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University of Southern California Dissertations and Theses
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Multiple functions of the PR-Set7 histone methyltransferase: from transcription to the cell cycle
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Multiple functions of the PR-Set7 histone methyltransferase: from transcription to the cell cycle
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Content
MULTIPLE FUNCTIONS OF THE PR-SET7 HISTONE METHYLTRANSFERASE:
FROM TRANSCRIPTION TO THE CELL CYCLE
by
Lauren Marie Congdon
A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(GENETIC, MOLECULAR AND CELLULAR BIOLOGY)
December 2012
Copyright 2012 Lauren Marie Congdon
ii
Dedication
In loving memory of Sarah Douglas Doyle.
iii
Acknowledgements
I would like to express my appreciation to my mentor Dr. Judd Rice for his
guidance. Thank you for always pushing me to do my best and encouraging me to think
critically and creatively. I would also like to thank my committee members, Drs. Amir
Goldkorn, Chih-Lin Hsieh and Woojin An, for their encouragement and helpful
suggestions over the years. I am also thankful to all of the members of the Rice Lab:
Jennifer, Tanya, Shumin, Sai, Creighton, Michael and Shawn. I am grateful not only for
all of the helpful scientific discussions but also for your friendships.
A huge thank you to my wonderful friends for being an incredible support system
and helping me to enjoy life to the fullest. To the South Bay crew and all my other
friends from New York to Arizona, you have all helped to make these last years some of
the best of my life. I would like to express my special appreciation to Ruzbeh, for his
encouragement and love, for supporting me every step of the way and for being my
biggest cheerleader.
Last, but certainly not least, I would like to thank my family. Mom, thank you for
everything you have done for me and continue to do: the advice, friendship, support and
unconditional love. Grandma and Grandpa, I am forever grateful for your boundless
generosity, enthusiasm for education and positive presence in my life. Dad, thank you for
sharing your passion for science and for all the great skiing. Jason and Eric, I feel so
lucky to count my brothers among my closest friends. Without all of your love and
support I would not be where I am today.
iv
Table of Contents
Dedication ii
Acknowledgements iii
List of Tables vi
List of Figures vii
Abstract ix
Introduction 1
Chromatin 1
Histone Methylation and Histone Methyltransferases (HMTs) 2
PR-Set7 and H4 Lysine 20 Monomethylation 3
Dual Roles of PR-Set7 in Transcription 3
PR-Set7 and Transcriptional Repression 5
PR-Set7 and Transcriptional Activation 8
Combinatorial Histone Modifications 12
Chapter 1: PR-Set7 and H4K20me1 function as transcriptional repressors 14
of specific human genes
1.1. Introduction 14
1.2. Results 15
1.2.1. H4K20me1 is preferentially enriched in gene bodies whereas the 15
majority of H4K20me3 is found in repetitive elements
1.2.2. H4K20me1 is required for transcriptional repression of specific 20
endogenous genes
1.2.3. H4K20me1-enriched genes exhibit lower expression at G2/M 31
compared to genes devoid of H4K20me1
1.3. Discussion and future directions 35
Chapter 2: PR-Set7 binds the Riz1 histone H3K9 methyltransferase to 37
establish a trans-tail histone code that may be important
for tumor suppression
2.1.Introduction 37
2.2. Results 38
2.2.1. PR-Set7 selectively and directly binds the Riz1 H3K9 38
methyltransferase
2.2.2. PR-Set7 and Riz1 specifically bind via their C-terminal domains 41
2.2.3. Riz1 predominantly functions as a histone H3K9 44
monomethyltransferase
v
2.2.4. Interaction with PR-Set7 is required for Riz1 recruitment to 48
chromatin
2.2.5. Direct binding to PR-Set7 is required for Riz1-mediated H3K9me1 50
2.2.6. Riz1 is dispensable for the transcription of PR-Set7 regulated genes 53
2.2.7. PR-Set7 interaction with Riz1 may be critical for Riz1 tumor 55
suppressor activity
2.3.Discussion and future directions 59
Chapter 3: PR-Set7 interacts with HDAC3 and this interaction may 62
promote transcriptional repression and epithelial to
mesenchymal transition
3.1 Introduction 62
3.2 Results 63
3.2.1 The PR-Set7 multi-protein complex contains histone deacetylase 63
activity
3.2.2 PR-Set7 specifically interacts with class I HDAC proteins 65
3.2.3 PR-Set7 directly binds the class I HDAC, HDAC3 65
3.2.4 Selective inhibition of class I HDAC activity results in the 67
de-repression of PR-Set7-regulated genes
3.2.5 Ectopic expression of PR-Set7 or HDAC3 is sufficient to alter 71
markers of epithelial-mesenchymal transition
3.3. Discussion and future directions 73
Chapter 4: JNK-dependent phosphorylation of Serine 29 stabilizes 76
PR-Set7 against UV-dependent degradation
4.1. Introduction 76
4.2. Results 78
4.2.1. Osmotic stress promotes JNK-mediated stabilization of PR-Set7 78
against UV-induced degradation
4.2.2. PR-Set7 is phosphorylated at Serine 29 after Osmotic Stress and 81
this modification may block UV-induced ubiquitination of PR-Set7
4.3.Discussion and future directions 84
Chapter 5: Methods 86
5.1. ChIP-cloning 86
5.2. Immunoprecipitation 87
5.3. Plasmids and shRNA 88
5.4. Protein expression and purification 89
5.5. Histone methyltransferase assay 93
5.6. Nuclear fractionation 94
5.7. Cell culture 95
5.8. Stress treatments 95
References 99
vi
List of Tables
Table 1. Identification of genomic locations of H4K20me1 in HeLa 18
cells by ChIP-cloning
Table 2. Identification of genomic locations of H4K20me3 in HeLa 19
cells by ChIP-cloning
Table 3. Genes whose expression change >2-fold in cells lacking 29
PR-Set7
Table 4. Summary of Riz1 mutations in human cancers 56
Table 5. Complete list of all primers used 96
vii
List of Figures
Figure 1. Proposed model of the dual roles of PR-Set7 in transcriptional 11
regulation
Figure 2. H4K20me1 is preferentially enriched within genes while 17
H4K20me3 is typically located within repetitive elements
Figure 3. Depletion of the PR-Set7 H4K20 monomethyltransferase results 21
in the selective de-repression of H4K20me1-associated genes
Figure 4. Loss of PR-Set7 or H4K20me1 does not affect the expression 23
of housekeeping and H4K20me1/me3-negative genes
Figure 5. H4K20me1 is essential for repression of endogenous 25
H4K20me1-associated genes
Figure 6. Both PR-Set7 WT and PR-Set7 CD are selectively targeted 27
to H4K20me1-postive loci
Figure 7. Quantitative real-time PCR validation of PR-Set7 target 30
genes identified by Illumina microarray analysis
Figure 8. FACS and western analysis of synchronized HeLa cells 32
Figure 9 H4K20me1-enriched genes exhibit lower expression at 34
G2/M compared to genes devoid of H4K20me1
Figure 10. PR-Set7 selectively binds Riz1 40
Figure 11. PR-Set7 and Riz1 interact via their C terminal domains 43
Figure 12. Riz1 possesses H3K9 monomethylation activity in vivo and 46
in vitro
Figure 13. The Riz1 C-terminal domain is necessary and sufficient for 47
proper cellular localization of Riz1
Figure 14. The Riz1 C-terminal domain is required for proper nuclear 50
localization of Riz1 and over-expression of this domain results
in global reduction in H3K9me1
Figure 15. Riz1 recruitment to chromatin is selectively mediated by 52
PR-Set7
viii
Figure 16. Riz1 is dispensable for transcriptional regulation of 54
PR-Set7-regulated genes
Figure 17. HCT116 cells contain a C-terminally-truncated Riz1, which 57
is unable to interact with PR-Set7
Figure 18. Ectopic Riz1 expression in HCT116 cells induces G2/M arrest 58
and apoptosis
Figure 19. The PR-Set7 multi-protein complex contains HDAC activity 64
Figure 20. PR-Set7 binds HDAC3 via its N-terminal domain in vitro 66
Figure 21. Inhibition of class I HDAC activity results in the 69
de-repression of PR-Set7 target genes
Figure 22. Over-expression of PR-Set7 or HDAC3 in MCF7 cells results 72
in the up-regulation of the mesenchymal marker N-cadherin
Figure 23. Osmotic Stress Promotes MAPK-mediated Stabilization of 79
PR-Set7 Against UV-induced Degradation
Figure 24. Osmotic Stress Promotes JNK-mediated Stabilization of 80
PR-Set7 Against UV-induced Degradation
Figure 25. PR-Set7 is Phosphorylated at Serine 29 after Osmotic Stress 82
Figure 26. Phosphorylation at S29 May Block UV-induced Ubiquitination 83
of PR-Set7
ix
Abstract
Within the eukaryotic nucleus, DNA is packaged via its interaction with histones
and non-histone proteins into a structure known as chromatin. Chromatin is dynamic in
nature, and can become more condensed or more accessible depending on, among other
things, the post-translational modifications present on the histone proteins. Histone-
modifying enzymes can alter the configuration of chromatin and are known to play
essential roles in DNA-templated processes, including gene transcription, cell cycle
progression and DNA damage repair pathways. This dissertation presents novel findings
on the functions and binding partners of the PR-Set7 histone H4 lysine 20
monomethyltransferase.
