressed transcription, DZNep may
still be clinically useful. HDAC inhibitors are also global inhibitors that affect the
deacetylation of other proteins as well as histones, but are still successfully used in the
treatment of cancer (Drummond et al., 2005; Su et al., 2008). Therefore, further
investigation of DZNep’s potential as an epigenetic therapeutic was warranted.
102
Figure 4.3. The effects for DZNep on global histone methylation. MCF7 cells were
treated with either 1 µM or 5 µM DZNep for 72 h. Cells were harvested and global
histone methylation levels were determined by western blot analysis. (Western blots
carried out by Christine B. Yoo). Histone methylation marks associated with either
inactive or active transcription are noted.
DZNep (µM) 0 1 5
SUZ12
EZH2
H3K27me3
H4K20me3
H3K9me2
H3K79me3
H3K9me3
H3K4me3
Histone H3
H3K9me1
H3K36me3
H3R2me2
Inactive
Active
103
Two distinct mechanisms are involved in the silencing of a single gene
It was previously shown that DZNep, but not 5-Aza-CdR, could cause re-
expression of genes silenced by DNA methylation without causing DNA demethylation
(Tan et al., 2007). Since it is atypical for a transcript containing a methylated CpG island
in its promoter to be re-expressed without demethylation of the CpG sites, further
investigation of this anomaly was needed. KRT7 was one of the genes which was shown
to be reactivated upon treatment with DZNep without undergoing demethylation of the
CpG island in its promoter (Tan et al., 2007), therefore making this gene interesting to
study.
A 5´-RACE was performed on mRNA isolated from MCF7 cells that had been
treated with either 5-Aza-CdR or DZNep for 72 h and two alternate transcripts of KRT7
were identified (Figure 4.4). The second transcript, which was identified in cells treated
with DZNep, had not been previously described. Primers specific to each transcript
revealed 5-Aza-CdR could induce the expression of the transcript generated from the
promoter containing the methylated CpG island (P1) (Figure 4.5a). In contrast, DZNep
was able only to induce the expression of the second transcript generated from a
downstream promoter located in a CpG poor region (P2) (Figure 4.5b).
It was next of interest to determine whether the second promoter’s transcript
normally occurs in an epithelial cell or if DZNep was activating a cryptic promoter. Both
MCF7 and T24 cell lines were treated with 5µM DZNep and the expression of both P1
and P2 KRT7 transcripts in these two cell lines were compared to the expression found in
the immortalized keratinocyte cell line HaCaT. Both the P1 and P2 transcripts were
104
Figure 4.4. Map of KRT7. 5´ RACE was performed on RNA purified from MCF7 cells
treated with either 1 µM 5-Aza-CdR or 5 µM DZNep for 72h. The two KRT7 transcripts
found by 5´ RACE are depicted. P1, promoter 1; P2, promoter 2; black boxes, exons;
gray box, CpG Island; tick marks, CpG sites; bent arrows, transcriptional start sites
determined by 5´RACE; vertical arrows, CpG sites analyzed by pyrosequencing; gray
hatched squares, ChIP PCR products. Black triangles represent the binding sites for the
primers specific to the promoter 1 (P1) transcript whereas the white triangles indicate the
binding sites for the primers specific to the promoter 2 (P2) transcript.
105
Figure 4.5. KRT7 P1 and P2 expression after drug treatments. MCF7 cells were
treated with either 1 µM 5-Aza-CdR or 5 µM DZNep for 72 h. Real time RT-PCR was
used to verify the activation of either A. KRT7 P1 or B. P2 promoter by 5-Aza-CdR and
DZNep. Error bars represent the range from two biological repeats.
KRT7 P1 Transcript Expression
0
0.003
0.006
0.009
0.012
0.015
PBS 1µM 5-Aza-CdR 5µM DZNep
Expression/ GAPDH
KRT7 P2 Transcript Expression
0
0.05
0.1
0.15
0.2
0.25
PBS 1µM 5-Aza-CdR 5µM DZNep
Expression/GAPDH
A.
B.
106
expressed in the HaCaT and T24 cell lines (Figure 4.6). MCF7 cells expressed only the
KRT7 P2 transcript after DZNep treatment. Therefore KRT7 P2 is not a cryptic promoter
and the transcript generated from this promoter is expressed in keratinocytes, yet
becomes silenced in some epithelial cancers and can be re-expressed upon DZNep
treatment.
Next, the effects of DZNep on DNA methylation were determined. DNA from
MCF7 cells treated with 5 µM DZNep were analyzed for DNA methylation by
pyrosequencing both KRT7 promoters. DZNep did not affect the levels of DNA
methylation within either the KRT7 P1 or P2 promoters, confirming previous studies (Tan
et al., 2007). However, 5-Aza-CdR caused a 27 % decrease in CpG methylation in the
KRT7 P1 promoter (Figure 4.7). This confirms that CpG methylation is the primary
mechanism through which expression of the P1 transcript, but not the P2 transcript, is
controlled.
These results show that there are two mechanisms for silencing one gene and
demonstrate the need for combinatorial drug treatments for maximum expression of a
gene. This study also highlights the importance of knowing the exact start site of the
transcript being studied when analyzing epigenetic modifications.
Decrease in H3K27me3 is correlated with an increase in the expression of the KRT7
P2 transcript
Chromatin immunoprecipitation (ChIP) was used to study the local changes in
histone modifications after treatment of MCF7 cells with either 5-Aza-CdR or DZNep
107
Figure 4.6. KRT7 P2 transcript occurs in immortalized keratinocytes and in
epithelial bladder cancer cells. RNA was isolated from the MCF7, T24 and HaCaT cell
lines. Real time RT-PCR was used to verify the presence of either the KRT7 P1 and P2
transcript.
KRT7 P2 Transcript Expression
0.00E+00
2.00E-02
4.00E-02
6.00E-02
8.00E-02
1.00E-01
1.20E-01
1.40E-01
MCF7 + PBS MCF7 + DZNep T24 + PBS T24 + DZNep HaCaT
KRT7 P2/GAPDH
KRT7 P1 Transcript Expression
0.00E+00
2.00E-03
4.00E-03
6.00E-03
8.00E-03
1.00E-02
1.20E-02
1.40E-02
1.60E-02
MCF7 + PBS MCF7 + DZNep T24 + PBS T24 + DZNep HaCaT
KRT7 P1/GAPDH
108
Figure 4.7. KRT7 P1 and P2 DNA methylation status after 5-Aza-CdR or DZNep
treatment. MCF7 cells were treated with either 1 µM 5-Aza-CdR or 5 µM DZNep for 72
h. Pyrosequencing was used to analyze the levels of DNA methylation at either the A.
KRT7 P1 or B. P2 promoter. Error bars represent the range from two biological repeats.
KRT7 P1
0
20
40
60
80
100
PBS 1µM 5-Aza-CdR 5µM DZNep
% DNA Methylation
KRT7 P2
0
20
40
60
80
100
PBS 1µM 5-Aza-CdR 5µM DZNep
%DNA Methylation
A.
B.
109
(Figure 4.8). Treatment with 5-Aza-CdR did not affect the H3K27me3 levels at either
the P1 or P2 promoter. However, treatment with DZNep resulted in approximately a
50% decrease in H3K27me3 at both promoters. Both 5-Aza-CdR and DZNep treatments
also caused a decrease in the inactive H3K9me2 mark at the P1 and P2 promoters. 5-
Aza-CdR generated an increase in H3K4me3 in the P1 promoter whereas treatment with
DZNep resulted in no change in relative enrichment of this mark. No change was
observed in the levels of acetylated H3, however, these levels were high initially in the
P2 promoter.
These results showed that a decrease in H3K27me3 levels occurs as the KRT7 P2
promoter is activated. Although DZNep also affected H3K9me2 levels at KRT7 P2, a
decrease in H3K9me2 alone cannot cause activation of the P2 transcript. This was
illustrated when MCF7 cells were treated with 5-Aza-CdR. 5-Aza-CdR caused a similar
decrease in H3K9me2 levels at KRT7 P2, but was unable to reactivate this transcript.
Together this data infers that H3K27me3 is the principal mechanism by which KRT7 P2
is silenced in MCF7 cells. (ChIPs were primarily performed by by Dr. Tina Miranda)
DZNep and 5-AzaCdR activate different sets of genes
Next, genes that can be activated by 5-Aza-CdR and genes that can be activated
by DZNep were compared. RNA extracted from MCF7 cells treated with either 1 µM 5-
Aza-CdR or 5 µM DZNep for 72 h was subjected to microarray analysis. In addition to
KRT7 (Figure 4.5), the LAMB3 and KRT17 genes were found to be upregulated by
110
Figure 4.8. KRT7 P1 and P2 ChIP analysis. ChIP analysis of the KRT7 P1 and P2
locus after treatment of MCF7 cells with either 1µM 5-Aza-CdR or 5µM DZNep for 72h.