Several lines of evidence have been reported implicating PR-Set7 in both
transcriptional repression and activation. Chapter 1 presents our efforts to more clearly
elucidate the role of PR-Set7 and H4K20me1 in transcription. In short, we found that
depletion of PR-Set7 or its catalytic activity resulted in the de-repression of newly
identified PR-Set7 target genes, strongly suggesting that PR-Set7 and H4K20me1
function in the transcriptional repression of specific genes.
A ‘histone code’ hypothesis has been proposed, in which specific combinations of
post-translational modifications on histone tails may function coordinately to regulate
distinct chromatin-templated processes. We previously reported a novel trans-tail histone
code involving monomethylated H4K20 and H3K9. We found that global H3K9
monomethylation requires PR-Set7 but does not require its catalytic activity. We
therefore predicted that PR-Set7 recruits an unidentified H3K9 methyltransferase to
x
establish this novel histone code. In Chapter 2 we identify this H3K9 methyltransferase
as Riz1 (PRDM2/KMT8). Riz1 is a tumor suppressor, and is frequently mutated in
human cancers by a frameshift mutation resulting in the expression of a truncated protein
lacking the C-terminal domain found to be required for PR-Set7 binding and proper
localization. Finally, forced ectopic expression of wild type Riz1 in cancer cell lines
carrying this Riz1 truncation mutation resulted in cell cycle arrest and apoptosis. These
data indicate that Riz1 is an important tumor suppressor and imply that direct Riz1
binding to PR-Set7 is required for proper Riz1 localization and function.
In Chapter 3 we demonstrate that PR-Set7 interacts with class I HDAC proteins in
vivo, and that treatment with class I HDAC inhibitors results in the selective de-
repression of PR-Set7 target genes. Furthermore, we found that PR-Set7 directly binds
HDAC3 in vitro and that this interaction may be functionally significant in epithelial-
mesenchymal transition. Together, these results suggest that PR-Set7 and class I HDACs
cooperate to regulate transcription of specific human genes.
Lastly, in Chapter 4 we present evidence that after osmotic stress, PR-Set7 is
phosphorylated at serine 29 by JNK kinase, and that this modification protects PR-Set7
from degradation after UV irriadiation. Collectively, this dissertation presents novel
findings regarding PR-Set7’s binding to other histone-modifying enzymes, Riz1 and
HDAC3. These findings help to improve our understanding of PR-Set7 function during
transcriptional repression and cell cycle progression, as well as provide insights into how
perturbation of PR-Set7 may contribute to cancer development and progression.
1
Introduction
Chromatin
The length of total DNA in a single human cell is more than 2 meters (Getzenberg
et al., 1991). In order for this sizeable amount of genetic material to fit within the nucleus
of a cell, DNA is greatly compacted through its interaction with various nuclear proteins
and packaged into a structure known as chromatin. The first level of compaction is the
nucleosome, a 10 nm fiber consisting of 147 bp of DNA wound around two copies of
each core histone protein, H2A, H2B, H3 and H4 (Luger et al., 1997). Nucleosomes are
separated by lengths of linker DNA and appear as a ‘beads on a string’ structure.
Chromatin can be further compacted by the binding of linker histone H1 and other
proteins to form 30 nm fibers consisting of multiple nucleosomes or to form the
metaphase chromosomes seen during cell division (Robinson and Rhodes, 2006). This
highly structured packaging of DNA means that chromatin and chromatin-modifying
enzymes play central roles in nearly all DNA-templated processes, including
transcription, replication and DNA damage repair.
Each core histone contains two domains; a highly structured C-terminal globular
domain and a flexible N-terminal ‘tail’ domain. The globular domains of the core
histones participate in histone-histone and histone-DNA interactions, while the
unstructured tail domains protrude outward into the nuclear environment (Luger et al.,
1997). Decades worth of research has documented a considerable variety of post-
translational modifications that occur on the tail domains of histones, including
acetylation, phosphorylation, methylation, ubiquitination, and ADP-ribosylation
2
(Jaskelioff and Peterson, 2003). The modifications of histone tail residues can
subsequently be recognized and bound by specific domains of ‘effector’ proteins
(Taverna et al., 2007). The modification of histone proteins and the subsequent
recruitment of the proteins that bind these modifications can define unique functional
states of chromatin and regulate DNA-templated processes.
Histone Methylation and Histone Methyltransferases (HMTs)
Over 60 different residues on histone proteins have been identified by various
methods as sites of post-translational modifications, including acetylation, methylation,
phosphorylation, ubiquitination, sumoylation and ADP ribosylation (Kouzarides, 2007).
Another level of complexity is added by the possibility that methylation at lysine or
arginine residues can be mono-, di- or tri-methyl on lysine, and mono- or di- (asymmetric
or symmetric) on arginine. The vast combinatorial possibilities of histone modifications
can function to disrupt chromatin contacts or to recruit non-histone proteins to chromatin.
Histone methylation was first discovered several decades ago, and has since been
shown to occur predominantly at specific lysine or arginine residues on histones H3 and
H4 (Jenuwein and Allis, 2001; Murray, 1964; Schreiber and Bernstein, 2002; Stallcup,
2001). Lysine methylation is a stable modification established by lysine
methyltransferase enzymes, which have very high specificity compared to
acetyltransferases (Kouzarides, 2007). In general a histone methyltransferase only targets
a single lysine. The same or different enzymes can catalyze the mono-, di- or
trimethylation of lysine residues. For example, monomethylation of histone H4 lysine 20
3
(H4K20me1) is catalyzed only by PR-Set7 in metazoans (Couture et al., 2005; Nishioka
et al., 2002; Xiao et al., 2005). The di- and tri-methylation of H4K20 (H4K20me2/me3)
are subsequently established by separate enzymes, SUV4-20H1/H2 (Schotta et al., 2004;
Yang et al., 2008). Interestingly, different degrees of methylation on the same residue can
function in distinct biological processes. For example, H4K20me2 plays an important
role in targeting the DNA repair protein 53BP1 to sites of DNA damage (Botuyan et al.,
2006), while H4K20me1 and H4K20me3 function in transcriptional regulation and as a
hallmark of pericentric heterochromatin, respectively (Congdon et al., 2010; Schotta et
al., 2004).
The attachment of methyl groups to histone proteins is stable yet dynamic and
therefore the removal of these modifications require specialized demethylase enzymes
(Agger et al., 2008). Precise regulation and balance between histone methyltransferases
and demethylases are crucial for proper transcriptional regulation, among other processes,
and disruption of coordination of histone methylation is implicated in the development
and progression of diseases such as cancer (Varier and Timmers, 2011).
PR-Set7 and H4 Lysine 20 Monomethylation
DUAL ROLES OF PR-SET7 IN TRANSCRIPTION
Chromatin can exist in two forms: euchromatin and heterochromatin.
Euchromatin is the more open and less compacted form of chromatin and is primarily
associated with genes that are actively transcribed. Conversely, heterochromatin is tightly
compacted and is primarily associated with repressed genes (Grewal and Jia, 2007). The
4
post-translational modification of histone tails has been shown to influence whether
chromatin adopts a euchromatic or heterochromatic state, thus implicating histone-
modifying enzymes as central players in transcriptional regulation (Grewal and Moazed,
2003; Vermaak et al., 2003).
PR-Set7 (SETD8 or KMT5A) is a member of the SET domain histone
methyltransferase family and is responsible for specifically catalyzing the
monomethylation of histone H4 at lysine 20 (H4K20me1) in a nucleosome-dependent
manner (Couture et al., 2005; Fang et al., 2002; Nishioka et al., 2002; Xiao et al., 2005).
PR-Set7 is an essential enzyme as loss of PR-Set7 results in early embryonic lethality at
the 8-cell stage, indicating that its function is critical for development (Oda et al., 2009).
Both PR-Set7 and H4K20me1 are tightly cell cycle regulated and proper PR-Set7
regulation is required for normal mitotic progression (Rice et al., 2002; Wu et al., 2010).
Additionally, deregulation of PR-Set7/H4K20me1 results in DNA repair defects, DNA
re-replication and genomic instability (Houston et al., 2008; Tardat et al., 2010). Another
prominent role for PR-Set7 and H4K20me1 is in transcriptional regulation. It was
originally demonstrated that PR-Set7 and H4K20me1 are associated with
transcriptionally repressed chromatin and it was suggested that H4K20me1 functions in
X chromosome inactivation (Fang et al., 2002; Karachentsev et al., 2005; Kohlmaier et
al., 2004; Nishioka et al., 2002). In contrast, other reports have found that H4K20me1 is
located within active genes, which would suggest that it is a mark of active transcription
(Barski et al., 2007; Talasz et al., 2005). In light of these contradictory reports, the role of
PR-Set7 and H4K20me1 in transcriptional regulation remains subject to debate.