ChIP analysis for histone H3K27me3, H3K9me2, H3K4me3, and acetyl H3. Error bars
represent the range between duplicate ChIPs.
H3K27me3
H3K9me2
H3K4me3
Acetyl H3
0
0.05
0.1
0.15
0.2
0.25
0.3
KRT7 P1 KRT7 P2
IP/H3
PBS
1µM 5-Aza-CdR
5µM DZNep
0
0.05
0.1
0.15
0.2
0.25
0.3
KRT7 P1 KRT7 P2
IP/H3
0
0.3
0.6
0.9
1.2
1.5
1.8
KRT7 P1 KRT7 P2
IP/H3
0
0.1
0.2
0.3
0.4
0.5
0.6
KRT7 P1 KRT7 P2
IP/H3
111
DZNep and were validated by real time RT-PCR (Figure 4.9). Scatter plots for treated
versus untreated cells (Figure 4.10) show that while 5-Aza-CdR works mostly to
upregulate genes, DZNep can both upregulate and downregulate genes. This may be the
result of DZNep’s ability to reduce H3K4me3 levels (Figure 4.3). The Venn diagram
(Figure 4.11) shows that DZNep treatment resulted in the activation of 154 genes
whereas treatment with 5-Aza-CdR resulted in the activation of only 68 genes (>2 fold)
in MCF7 cells. Only seven genes were commonly upregulated by both drugs. Further
analysis is necessary to determine whether the commonly regulated genes are in fact the
result of different transcripts as has been demonstrated for KRT7.
In order to determine whether 5-Aza-CdR and DZNep turn on genes involved in
different functional categories, an EASE analysis was performed (by Tina Miranda). 5-
Aza-CdR was found to affect genes mostly involved in defense responses to parasites and
wounding whereas DZNep activated genes involved in organogenesis and epidermal and
ectoderm development (Figure 4.12). Since MCF7 is an epithelial cancer cell line it is
exciting that DZNep can cause re-expression of epidermal and ectoderm genes that have
been silenced in cancer. It will be interesting to determine if DZNep can cause genes that
are specific to blood cells to be re-expressed in lymphomas or genes that are specific to
muscle development to become activated in muscle cancer cells.
Next a time course was performed to determine the heritability of DZNep’s
effects. MCF7 cells were treated with 10 µM DZNep or 1 µM 5-Aza-CdR for 24h.
Twenty-four hour treatments were chosen to be comparable to previous heritability
studies using 5-Aza-CdR (Cheng et al., 2004). In order to increase the effects of DZNep
112
Figure 4.9. Microarray validation. RNA extracted from MCF7 cells treated with either
1 µM 5-Aza-CdR or 5 µM DZNep for 72 h was subjected to microarray analysis. Real
time RT-PCR was used to validate the expression of KRT17 and LAMB3. Expression
was normalized to GAPDH and error bars on LAMB3 data represent the range from two
biological repeats.
LAMB3 Expression
0
0.01
0.02
0.03
0.04
0.05
PBS 1µM 5-Aza-CdR 5µM DZNep
Expression/GAPDH
KRT17 Expression
0
0.005
0.01
0.015
0.02
0.025
0.03
PBS 1µM 5-Aza-CdR 5µM DZNep
KRT17/GAPDH
113
Figure 4.10. Scatter plots from expression microarray. Genes upregulated or
downregulated more than two-fold upon treatment of MCF7 cells treated with either 1
µM 5-Aza-CdR (top) or 5 µM DZNep (bottom) for 72 h when compared to untreated
MCF7 cells. Gene expression was analyzed using Illumina human 6 microbead array.
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
10
2
10
3
10
4
10
5
Scatter Plot of Genes Affected by DZNep
Scatter Plot of Genes Affected by 5-Aza-CdR
1µM 5-Aza-CdR AVG_Signal 5µM DZNep AVG_Signal
untreated AVG_Signal
untreated AVG_Signal
114
Figure 4.11. Venn diagram showing the overlap of genes upregulated in MCF7 cells
treated with 5-Aza-CdR or DZNep. MCF7 cells were treated with either 1 µM 5-Aza-
CdR or 5 µM DZNep for 72 h. Gene expression was analyzed using Illumina human 6
microbead array.
115
Figure 4.12. EASE analysis showing the categories of genes turned on by DZNep or
5-Aza-CdR. The Bonferroni score was used to determine the significance.
0 10 20 30 40 50 60 70
cellular defense response
response to wounding
response to pest/pathogen/parasite
metal ion binding
development
Gene Catagory
Percent of Represented Genes
5-Aza-CdR
0 10 20 30 40 50 60 70
ectoderm development
organogenesis
epidermal differentiation
intermediate filament cytoskeleton
cytoplasm
cytoskeleton
pathogenesis
Gene Catagory
Percent of Represented Genes
DZNep
116
during the shorter 24hr incubation period, a 10 µM DZNep concentration was used
instead of 5 µM. The heritability of re-expression of the KRT7 gene after treatment with
DZNep or 5-Aza-CdR (Figure 4.13a and b) was examined. It was found that while
DZNep could not activate the KRT7 P1 transcript, the P2 transcript was activated on
day 1. After the DZNep was removed, expression from the KRT7 P2 was lost after 24 h.
When the cells were treated with 5-Aza-CdR, the KRT7 P1 transcript expression levels
gradually raised with the maximum amount of expression observed on day 7 followed by
a gradual decrease. The global changes in gene expression were examined by microarray
analysis on day 1 and day 7 upon treatment of MCF7 cells with either DZNep or 5-Aza-
CdR (Figure 4.13c). The genes that showed an increase in expression by 2-fold or more
after DZNep treatment were higher on day 1 than on day 7. This suggests that other
factors, besides the repressive histone marks, are predisposing these genes to
transcriptional silencing. In contrast, there were many more genes that were upregulated
by more than 2-fold by 5-Aza-CdR on day 7 than on day 1. This microarray data as well
as the data for the KRT7 gene shows that the effects of DZNep are not heritable.
117
Figure 4.13. Comparison of the heritability of gene expression upon treatment of
MCF7 cells with either 5-Aza-CdR or DZNep. MCF7 cells were seeded and the next
day (day 0) were treated with either 1 µM 5-Aza-CdR or 10 µM DZNep for 24 h. Cells
were harvested for RNA extraction every day for 14 days. Real time RT-PCR showing
the heritability of A. KRT7 P1 transcript and B. KRT7 P2 transcript upon drug treatments.
C. Illumina microarray expression showing the numbers of genes upregulated on day 1
and day 7 by the indicated drug treatments.
0
0.001
0.002
0.003
0.004
unt day1 day2 day4 day5 day6 day7 day10 day14
KRT7/ GAPDH
5-Aza-CdR
DZNep
0
0.02
0.04
0.06
0.08
unt day1 day2 day4 day5 day6 day7 day10 day14
KRT7/GAPDH
0
30
60
90
120
150
day 1 day 7
Day cells were harvested
# of genes upregulated
more than 2-fold
A.
B.
C.
118
DISCUSSION
It was previously reported that DZNep was a selective inhibitor of H3K27 and
H4K20 trimethylation (Tan et al., 2007). However, in that particular study the authors
focused only on the H3K27me3, H3K9me3, and H4K20me3 marks, therefore it was the
intention of this study to expand the panel of histone methylation marks investigated to
determine the effects of DZNep. DZNep was not found to be a selective inhibitor of
H3K27me3 and H4K20me3 as previously reported (Tan et al., 2007). Instead DZNep
globally inhibited both repressive as well as active histone methylation marks, similar to
other AdoHcy hydrolase and global methyltransferase inhibitors. In addition, the
inhibition of EZH2 was not found to be specific to DZNep since other AdoHcy hydrolase
and global methyltransferase inhibitors had the same effect on the histone methylation
marks.
It had been reported that that DZNep could turn on genes containing methylated
CpG islands within their promoters without demethylation of the CpG sites, whereas 5-
Aza-CdR was incapable of turning on these genes (Tan et al., 2007). Since this finding
was surprising in view of the current understanding of epigenetic therapy, KRT7 was
further studied. 5´-RACE and real time RT-PCR showed that DZNep activated a novel
transcript that was located further downstream from the CpG island. 5-Aza-CdR
activated the transcript containing the methylated CpG island and this activation was
accompanied by a 27% decrease in methylation of this promoter as measured by
pyrosequencing contrary to the findings in the previous study (Tan et al., 2007).