5
PR-Set7 and Transcriptional Repression
To date, several groups have presented evidence that would indicate that PR-Set7
and H4K20me1 function in transcriptional repression. The first line of evidence comes
from studies conducted in Drosophila in which staining of polytene chromosomes using a
methyl-H4K20 antibody demonstrated that H4K20 methylation is very low or absent at
transcriptionally competent regions (Nishioka et al., 2002). At that time, it was
hypothesized that methylation of H4K20 maintains silent chromatin, in part, by
precluding acetylation at H4K16, a transcriptionally activating modification. Lending
support to this hypothesis, recent analysis of ENCODE (Encyclopedia of DNA
Elements)(Ernst et al., 2011) data by Chendhore Veerappan in our lab demonstrated that
genomic regions of enrichment of H4K20 methylation or H4K16 acetylation are mutually
restrictive (data not shown). Subsequent studies established that the reduction of PR-Set7
in Drosophila suppresses position effect variegation (PEV), also suggesting that PR-Set7
functions as a repressor of gene activation (Karachentsev et al., 2005).
More recent work in our and others labs provide further evidence to support that
PR-Set7 and H4K20me1 function to repress transcription of specific genes under basal
cellular conditions. Chapter 1 of this dissertation will detail these studies from our lab.
Briefly, using ChIP-chip technology we identified specific genes on human chromosomes
21 and 22 that are enriched with the H4K20me1 modification (Congdon et al., 2010). We
demonstrated that the expression of H4K20me1-associated genes, but not control genes,
increased ~2 fold after shRNA-mediated depletion of PR-Set7 or over-expression of a
dominant negative catalytically dead PR-Set7, indicating that both PR-Set7 and the
6
H4K20me1 modification function to selectively repress specific genes. Additionally we
performed whole genome expression analysis in HeLa cells transfected with control or
PR-Set7 shRNA plasmids (Spektor et al., 2011). Computational comparison of the
averaged expression of control versus PR-Set7 shRNA samples revealed that >95% of
genes with significantly altered expression were up-regulated in the absence of PR-Set7.
These findings also strongly suggest that PR-Set7 functions predominantly in the
repression of specific genes under normal cellular conditions.
Several reports have proposed that H4K20me1 promotes transcriptional
repression via recruitment of the ‘effector’ protein L3MBTL1, the human homolog of the
Drosophila polycomb group (PcG) protein L(3)MBT. The 3xMBT repeats of L3MBTL1
selectively interact with H4K20me1(Kalakonda et al., 2008). It is believed that
L3MBTL1 inhibits transcription by facilitating chromatin compaction, thus blocking
access of transcription factors and polymerases. Importantly, it was shown that the
presence of H4K20me1 on nucleosome arrays is sufficient to recruit L3MBTL1 and
promote chromatin compaction (Trojer et al., 2007). Using a luciferase reporter assay, it
was shown that PR-Set7 alone does not significantly repress expression, however when
co-expressed with L3MBTL1, PR-Set7 induces a dose-dependent repression of
luciferase. It was demonstrated that the H4K20me1 modification is required for the
observed repression, as co-expression of a catalytically dead PR-Set7 along with
L3MBTL1 does not repress luciferase expression (Kalakonda et al., 2008). Additionally,
siRNA-mediated reduction of PR-Set7 led to a reduction of both H4K20me1 and
L3MBTL1 at an endogenous gene regulated by PR-Set7. The loss of L3MBTL1 and
7
H4K20me1 at the promoter of this gene corresponded with an increase in mRNA levels.
Collectively, this data provides evidence that H4K20me1 recruits L3MBTL1 to gene
promoters to facilitate transcriptional repression.
PHF8 was recently shown to be capable of demethylating H4K20me1 (Liu et al.,
2010; Qi et al., 2010). ChIP sequencing (ChIP-seq) of PHF8 in HeLa cells found that
>72% of PHF8 peaks localized on promoters (Liu et al., 2010). Upon PHF8 depletion,
there was an increase in H4K20me1 levels at promoters bound by PHF8. The increase in
H4K20me1 was concomitant with increased promoter binding by L3MBTL1. An
independent group found by ChIP-seq and gene expression microarray data that PHF8-
binding events are positively correlated with gene expression (Qi et al., 2010).
Additionally, they confirmed that depletion of PHF8 led to the down-regulation of gene
expression that was accompanied by increases in H4K20me1 and L3MBTL1 occupancy.
Taken together, these studies indicate that PHF8 positively regulates transcription by
demethylating H4K20me1.
We previously demonstrated that PR-Set7-mediated gene repression may function
to regulate genes during specific differentiation pathways. We found that under basal
conditions PR-Set7 represses the transcription of AML1/RUNX1, a master regulator of
hematopoietic differentiation (Sims and Rice, 2008). Inducing K562 cells to differentiate
into either erythrocytes (RUNX1 -) or megakaryocytes (RUNX1 +) resulted in the loss of
H4K20me1 and L3MBTL1 at the RUNX1 promoter, increased RUNX1 expression and
the selective differentiation toward the megakaryocytes lineage. Strikingly, it was
demonstrated that the absence of catalytically active PR-Set7 and H4K20me1 resulted in
8
spontaneous megakaryocytic differentiation of K562 cells. These results underscore the
functional significance of PR-Set7-mediated repression, and also suggest that PR-Set7 is
likely to function similarly in other multipotent cell types to regulate differentiation.
PR-Set7 and Transcriptional Activation
Contrary to the previously summarized studies, in recent years there have also
been several reports that suggest that PR-Set7 is important for transcriptional activation.
One such study in adipocytes found that PR-Set7 transcript levels increase as adipocyte
differentiation progresses (Wakabayashi et al., 2009). 3T3-L1 fibroblast cells transfected
with siRNA for PR-Set7 and then induced to differentiate into adipocytes showed
decreased lipid accumulation, suggesting that the up-regulation of PR-Set7 is necessary
for normal adipocytic differentiation. Many genes gained the H4K20me1 modification
during adipogenesis and it was therefore suggested that PR-Set7 promotes transcription
during this process. However, this hypothesis was not directly tested. Studies employing
a catalytically dead PR-Set7 to determine whether a lack of H4K20me1 can directly
inhibit lipid accumulation and up-regulation of adipogenic factors would be informative
as to whether PR-Set7 and H4K20me1 are functioning in gene activation during
adipogenesis.
More evidence that PR-Set7 may function in transcriptional activation came from
a recent study using an inducible conditional knockout mouse model for PR-Set7 in skin
(Driskell et al., 2011). When both alleles of PR-Set7 were deleted, the epidermis was
severely disrupted. It was found that in skin cells lacking PR-Set7 there is a decrease in
9
p63, one of the most important transcription factors that regulates skin homeostasis.
Reporter assays confirmed that PR-Set7 could stimulate transcription driven by the p63
promoter. It remains unclear, however, whether PR-Set7 is directly recruited to the
endogenous p63 promoter to catalyze H4K20me1 and facilitate transcriptional activation.
ChIP experiments would be informative for showing the direct involvement of PR-Set7
in regulating the transcription of this key skin transcription factor.
Additional support for a role of PR-Set7 in transcriptional activation comes from
studies revealing PR-Set7 interaction with transcription factors, such as estrogen receptor
α (ERα), TCF4 and TWIST (Li et al., 2011a; Li et al., 2011b; Yang et al., 2011). A
recent report demonstrated that upon estradiol (E
2
)
induction, H4K20me1 is elevated at
both the promoter and 3’-end of the coding region of an ERα−target gene, suggesting that
H4K20me1 is implicated in both transcription initiation and elongation (Li et al., 2011a).
Over-expression or depletion of PR-Set7 resulted in the up- or down-regulation,
respectively, of several endogenous ERα target genes. Immunoprecipitation experiments
showed that PR-Set7 interacts with ERα in vivo and in vitro. Co-immunoprecipitation
experiments were performed in the absence of E
2
, therefore it is unclear how the PR-
Set7/ ERα interaction is stimulated or increased upon E
2
induction to selectively target
PR-Set7 to ERα responsive genes. Finally, it was found that PR-Set7 interacts with the
transcriptionally competent form of RNA polymerase II, potentially linking PR-Set7 to
transcriptional elongation. Similarly, another group reported that PR-Set7 and
H4K20me1 are involved in the transcriptional activation of Wnt target genes (Li et al.,
2011b). It was shown that endogenous PR-Set7 forms a complex with the TCF4 family
10
transcription factors only after Wnt3a stimulation, providing a mechanism by which PR-
Set7 would be selectively targeted to a set of genes to promote transcription, only under
particular conditions, such as activation of a signal transduction cascade.