119
However, in the earlier study, the authors used a different, less potent DNA
methyltransferase inhibitor, 5-Aza-CR, which could account for the discrepancy (Cheng
et al., 2004). Nonetheless, the authors observed no decrease in DNA methylation at any
of the CpG sites they studied upon treatment with 5-Aza-CR suggesting that their
treatments were ineffective. This study therefore stresses the importance of identifying
the exact start site of the transcripts being analyzed when conducting epigenetic studies.
The use of DZNep as a global inhibitor has allowed for the effect of chromatin
modifications on gene expression to be determined. This study has shown that although
histone repressive marks H3K9me2 and H3K27me3 are reversed, a transcript is unable to
be re-expressed if it contains a methylated CpG island. This is in accordance with the
data in which knockdowns of the H3K9me2 methyltransferase or of EZH2 failed to cause
a re-expression of methylated genes (McGarvey et al., 2007; Kondo et al., 2008a). In
addition, this study shows that one gene, KRT7, does not need H3K4me3 methylation in
order to be activated (Figure 4.8) and only the repressive marks only have to be removed.
This does not mean a greater increase in expression of this transcript would be observed
if the active mark was not inhibited.
A compound, such as DZNep, that inhibits multiple repressive marks may be
useful in epigenetic therapy. It has been shown that SUV39H1 and G9a, both H3K9
methyltransferases, are required to perpetuate the malignant phenotype seen in prostate
cancer cells PC3 and that targeting these histone methyltransferases may be of
therapeutic benefit in cancers (Kondo et al., 2008a). Therefore, having a single drug that
can cause in decrease in both H3K27me3 and H3K9me2 could be powerful. However,
120
this study showed DZNep could also inhibit the active histone mark H3K4me3.
Although induction of transcription was found even upon the inhibition of H3K4me3, it
is still unclear what would happen if drugs were available that inhibited the repressive
marks alone and did not affect the active histone marks. It is therefore useful to continue
looking for inhibitors to specific histone methyltransferases.
Since there are different epigenetic mechanisms that silence genes during
carcinogenesis, it is important to develop combination therapies targeting these pathways.
HDAC inhibitors have been shown to act synergistically with both 5-Aza-CdR and
DZNep (Cameron et al., 1999; Egger et al., 2007). It is also possible that DZNep, in
combination with H3K4 demethylase inhibitors, could work together providing a more
powerful epigenetic therapy, however more research is needed (Lee et al., 2006;
Szewczuk et al., 2007). It is also necessary to develop therapies that simultaneously
target DNA methyltransferases and EZH2. This data suggests that combination treatment
with both 5-Aza-CdR and DZNep may be beneficial for cancer treatment.
121
CHAPTER 5
SUMMARY AND CONCLUSIONS
Genome wide studies have revealed the patterns of methylation across the whole
epigenome both at CpG islands and at non CpG islands. While many CpG rich regions
are unmethylated, the methylated CpG island genes tend to be expressed in a tissue
specific manner indicating that DNA methylation plays a role in tissue specific
expression (Eckhardt et al., 2006). Furthermore, these studies have shown that regions
that do not qualify as CpG islands may be epigenetically regulated and important in cell
differentiation (Rakyan et al., 2008), however this area of research is still at an early
stage.
In Chapter 2, the epigenetic mechanisms that aid in the regulation of the RUNX3
gene were explored. This gene has two promoters, RUNX3 P1, which in a non CpG
island promoter, and RUNX3 P2, which is surrounded by a CpG island. Although the
DNA hypermethylation of the CpG island promoter of RUNX3 P2 has been implicated in
many cancers (Kang et al., 2004; Wada et al., 2004; Ito et al., 2005; Jiang et al., 2008;
Rakyan et al., 2008). RUNX3 P1 has not been studied in great detail, and not in the
context of epigenetic regulation. It was unknown whether DNA methylation would play
any role in the transcriptional regulation of RUNX3 P1 due to the limited number of CpG
sites in its promoter. However upon further examination of the DNA methylation
patterns in various cell types, surprisingly this non CpG island promoter was shown to
have a tissue specific methylation and expression pattern.