Based on the available data, we propose a model (Figure 1) wherein under normal
cellular conditions PR-Set7 promotes transcriptional repression of genes though
monomethylation of H4K20, recruitment of L3MBTL1 and subsequent chromatin
compaction. Similarly, PHF8 facilitates transcriptional activation via the removal of
H4K20me1 and loss of L3MBTL1 recruitment. Alternatively, during differentiation or
under various extra-cellular stimuli, PR-Set7 may selectively interact with other
transcription factors and be targeted to specific sets of target genes to facilitate full
transcriptional activation. The molecular mechanisms that allow PR-Set7 and H4K20me1
to contribute to transcriptional activation remain unclear. It is possible that upon specific
cellular stimuli or stresses, or depending on other loci-dependent factors, PR-Set7 and/or
H4K20me1 bind and recruit yet unidentified transcription factors or chromatin
remodeling factors that promote transcriptional activation. Much work remains to be
done to determine if and what these factors are. Additionally, it is also unknown how the
recruitment of L3MBTL1 and subsequent chromatin compaction is inhibited at the
promoters of genes that require PR-Set7 and H4K20me1 for their transcriptional
activation. It seems likely that PR-Set7 and H4K20me1 function as part of a ‘histone
code’ in which many other transcription factors, chromatin modifying enzymes and
histone modifications participate in context-dependent combinations to either repress or
activate transcription of specific human genes.
11
Figure 1: Proposed model of the dual roles of PR-Set7 in transcriptional regulation
Under basal cellular conditions (left) PR-Set7 promotes transcriptional repression of
genes though monomethylation of H4K20, recruitment of L3MBTL1 and subsequent
chromatin compaction. Similarly, PHF8 facilitates transcriptional activation via the
removal of H4K20me1 and loss of L3MBTL1 recruitment. Alternatively, during
differentiation or under various extra-cellular stimuli (right), PR-Set7 may selectively
interact with other transcription factors (TF) and be targeted to specific sets of target
genes via transcription factor binding elements (TF-BE) to facilitate full transcriptional
activation.
L3MBTL1
PR-Set7
PHF8
TF-BE
PR-Set7
RNAPII-pS2
Activation Repression
TF
12
Combinatorial Histone Modifications
It has been proposed that different histone modifications, on one or more tails, can
function together to create a ‘histone code’ that is read by other proteins to effect
downstream DNA-templated processes (Jenuwein and Allis, 2001; Strahl and Allis,
2000). This ‘code’ is read, in part, by effector proteins that selectively recognize specific
modifications. For example, proteins containing a bromodomain motif can selectively
bind acetylated lysine residues, while proteins containing a chromodomain instead bind
methylated lysine residues (Jacobs and Khorasanizadeh, 2002; Zeng and Zhou, 2002).
There is also evidence that one histone modification can influence another, by either
blocking or recruiting another histone modifying enzyme. One such example of a
combinatorial histone code was described involving H3K9 and H4K20 trimethylation
(me3) (Schotta et al., 2004). In this pathway, Suv39h is first targeted to pericentric
heterochromatin to establish H3K9me3. Next, HP1α and HP1β bind to the H3K9me3
nucleosomes (Bannister et al., 2001) and in turn recruit Suv4-20H1/H2 to establish
H4K20me3. This sequential recruitment of HMTs comprises an important repressive
pathway that demarcates pericentric heterochromatin.
Similar to what was shown for H4K20me3 and H3K9me3, our lab has previously
reported a novel trans-tail histone code involving monomethylated H4K20 and H3K9
(Sims et al., 2006; Sims and Rice, 2008). We demonstrated that global H3K9me1
requires PR-Set7 but does not require its catalytic activity. These findings suggested that
PR-Set7, itself, recruits an unknown H3K9 monomethyltransferase to establish this novel
histone code, and served as the impetus for the research presented in Chapter 2. We
13
found that this H4K20me1/H3K9me1 histone code is mediated by PR-Set7 binding to the
H3K9 methyltransferase, Riz1 (PRDM2/KMT8). Over a decade of research has provided
strong evidence that Riz1 is an important tumor suppressor. Riz1 is frequently mutated in
human cancers by a frameshift mutation resulting in the expression of a truncated protein
lacking the C-terminal domain. Interestingly, we found the C-terminal domain of Riz1 to
be required for PR-Set7 binding, and demonstrated that the mutant truncated Riz1 protein
has abolished PR-Set7 binding capabilities. These data strongly suggest that direct Riz1
binding to PR-Set7 is required for proper Riz1 localization and function.
14
Chapter 1. PR-Set7 and H4K20me1 function as transcriptional
repressors of specific human genes
1.1. Introduction
The role of PR-Set7 and H4K20me1 in transcription has been the subject of
considerable debate. It has been demonstrated that H4K20me1 can directly promote
chromatin compaction, in part by recruitment of the transcriptionally repressive
L3MBTL1 protein (Oda et al., 2009; Trojer et al., 2007). In Drosophila H4K20me1 was
found to be exclusively localized in transcriptionally inactive regions and loss of PR-Set7
led to a suppression of variegation phenotype, implicating PR-Set7 and H4K20me1 in
gene silencing (Karachentsev et al., 2005; Nishioka et al., 2002). Supporting a role for
PR-Set7 in transcriptional repression, studies in mouse cells revealed that H4K20me1 is
enriched on the inactive X chromosome (Kohlmaier et al., 2004). PHF8 was recently
found to be an H4K20me1 demethylase. Also supporting a role for H4K20me1 in
transcriptional repression, the loss of PHF8 resulted in the decreased expression of
specific genes concomitant with increased H4K20me1 and L3MBTL1 occupancy (Liu et
al., 2010).
Conversely, genome wide studies have shown that H4K20me1 enrichment at the
5’-end of gene bodies correlates with genes that are highly transcribed (Barski et al.,
2007; Cui et al., 2009). Additionally, H4K20me1 and PR-Set7 have been implicated in
the transcriptional activation of estrogen-responsive and wnt3a-responsive genes, as well
as in both the activation and repression of genes involved in epithelial-mesenchymal
transition (Li et al., 2011a; Li et al., 2011b; Yang et al., 2011).
15
Chapter 1 will summarize our efforts to gain a clearer understanding of how PR-
Set7 and H4K20me1 function to regulate gene expression. We first sought to identify
genes that are transcriptionally regulated by H4K20me1 and PR-Set7 by using two
independent approaches: ChIP-chip to identify genes enriched with PR-Set7-mediated
H4K20me1 and gene expression profiling to identify genes whose expression
significantly changes in the absence of PR-Set7. These studies provided us with an
extensive panel of putative PR-Set7 target genes, representing a wide spectrum of basal
expression levels. PR-Set7 knockdown studies demonstrated that in the absence of PR-
Set7 and/or H4K20me1, the expression of PR-Set7 target genes was increased, strongly
suggesting that PR-Set7 and H4K20me1 function in the transcriptional repression of
specific genes regardless of their basal transcriptional state.
1.2. Results
1.2.1. H4K20me1 is preferentially enriched in gene bodies whereas the majority of
H4K20me3 is found in repetitive elements
In order to gain a better understanding of the role of monomethylated H4K20 in
transcription, chromatin immunoprecipitation experiments were performed using
antibodies that selectively recognize monomethyl or trimethyl H4K20. The purified
H4K20me1 and H4K20me3-associated DNA was blunted, PCR amplified, labeled and
hybridized to a NimbleGen oligonucleotide genome tiling microarray containing the
entire length of chromosomes 21 and 22, with input DNA used as a reference for
16
enrichment. Analysis of the ChIP-chip data revealed that H4K20me1 is preferentially
enriched within the body of genes (75%), whereas H4K20me3 is predominantly found in
intergenic regions (54%) (Figure 2A). This data suggest that H4K20me1 is specifically
targeted to genes compared to H4K20me3. Since repetitive elements were excluded from
the microarray, however may comprise over two-thirds of the human genome (de Koning
et al., 2011), we sought to investigate if H4K20me1 and H4K20me3 were also targeted to
repetitive elements. To do so, ChIPs were performed using H4K20me1 and H4K20me3-
specific antibodies and the DNA was used to create small bacterial libraries for direct
sequencing (ChIP-cloning) (Weinmann and Farnham, 2002). Due to the large quantity of
repetitive elements in the human genome, if H4K20me1 or H4K20me3 were targeted to
repetitive elements then we would expect to find the vast majority of sequences
containing repeats. Instead, it was revealed that almost half (42%) of H4K20me1-
enriched sequences (Table 1) corresponded to non-repetitive elements (Figure 2B). In
contrast, the majority (77%) of H4K20me3-enriched sequences contained repeats (Table
2), predominantly SINE elements (43%). These findings demonstrate that H4K20me1 is
typically enriched within gene bodies, whereas H4K20me3 is preferentially enriched
within repetitive elements, and imply an important role for H4K20me1 in transcription.
17
Figure 2. H4K20me1 is preferentially enriched within genes while H4K20me3 is
typically located within repetitive elements
(Congdon et al., 2010) (A) Chromatin immunoprecipitations (ChIPs) were performed in
HeLa cells to isolate H4K20me1- and H4K20me3-associated DNA. The ChIPed material
was hybridized to a NimbleGen oligonucleotide genome tiling microarray containing the
entire length of human chromosomes 21 and 22. Statistically significant peaks (P<0.005)
of enrichment were identified using MA2C software. The percentages of peaks located in
specific genomic regions are represented. Proximal is defined as the regions 5kb
upstream and 500bp downstream of the gene body. (B) ChIPed material was cloned and
86 insert positive H4K20me1-associated clones and 57 H4K20me3-associated clones
were directly sequenced. The general distribution of the sequences and the different
classes of corresponding repetitive elements are shown.
18
Genomic Position Genomic Position
1 2:173,458,748-173,458,952 44 3:105,564,249-105,564,458
2 10:31,518,665-31,518,946 45 16:86,728,076-86,728,225
3 12:10,257,472-10,257,748 46 7:61,607,745-61,607,846
4 16:29,449,716-29,449,984 47 10:41,713,062-41,714,334
5 22:41,548,340-41,548,606 48 5:166,074,141-166,074,387
6 6:18,562,332-18,562,463 49 3:198,588,765-198,589,035
7 11:25,929,371-25,929,671 50 16:75,262,107-75,262,239
8 2:169,058,832-169,059,096 51 13:98,176,413-98,176,455
9 X:44,182,228-44,182,568 52 3:139,282,371-139,282,584
10 13:48,051,750-48,052,072 53 3:63,110,558-63,110,806
11 2:234,919,762-234,919,993 54 5:163,852,084-163,852,339
12 19:58,183,392-58,183,680 55 3:167,449,562-167,449,728
13 1:179,223,001-179,223,199 56 5:175,056,419-175,056,500
14 14:46,952,743-46,953,083 57 18:98,722-98,884
15 5:12,101,473-12,101,705 58 2:91,163,898-91,164,056
16 6:31,591,240-31,591,551 59 2:61,089,121-61,089,223
17 15:25,766,745-25,766,862 60 19:16,854,006-16,854,096
18 8:1,139,293-1,139,399 61 X:149,705,784-149,705,966
19 6:159,042,268-159,042,484 62 7:96,154,795-96,154,974
20 22:31,402,997-31,403,183 63 3:178,313,285-178,313,483
21 1:219,131,408-219,131,702 64 8:133,949,938-133,950,256
22 11:85,502,216-85,502,422 65 11:32,785,151-32,785,400
23 3:76,243,133-76,243,378 66 4:191,235,573-191,235,742
24 22:37,430,776-37,431,094 67 1:121,186,060-121,186,312
25 7:157,929,326-157,929,455 68 1:57,634,352-57,634,385
26 1:121,186,829-121,186,894 69 16:87,238,775-87,239,028
27 10:73,581,734-73,581,814 70 17:38,737,116-38,737,247
28 18:100,598-100,692 71 19:57,652,736-57,653,038
29 13:67,830,087-67,830,196 72 17:19,252,732-19,252,894
30 10:66,813,181-66,813,382 73 12:88,637,110-88,637,320
31 6:13,449,869-13,450,070 74 11:48,685,542-48,685,772
32 9:117,818,323-117,818,507 75 8:34,610,489-34,610,732
33 19:3,973,595-3,973,746 76 10:38,844,776-39,192,159
34 15:82,619,147-82,619,301 77 3:10,304,188-10,304,414
35 18:32,022,992-32,023,237 78 17:73,067,192-73,067,320
36 2:234,919,809-234,919,993 79 6:21,424,534-21,424,683
37 10:51,695,309-51,695,889 80 12:52,967,799-52,967,975
38 20:60,411,516-60,411,743 81 8:145,273,486-145,273,604
39 19:13,027,946-13,028,120 82 18:16,773,828-16,774,026
40 1:85,114,574-85,114,795 83 6:448,810-449,034
41 3:54,739,347-54,739,561 84 16:13,425,633-13,425,901
42 2:239,463,815-239,464,050 85 6:165,205,348-165,205,541
43 2:169,058,877-169,059,093 86 9:138,211,386-138,211,578
Table 1. Identification of genomic locations of H4K20me1 in HeLa cells by ChIP-
cloning
Chromatin immunoprecipitations (ChIPs) were performed in HeLa cells to isolate
H4K20me1-associated DNA. The ChIPed material was ligated into the pGEM-T-Easy
TA cloning vector and transformed into E. coli. 86 insert positive clones were directly
sequenced and aligned to the human genome using the UCSC Genome Browser.
19
Genomic Position Genomic Position
1 18:60,503,913-60,504,258 30 X:105,937,011-105,937,011
2 17:73,593,871-73,595,598 31 6:139,538,328-139,539,328
3 1:14,603,202-14,603,571 32 22:20,062,165-20,062,472
4 5:7,830,865-7,831,370 33 3:84,779,755-84,780,160
5 9:69,000,671-69,001,148 34 17:29,507,633-29,507,843
6 16:81,089,538-81,089,944 35 5:2,198,554-2,199,131
7 X:55,514,289-55,514,436 36 7:71,363,846-71,364,168
8 5:33,654,542-33,654,675 37 8:87,138,882-87,139,215
9 2:205,960,012-205,961,433 38 6:39,157,896-39,158,599
10 2:241,275,096-241,275,749 39 5:25,766,869-25,767,468
11 19:49,696,901-49,697,497 40 10:41,719,880-41,720,647
12 2:74,306,497-74,307,052 41 1:22,008,980-22,009,210
13 2:230,729,404-230,729,995 42 11:44,250,882-44,251,261
14 21:35,853,509-35,854,259 43 6:3,001,046-3,001,525
15 22:32,883,148-32,883,584 44 11:5,222,262-5,222,951
16 2:64,519,679-64,520,178 45 6:38,218,297-38,218,757
17 17:22,168,811-22,173,854 46 11:87,880,988-87,881,470
18 9:74,072,355-74,072,924 47 11:47,119,430-47,119,722
19 18:97,784-98,302 48 1:74,760,487-74,760,728
20 7:31,497,822-31,498,155 49 12:125,482,550-125,482,872
21 13:113,006,889-113,007,561 50 5:41,385,205-41,385,687
22 10:41,704,897-41,705,391 51 4:81,402,281-81,402,939
23 4:97,520,842-97,522,655 52 2:153,664,965-153,665,242
24 19:9,439,599-9,440,741 53 5:179,702,571-179,703,338
25 8:13,507,257-13,508,044 54 8:8,862,275-8,625,624
26 2:119,597,708-119,600,073 55 3:78,708,510-78,709,169
27 19:32,424,918-32,432,178 56 X:17,060,097-17,060,568
28 3:164,787,677-164,788,208 57 20:58,583,777-58,584,576
29 3:115,452,268-115,452,816
Table 2. Identification of genomic locations of H4K20me3 in HeLa cells by ChIP-
cloning
Chromatin immunoprecipitations (ChIPs) were performed in HeLa cells to isolate
H4K20me3-associated DNA. The ChIPed material was ligated into the pGEM-T-Easy
TA cloning vector and transformed into E. coli. 57 insert positive clones were directly
sequenced and aligned to the human genome using the UCSC Genome Browser.
20
1.2.2. H4K20me1 is required for transcriptional repression of specific endogenous
genes
Upon identification of several H4K20me1-associated genes by ChIP-chip, we
sought to determine the role of H4K20me1 in transcriptional regulation since this has
been controversial. To do so, HeLa cells were transfected with a control shRNA plasmid
or a PR-Set7 shRNA plasmid in order to reduce H4K20me1 without affecting
H4K20me2 or H4K20me3 levels (Figure 3A). To confirm that depletion of PR-Set7 also
resulted in the local reduction of H4K20me1 at H4K20me1-associated genes, ChIPs were
performed using an H4K20me1-specific antibody in control and PR-Set7 shRNA HeLa
cells. PCR was performed by using increasing amounts of ChIPed DNA for amplification
of two loci within BRD1 found to be enriched for H4K20me1. ChIP-PCR confirmed that
upon depletion of PR-Set7 there was a local reduction in H4K20me1 at both H4K20me1-
enriched loci in BRD1 (Figure 5B). RNA from control and PR-Set7 shRNA cells was
harvested for quantitative real-time PCR (qRT-PCR) analysis. To normalize expression
changes between samples, several expressed genes were identified from the ChIP-chip
data that lacked both H4K20me1 and H4K20me3. The expression of these genes was
unchanged upon depletion of PR-Set7 and MTMR3 was subsequently chosen for use as
an internal normalization control between samples (Figure 4).
21
Figure 3. Depletion of the PR-Set7 H4K20 monomethyltransferase results in the
selective de-repression of H4K20me1-associated genes
(Congdon et al., 2010) HeLa cells were transfected with a control shRNA plasmid or a
PR-Set7 shRNA plasmid. (A) Western analysis of whole cell lysates using the indicated
antibodies. (B) qRT-PCR analysis was performed to analyze the expression levels of
seven H4K20me1-associated genes or four H4K20me3-associated genes (C) in cells
containing the control shRNA (white) or PR-Set7 shRNA (gray) plasmid. Results are
plotted relative to MTMR3 expression (y-axis). Three independent biological replicates
were performed to generate the standard deviation in each experiment. The Student’s t-
test was used to determine statistically significant changes at * P <0.01.