122
RUNX3 P1 had high levels of DNA methylation in many of the tissues analyzed
including the DNA from sperm, while lower methylation levels were seen in DNA from
lung tissue and white blood cells. Although the presence of DNA methylation at a gene
in sperm usually is indicative of an imprinted gene, transcripts from both RUNX3 P1
alleles were detected using a SNP present in the coding region revealing that RUNX3 P1
was not imprinted. Subcloning experiments, with B lymphocytes, that tested for mono-
allelic methylation and expression were inconclusive, however they did suggest that
RUNX3 P1 was important to the survival and growth of B lymphocytes. RUNX3 is
imperative to the function of T lymphocytes, and since the DNA from white blood cells
displayed a low level of methylation, whole blood was fractionated to determine the
origin of the unmethylated strands. CD4
+
and CD8
+
T cells were further purified from
the fractionated lymphocytes and when analyzed for DNA methylation, the T cells were
found to be devoid of methylation at RUNX3 P1. The transcript from RUNX3 P1 is
known to be very important in the development of CD8
+
T cells (Chung et al., 2007),
therefore providing a link between the expression from P1 and the importance of an
unmethylated RUNX3 P1 in T cells.
The methylation status was examined at both promoters in bladder, colon and
prostate cancer cell lines as well as primary tumor matched sets. Only RUNX3 P2 was
found to have a cancer specific DNA methylation pattern with low levels of methylation
in normal fibroblasts, and hypermethylation in the cancer cell lines as well as in primary
tumors. The induction of both RUNX3 P1 and P2 transcripts after 5-Aza-CdR treatment
in the T24 bladder cancer cell line further supported the role of methylation in the
123
silencing of both promoters. Additionally, when RUNX3 P1 was analyzed for histone H3
acetylation, the presence of this mark coincided with active expression of this transcript
suggesting that histone acetylation, along with DNA methylation can influence the
transcriptional regulation of a non CpG island gene. The study of RUNX3 P1 in normal
tissues supports that promoters that do not contain CpG islands can be regulated by
methylation, while the study of RUNX3 P2 demonstrated that this transcript is silenced in
bladder cancer by DNA hypermethylation contributing to the cancer phenotype.
To further investigate how DNA methylation and histone modifications affect a
non CpG island gene, the LAMB3 gene was studied. Similar to RUNX3 P1, the LAMB3
promoter had a tissue specific methylation pattern, with high levels of DNA methylation
in most normal tissues and little DNA methylation in keratinocytes. Also like RUNX3
P1, DNA from sperm was methylated at the LAMB3 promoter. It is speculated that these
types of non CpG island genes that are methylated in sperm undergo a demethylation
process as differentiation occurs, allowing for their expression in the differentiated cell.
It is still not understood as to why these genes would be methylated in sperm since genes
that are methylated in the germline are most susceptible to CpG deamination and
eventual mutation (Weber et al., 2007). These are still issues that have not been greatly
explored in the scientific literature.
LAMB3 was also shown to have an inverse correlation between DNA methylation
and expression in cell lines. Additionally, when 5-Aza-CdR was administered to a cell
line methylated at LAMB3, expression of the LAMB3 transcript could be induced. Also,
active histone marks were found in unmethylated cell lines, and inactive histone marks in
124
methylated cell lines. When primary bladder tumors and the paired adjacent normal
tissues were analyzed for DNA methylation at the LAMB3 promoter, strikingly there
were many matched sets that displayed hypomethylation in the tumors. This was
contrary to what had been published previously by another group that reported
hypermethylation of the same region (Sathyanarayana et al., 2004). To confirm our
results we bisulfite treated DNA from normal bladder tissue from a bladder cancer free
patient as well as the DNA from one of the paired matched sets that had showed
hypomethylation. The DNA from normal bladder tissue showed high levels of
methylation, as did the DNA from the adjacent normal, while the DNA from bladder
tumor showed many DNA strands that had become demethylated. It is known that during
carcinogenesis a global DNA hypomethylation occurs, and it is possible that the tissue
specific non CpG island genes such as LAMB3 may also become hypomethylated and
lead to the aberrant expression of LAMB3. Since LAMB3 codes for the protein that is an
important component of the laminin 5 complex, which aids in cell migration, the aberrant
expression of LAMB3 could cause an increase in cell motility thereby contributing to
metastasis (Katayama and Sekiguchi, 2004). Here we give additional support of another
non CpG island gene in which DNA methylation and histone modifications play roles in
its transcriptional activity.