22
Quantitative RT-PCR revealed that the H4K20me1-associated genes analyzed
displayed an ~2-fold increase in gene expression in PR-Set7 shRNA cells compared to
control shRNA, indicating that the role of PR-Set7 and H4K20me1 is to repress gene
expression (Figure 3B). In contrast, the expression of a set of genes that we identified as
being enriched with trimethyl H4K20 was analyzed in the PR-Set7 depleted cells and no
changes in expression were observed (Figure 3C). Additionally, gene expression changes
in several housekeeping genes were not observed between control and PR-Set7 shRNA
cells (Figure 4).
23
Figure 4. Loss of PR-Set7 or H4K20me1 does not affect the expression of
housekeeping and H4K20me1/me3-negative genes
(Congdon et al., 2010) HeLa cells were transfected with control shRNA, PR-Set7
shRNA, control expression or PR-Set7 CD expression plasmids. The control and PR-Set7
shRNA treated cells were harvested 6 days post-transfection while the control and PR-
Set7 CD over-expressing cells were harvested 4 days post-transfection. qRT-PCR was
performed to analyze the expression levels of four different housekeeping genes and three
different genes that were identified as lacking both monomethylated and trimethylated
H4K20. The mean cycle threshold values for amplification of each of the genes in the
four experimental samples are plotted on the y-axis. Three independent biological
replicates were performed to generate the standard deviation. These results demonstrate
that the loss of PR-Set7 and H4K20me1 does not affect the expression of these
housekeeping genes. Additionally, the data clearly indicates that the expression of genes
lacking both mono- and trimethylated H4K20 are also unaffected by the loss of PR-Set7
and H4K20me1 and, therefore, can be used to normalize gene expression differences
between the various experimental samples.
Supplemental Figure 1
Supplemental Figure 1. HeLa cells were transfected with either control shRNA, PR-Set7 shRNA,
control expression or PR-Set7 CD expression plasmids. The control and PR-Set7 shRNA treated cells
were harvested 6 days post-transfection while the control and PR-Set7 CD overexpressing cells were
harvested 4 days post-transfection. qRT-PCR was performed to analyze the expression levels of four
different housekeeping genes and three different genes that were identified as lacking both mono-
methylated and trimethylated H4K20. The mean cycle threshold values for amplification of each of the
genes in the four experimental samples is plotted on the y-axis. Three independent biological replicates
were performed to generate the standard deviation. These results demonstrate that the loss of PR-Set7
and H4K20me1 does not affect the expression of these housekeeping genes. Additionally, the data clearly
indicates that the expression of genes lacking both mono- and trimethylated H4K20 are also
unaffected by the loss of PR-Set7 and H4K20me1 and, therefore, can be used to normalize gene
expression differences between the various experimental samples.
0.00
5.00
10.00
15.00
20.00
25.00
30.00
35.00
40.00
GAPDH
18S
Lamin
Cyclophilin
CYYR1
MTMR3
RBM9
Mean Cycle Threshold
Control shRNA
PR-Set7 shRNA
Control
PR-Set7 CD
Housekeeping Genes H4K20me1/me3
negative genes
Primer F sequence R sequence
GAPDH CAGCCGAGCCACATCGCTCAGACA TGAGGCTGTTGTCATACTTCTC
18S AACTTTCGATGGTAGTCGCCG CCTTGGATGTGGTAGCCGTTT
Lamin CAAGCTTGAGGCAGCCCTAG CTCACGCAGCTCCTCACTGTA
Cyclophilin TATTAGCCATGGTCAACCCCAC TCTGCTGTCTTTGGGACCTTGT
CYYR1 TGCAAATCTTACTGCTGTGATG GATAGGAGGAGACGGTGTTGAT
MTMR3 TCATGGAGAGAGCACAGAGTTT GATTGCGTTGTTCAGTCTCTTC
RBM9 CTTTAGTTCCTGGCTTCCCTTA TATCCACCATAGAGGTCAGCAC
24
We next sought to determine if the observed de-repression of genes was due to a
loss of PR-Set7 and/or the H4K20me1 modification it creates. To do so, we employed a
PR-Set7 R265G point-mutant that is catalytically dead (CD) and acts as a dominant
negative to deplete H4K20me1 without reducing PR-Set7 (Figure 5A) (Houston et al.,
2008; Sims and Rice, 2008). ChIP experiments were performed to confirm that over-
expression of PR-Set7 CD resulted in the local reduction of H4K20me1 at the BRD1
H4K20me1-postive loci (Figure 5B). Additionally, ChIP experiments against FLAG-PR-
Set7 WT or FLAG-PR-Set7 CD demonstrated that there is no defect in the recruitment of
PR-Set7 CD to H4K20me1-postive loci, and that both PR-Set7 WT and PR-Set7 CD are
selectively targeted to these loci (Figure 6). The RNA from control or PR-Set7 CD HeLa
cells was harvested for qRT-PCR analysis. Similar to what was observed in PR-Set7
shRNA cells, the over-expression of PR-Set7 CD resulted in ~2-fold increase in the
expression of H4K20me1 genes compared to control cells (Figure 5C). Furthermore, no
changes in expression were observed in the H4K20me3-associated genes (Figure 5D) or
housekeeping genes (Figure 4). Taken together, this data indicates that PR-Set7 and
H4K20me1 are targeted to specific genes to facilitate transcriptional repression.
25
Figure 5. H4K20me1 is essential for repression of endogenous H4K20me1-associated
genes
(Congdon et al., 2010) HeLa cells were transfected with a control eukaryotic plasmid or a
plasmid expressing a catalytically dead (CD) dominant negative PR-Set7 point mutant.
(A) Western analysis of whole cell lysates using the indicated antibodies. (B) ChIPs were
performed on the HeLa cells transfected with a control (top), a PR-Set7 CD (middle), or a
(Figure 5, continued) PR-Set7 shRNA expression plasmid (bottom) using either the
H4K20me1-specific antibody, a histone H3 general antibody (positive control), or rabbit
IgG (negative control). Thirty cycles of PCR amplifications were performed each time
for the two H4K20me1 positive regions of BRD1 using 0.1, 1, or 3 µl of ChIPed DNA
from each sample (triangle). (C) qRT-PCR analysis was performed to analyze the
expression levels of seven H4K20me1-associated genes or four H4K20me3-associated
genes (D) in cells expressing the control (white) or PR-Set7 CD (gray) plasmid. Results
are plotted relative to MTMR3 expression (y-axis). Three independent biological
replicates were performed to generate the standard deviation in each experiment. The
Student’s t-test was used to determine statistically significant changes at * P <0.01.
26
27
Figure 6. Both PR-Set7 WT and PR-Set7 CD are selectively targeted to H4K20me1-
postive loci
(Congdon et al., 2010) HeLa cells were transfected with pQCXIP FLAG-PR-Set7 wild
type (WT), FLAG-PR-Set7 CD, or FLAG-GFP. ChIP lysates were prepared 4 days post-
transfection. ChIPs were performed using anti-FLAG antibody, a histone H3 general
antibody (positive control) or rabbit IgG (negative control). Thirty cycles of PCR
amplifications were performed each time for the two H4K20me1 positive regions of
BRD1 or the H4K20me1 negative region of RUNX1 using 0.1, 1 or 3 µl of ChIPed DNA
from each sample (triangle). These results demonstrate that ectopically expressed PR-
Set7 is specifically targeted to H4K20me1-associated regions.
Our initial studies on the role of PR-Set7 in transcription were restricted to genes
on chromosomes 21 and 22. We next sought to broaden our analysis and examine how
PR-Set7 functions in transcriptional regulation genome wide. To do so, HeLa cells were
again transfected with a PR-Set7 shRNA or a null shRNA plasmid and total RNA was
harvested from cells after 5 days. Whole genome expression from two independent
biological replicates of control and PR-Set7 shRNA samples was determined using
Illumina BeadChip expression arrays. Computational comparison of the averaged
expression of control versus experimental samples revealed 43 genes whose expression
was significantly altered in cells lacking PR-Set7 (>2-fold, p<0.005) (Table 3)(Spektor et
al., 2011). Of these, only 2 genes displayed decreased expression: PR-Set7 and
28
HERPUD1, which was found to be a false positive (Figure 7). The remaining 41 genes
displayed increased expression. Quantitative RT-PCR of 11 of these genes confirmed 8
whose expression was significantly increased (p<0.05) in the absence of PR-Set7 (Figure
7). Consistent with the analysis of genes on chromosomes 21 and 22, these findings
strongly suggest that PR-Set7 functions predominantly in the repression of specific genes.
29
Genes Up-Regulated in the Absence of PR-Set7
Gene Name Probe ID Avg. log2Δ(Null/PR-Set7 shRNA) Avg. Fold Δ p-value
CSAG3A 4010095 -3.415 10.666 0
CSAG2 6900377 -2.99 7.945 0
LOC653297 5670563 -2.773 6.835 0
LOC643425 5860504 -2.352 5.105 0
HES5 6590300 -2.127 4.368 0
UBE2L6 2070170 -2.103 4.296 1.00E-05
EPSTI1 5700725 -2.072 4.205 0
MAFA 5090241 -1.997 3.992 1.00E-05
PRIC285 5960343 -1.834 3.565 0
FLJ20035 7610053 -1.72 3.294 0
NFS1 7570411 -1.71 3.272 0.00166
REC8L1 70541 -1.675 3.193 0
CDA 5090372 -1.671 3.184 4.00E-05
FLJ11000 4670414 -1.533 2.894 0
MGC4677 6350189 -1.529 2.886 0.00109
LOC442578 3710711 -1.518 2.864 0.00021
CALCB 2230053 -1.478 2.786 7.00E-05
SAMD9 1240142 -1.432 2.698 1.00E-05
CD24 610437 -1.386 2.614 6.00E-05
CYP4X1 3290048 -1.276 2.422 1.00E-05
HERC6 6860482 -1.189 2.280 2.00E-04
LOC619383 6220112 -1.182 2.269 4.00E-05
ANKRD19 2480040 -1.163 2.239 0.00013
LOC440157 4280093 -1.158 2.231 0.00034
ARL14 3400209 -1.138 2.201 0.00182
FRAS1 270358 -1.131 2.190 0.0016
LOC132241 4200551 -1.126 2.183 0.00077
C21orf58 2070220 -1.122 2.176 5.00E-05
HOXC8 4640059 -1.098 2.141 7.00E-05
HS.154336 7040731 -1.07 2.099 0.00242
LOC642477 4570041 -1.063 2.089 0.00499
NRBP2 4490142 -1.043 2.061 1.00E-05
NFKBIZ 2470348 -1.037 2.052 0.00015
DHRS1 1470017 -1.031 2.043 0.00033
C14orf153 7160747 -1.03 2.042 0.0047
KIAA1641 4570075 -1.02 2.028 0.0012
VAMP1 6650639 -1.017 2.024 0.00103
HS.514745 4610041 -1.012 2.017 0
ADM 5670465 -1.01 2.014 0.00013
MCOLN2 5490068 -1.009 2.013 8.00E-05
EPDR1 6400044 -1.004 2.006 0.00022
Genes Down-Regulated in the Absence of PR-Set7
Gene Name Probe ID Avg. log2Δ(Null/PR-Set7 shRNA) Avg. Fold Δ p-value
HERPUD1 4570458 1.186 0.440 7.00E-05
SETD8 2350735 1.938 0.261 0
Table 3. Genes whose expression change >2-fold in cells lacking PR-Set7
(Spektor et al., 2011)
30
Figure 7. Quantitative real-time PCR validation of PR-Set7 target genes identified
by Illumina microarray analysis
Quantitative RT-PCR expression analysis of putative PR-Set7-regulated genes (Table 3)
from PR-Set7 shRNA transfected HeLa cells. Results are plotted relative to 18S
expression and normalized to control shRNA levels (dotted line), which were set at 1 (y-
axis). Error bars represent the standard error from three or more independent biological
replicates. The student t-test was used to determine statistically significant changes at
p<0.05(*). Eight of 11 identified genes that were significantly up-regulated in the
absence of PR-Set7 according to the expression arrays (Table 3) were validated whereas
the only identified down-regulated gene (HERPUD1) was a false positive.
EPSTI1
REC8L1
HES5
ARL14
VAMP1
UBE2L6
NFKBIZ
ADM
CDA
CALCB
NFS1
HERPUD1
CBR1
MORC2
TUG1
PR-Set7
0
1
2
3
4
5
10
12
14
16
18
Expression Fold Change (relative to 18S)
Gene Expression in PR-Set7 shRNA HeLa Cells
False Positives H4K20me3 genes
*
*
*
*
*
*
*
*
31
1.2.3. H4K20me1-enriched genes exhibit lower expression at G2/M compared to
genes devoid of H4K20me1
Since it is well documented that PR-Set7 and H4K20me1 are tightly cell cycle
regulated (Rice et al., 2002; Wu and Rice, 2011), we next sought to investigate how
H4K20me1 regulates transcription of genes at different stages of the cell cycle. To do so,
HeLa cells were synchronized at G2/M using nocodazole and subsequently released and
harvested at various time points corresponding to distinct phases of the cell cycle. Flow
cytometry analysis for DNA content and western blot analysis confirmed that the cells
were uniformly synchronized (Figure 8).
32
Figure 8. FACS and western analysis of synchronized HeLa cells
HeLa cells were treated with 75 ng/ml nocodazole for 12 hours to arrest cells in the G2/M
phase of the cell cycle. Cells were then washed thoroughly before releasing into fresh
medium and harvesting at the indicated time points. Cells were analyzed for (A) DNA
content by propidium iodide staining and fluorescence-activated cell sorting (FACS)
analysis and (B) western blot analysis to determine the phase of cell cycle and
corresponding levels of PR-Set7 and H4K20 methylation.
RNA was extracted from the cells at the indicated time points and used for qRT-
PCR analysis of genes that were identified from ENCODE data as having high levels of
H4K20me1 or those devoid of H4K20me1 (Ernst et al., 2011)(Chendhore
Veerappan)(Figure 9). Upon analysis of either the pre-mRNA or mature mRNA transcript
levels, it was observed that both H4K20me1 positive and negative genes exhibited
increased expression at G1 (3 h) and S phase (11 h) compared to expression during G2/M
33
(nocodazole-0.5h) (Figure 9A-B). However, a closer examination of gene expression
during G2/M relative to expression in asynchronous cells revealed that genes enriched in
H4K20me1 have reduced expression during G2/M compared to genes devoid of
H4K20me1 (Figure 9C). This result is likely due to the fact that PR-Set7 and H4K20me1
levels peak during G2/M. We hypothesize that during G2/M, PR-Set7 and H4K20me1
function to repress the transcription of specific genes in order to promote proper mitotic
progression, and possibly to ensure proper origin licensing and prevent re-replication
(Brustel et al., 2011; Tardat et al., 2010). We hypothesize that without PR-Set7
H4K20me1-enriched genes will be de-repressed at inappropriate times during the cell
cycle and contribute to improper cell cycle progression. Future experiments will be aimed
at testing this hypothesis by depleting cells of PR-Set7 and monitoring expression of
H4K20me1-enriched genes during the cell cycle.
34
Figure 9. H4K20me1-enriched genes exhibit lower expression at G2/M compared to
genes devoid of H4K20me1
Whole RNA was extracted from nocodazole synchronized cells corresponding to distinct
phases of the cell cycle (Figure 8) and qRT-PCR analysis was performed to analyze the
mature and pre-mRNA expression levels of (A) genes enriched in H4K20me1
(ENCODE, (Ernst et al., 2011)) or (B) genes lacking H4K20me1. Results are plotted
relative to the expression of an H4K20me3-enriched gene, MORC2 (Figures 3 and 5),
and normalized to asynchronous expression levels, which were set at 1 (y-axis). (C)
Mature and pre-mRNA expression levels (relative to MORC2) in nocodazole-arrested
cells relative to expression in asynchronous cells were plotted for individual H4K20me1-
enriched or H4K20me-negative genes. The horizontal bars represent the averaged relative
gene expression during G2/M. Each data point represents the cycle threshold average
from 3 triplicate PCR reactions, N=1.
35
1.3. Discussion and future directions
We sought to clarify the role of PR-Set7 and H4K20me1 in transcription by
identifying PR-Set7 target genes. Using independent parallel approaches of H4K20me1
ChIP-chip and PR-Set7 knockdown gene expression analysis, we compiled an extensive
panel of putative target genes (Congdon et al., 2010; Spektor et al., 2011)(Table 3). The
basal expression levels of these target genes varies greatly and includes highly expressed
genes, supporting ChIP-sequencing studies that H4K20me1 correlates with genes that are
highly transcribed (Barski et al., 2007; Cui et al., 2009). Regardless of basal transcription
state, the loss of PR-Set7 or its catalytic activity typically led to a significant increase in
expression of H4K20me1-enriched genes and PR-Set7 target genes (Figures 3, 5, 7).
These results lend support to the view that PR-Set7, and importantly H4K20me1,
function to repress transcription of specific genes.
Studies from our lab and others have demonstrated that a key mechanism by
which H4K20me1 induces transcriptional repression is through the specific recruitment
of the transcriptional repressor protein, L3MBTL1 (Kalakonda et al., 2008). L3MBTL1
can selectively bind H4K20me1-containing nucleosome arrays and induce compaction of
the chromatin, subsequently limiting access for binding of transcription machinery
(Trojer et al., 2007). Additionally, loss of L3MBTL1 resulted in the de-repression of
H4K20me1-enriched genes including c-myc and RUNX1 (Sims and Rice, 2008; Trojer et
al., 2007). These studies suggest that L3MBTL1 would also be recruited to the novel PR-
Set7 target genes presented in Chapter 1. Future experiments will be focused on
36
determining whether L3MBTL1 is indeed recruited to these newly identified targets and,
if so, if L3MBTL1 is required for the observed PR-Set7-mediated gene repression.