The project in Chapter 4 was done in collaboration with Dr. Tina Miranda who
performed many of the ChIPs and microarray analysis, and Dr. Christine Yoo who
carried out the various western blots. The goal of epigenetic therapy is to take advantage
of the reversibility of epigenetic marks that in cancer cause the aberrant silencing of
125
genes normally expressed in a given cell type. The drug DZNep, which is specifically an
AdoHcy hydrolase inhibitor, was compared to other methyltransferase inhibitors to better
comprehend its mechanism. Similar to the other compounds, DZNep was found to be
able to indirectly inhibit the methyltransferase EZH2 and subsequently cause a decrease
in H3K27me3. To test if the effects of DZNep were specific to EZH2 and the
H3K27me3 mark, many other histone marks were also examined after MCF7 breast
cancer cells were treated with DZNep. DZNep was found to inhibit both active and
inactive histone marks. Although these global effects might be questioned when
pursuing a drug for the treatment of cancer, it is important to remember that there are
other drugs, such as the HDAC inhibitors, that also work on a global level but still have
beneficial outcomes.
To determine what genes DZNep could affect, MCF7 breast cancer cells were
treated with either DZNep or 5-Aza-CdR, and gene expression was analyzed by
microarray analysis. Interestingly, the genes that were affected by DZNep were mostly
different from the genes that were affected by 5-Aza-CdR suggesting that two different
mechanisms of silencing genes are involved. The genes that are activated only by
DZNep are most likely primarily governed by histone modifications and their expression
is not dependant on DNA methylation. In contrast, the genes that were only activated by
5-Aza-CdR are DNA methylation dependant. The two scenarios were exemplified by the
KRT7 gene, which has one promoter within a CpG island and another promoter that
contained only 4 CpG sites. The CpG island promoter was activated by 5-Aza-CdR and
was accompanied by a decrease in DNA methylation. Conversely, the second KRT7
126
promoter was found to be unmethylated at CpG sites before DZNep treatment, and
expression was activated only after DZNep treatment. If there are genes that become
silenced in cancer that are not due to DNA methylation, histone methyltransferase
inhibitors may be able to activate their expression. Therefore double treatments of DNA
methylation inhibitors and histone methyltransferase inhibitors may also be beneficial for
the treatment of cancer.
The studies in this thesis have important biological and clinical implications since
better cancer drugs can be developed as more is discovered about how the epigenome as
a whole is affected in cancer, not just regions where there are CpG islands. The purpose
of these studies was to determine whether epigenetic mechanisms regulate non CpG
island genes therefore warranting cancer studies to be extended to non CpG island genes.
As mentioned earlier, DNA methylation and histone modifications are interconnected and
together can influence the regulation of genes (Egger et al., 2007). This thesis has shown
two examples of non CpG island promoters, RUNX3 P1 and LAMB3, where DNA
methylation and histone modifications play a role in gene regulation. However, it has
been shown in some cases that changes in post-translational modifications can play a
greater role in gene expression than DNA promoter methylation (Richon et al., 2000;
Sigalotti et al., 2007). It is possible that transcriptional regulation is not always
influenced by DNA methylation, but rather is primarily dictated by the surrounding
chromatin configuration as seen by the second KRT7 promoter. In addition, by studying
inhibitors that target genes where DNA methylation may not heavily influence gene
expression further opens up an avenue where much can be discovered.
127
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Abstract (if available)
Abstract
DNA methylation and post-translational modifications of histones can specify transcriptional competency in both normal and cancer cells. However these epigenetic processes have largely been studied in the context of genes that contain CpG islands in their promoters. Therefore the epigenetic regulation of genes without CpG islands was explored in order to understand how epigenetics controls the expression of the remainder of the genome and provide insight into what aberrancies occur at these regions during cancer. Additionally, because there is an ongoing quest to find drugs that target the aberrancies that occur in the epigenome during cancer, methyltransferase inhibitors were also studied to test their potential as an epigenetic therapeutic.
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Creator
Cortez, Connie Christina
(author)
Core Title
Epigenetic regulation of non CPG island gene promoters
School
Keck School of Medicine
Degree
Doctor of Philosophy
Degree Program
Biochemistry and Molecular Biology
Degree Conferral Date
2008-12
Publication Date
12/03/2008
Defense Date
10/27/2008
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University of Southern California
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DNA methylation,epigenetics,LAMB3,OAI-PMH Harvest,RUNX3
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English
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DNA methylation
epigenetics
LAMB3
RUNX3