Since PR-Set7 is tightly cell cycle regulated, with high levels in G2/M and near-
absent levels during S phase, we began studies to determine how PR-Set7 and
H4K20me1 regulate transcription during distinct phases of the cell cycle. Preliminary
studies demonstrate that during G2/M, when PR-Set7 and H4K20me1 levels are highest,
H4K20me1-enriched genes are generally expressed at lower levels than genes lacking
H4K20me1 (Figure 9C). In order to rule out the possibility that these results are an
artifact of nocodazole drug treatment, future experiments will focus on repeating these
experiments using different methods of synchronization to obtain a homogenous
population of cells in G2/M. In order to determine whether PR-Set7 and H4K20me1 are
directly responsible for the decreased expression of H4K20me1-enriched genes during
G2/M, future experiments will be performed to deplete cells of PR-Set7 and subsequently
monitor changes in expression of H4K20me1-enriched genes during the cell cycle. It is
expected that these experiments will be difficult to perform and precise timing will be
important in order to account for cell-cycle defects that are incurred upon depletion of
PR-Set7. A possible role of PR-Set7 and H4K20me1 during G2/M is to repress the
transcription of specific genes to promote proper mitotic progression. The identification
of PR-Set7 target genes outlined in Chapter 1, will serve as an important resource for
testing this possibility.
37
Chapter 2. PR-Set7 binds the Riz1 histone H3K9 methyltransferase to
establish a trans-tail histone code that may be important for tumor
suppression
2.1. Introduction
Through immunofluorescence studies, our lab previously discovered significant
overlap and exclusion between the specific states of H4K20 and H3K9 methylation (Sims
et al., 2006). As reported by others, trimethyl H4K20 and H3K9 were both selectively
enriched at pericentric heterochromatin (Schotta et al., 2004). Similarly, monomethyl
H4K20 and H3K9 co-localized within the nucleus. Significantly, it was found that
H4K20me1 and H3K9me1 were preferentially and selectively enriched on the same
nucleosome particle in vivo. Subsequent work in our lab demonstrated that a bulk of
cellular H3K9 monomethylation is dependent upon PR-Set7 but independent of its
catalytic function (Sims and Rice, 2008). Based on these studies, we hypothesized that
PR-Set7 recruits an unidentified H3K9 monomethyltransferase to establish this trans-tail
histone code. In support of this hypothesis, we have discovered that PR-Set7 binds the
H3K9 methyltransferase, Riz1. Riz1 has been found to possess histone methyltransferase
activity and specifically methylate histone 3 lysine 9 (Kim et al., 2003). However, it
remained unclear whether Riz1 was capable of mono-, d- or tri-methylating its substrate.
We provide evidence that indicates that while Riz1 possesses both mono- and di-
methyltransferase activities in vitro, it functions predominantly as a
monomethyltransferase in cells. We have determined that Riz1 and PR-Set7 bind via
their C-terminal domains, and the C-terminal of Riz1 is required for proper recruitment to
chromatin. Interestingly, Riz1 gene expression is frequently silenced in human cancers,
38
including breast, liver, colon and lung cancers, and neuroblastoma, melanoma,
osteosarcoma and malignant meningiomas (Chadwick et al., 2000; He et al., 1998; Jiang
et al., 1999; Liu et al., 2012; Steele-Perkins et al., 2001). The Riz gene also frequently
contains inactivating missense mutations targeting the PR domain in human cancers and
cell lines (Steele-Perkins et al., 2001). Intriguingly, in micro-satellite instable tumors, the
Riz gene commonly contains frameshift mutations resulting in a truncated Riz1 protein
lacking the majority of the C-terminal domain that is critical for PR-Set7 binding and
proper recruitment to chromatin (Chadwick et al., 2000; Piao et al., 2000). Furthermore,
Riz1-deficient mice develop a wide variety of tumors (Steele-Perkins et al., 2001).
Combined, these data strongly suggest that Riz1 is a tumor suppressor, link Riz1
inactivation to tumor formation in mammals, and imply that direct Riz1 binding to PR-
Set7 is required for proper Riz1 localization and function.
2.2. Results
2.2.1. PR-Set7 selectively and directly binds the Riz1 H3K9 methyltransferase
Consistent with our previous reports (Sims and Rice, 2008), depletion of the PR-
Set7 H4K20 monomethyltransferase by RNAi resulted in the specific reduction of bulk
levels of histone H3K9me1 in HeLa cells (Figure 10A). Furthermore, ectopic expression
of a catalytically dead PR-Set7 point mutant resulted in a dominant negative phenotype
by reducing global H4K20me1 levels but H3K9me1 levels were not altered. Taken
together, these findings strongly suggested that PR-Set7 interacts with an unknown H3K9
39
monomethyltransferase and that this interaction is required for a significant fraction of
H3K9me1 in human cells. To identify this enzyme, HeLa cells were co-transfected with a
myc-tagged PR-Set7 plasmid and epitome tagged plasmids of three different well
characterized H3K9 methyltransferases: G9a, GLP-1 and SetDB1 (Schultz et al., 2002;
Shinkai and Tachibana, 2011). Western analysis for myc-PR-Set7 following
immunoprecipitation for each H3K9 methyltransferase demonstrated that PR-Set7 does
not interact with G9a, GLP-1 or SETDB1 in cells (Figure 10B).
It was previously reported that the Riz1/PRDM2/KMT8 tumor suppressor protein
also possesses H3K9 methyltransferase activity in vitro (Kim et al., 2003). Therefore,
HeLa cells were co-transfected with a full length Riz1 plasmid (Steele-Perkins et al.,
2001) and either a HA-PR-Set7 plasmid or a HA-p53 negative control plasmid. Western
analysis for Riz1 following HA-immunoprecipitation revealed that PR-Set7 selectively
interacted with Riz1 in cells (Figure 10B). To confirm these findings, HeLa nuclear
lysates expressing HA-PR-Set7 or HA-p53 were incubated with in vitro translated
35
S-
Riz1 prior to HA-immunoprecipitation. Autoradiography of the SDS-PAGE fractionated
eluted material revealed that Riz1 selectively bound PR-Set7 but not the negative control
p53 (Figure 10C). In addition, recombinant S-tag-His-PR-Set7 was incubated with either
in vitro translated
35
S-Riz1 or
35
S-G9a prior to S-tag-immunoprecipitation. Consistent
with the above results, autoradiography of the SDS-PAGE fractionated eluted material
demonstrated that PR-Set7 selectively bound Riz1 but not the negative control G9a
(Figure 10D). Collectively, these findings indicate that PR-Set7 selectively and directly
binds the Riz1 H3K9 methyltransferase both in vitro and in cells.
40
Figure 10. PR-Set7 selectively binds Riz1
(A) HeLa cells were transfected with a control shRNA or a PR-Set7 shRNA construct
(left) or either an empty eukaryotic expression vector or one encoding a catalytically dead
(CD) PR-Set7 (right). Whole cell lysates were prepared 5 days post-transfection. Western
analysis was preformed using a PR-Set7 antibody or antibodies specific for the varying
methyl states of H4K20 an H3K9. (B) HeLa cells were co-transfected with the specified
combinations of plasmids encoding epitope tagged histone methyltransferases. 24 hours
post-transfection IP lysates were incubated with HA- or FLAG-conjugated beads prior to
washing and eluting bound proteins. (C) Nuclear extracts from HeLa cells over-
expressing either HA-PR-Set7 or HA-p53 were incubated with FLAG-conjugated beads
and in vitro translated
35
S-labeled Riz1. Beads were washed before eluting the bound
material. (D) Recombinant PR-Set7 was incubated with Protein A Dynabeads and α-S-
tag antibody prior to the addition of in vitro translated
35
S-labeled protein.
41
2.2.2. PR-Set7 and Riz1 specifically bind via their C-terminal domains
To define the specific Riz1 protein domain required for binding PR-Set7,
recombinant truncations of Riz1 were expressed in either E. coli bacteria or Sf9 insect
cells prior to affinity purification of the recombinant proteins (Figure 11A). Recombinant
wild type S-tag-His-PR-Set7 was incubated with the different recombinant FLAG-tagged
Riz1 truncated proteins prior to S-tag-immunoprecipitation. Western analysis revealed
that only the fragment containing the last 323 amino acids of the Riz1 C-terminal region
(aa 1397-1719) specifically bound PR-Set7 (Figure 11A). To determine the specific PR-
Set7 protein domain required for binding the Riz1 C-terminus, recombinant truncations
of His-PR-Set7 containing either the N- or C-terminal fragments were expressed and
affinity purified from E. coli (Figure 11B). Recombinant GST-Riz1 C protein (aa 1397-
1719) was incubated with the different recombinant His-PR-Set7 truncations prior to
GST-immunoprecipitation. Western analysis revealed that the C-terminal portion of PR-
Set7 containing the catalytic SET domain specifically bound the Riz1 C-terminal
fragment (Figure 11B). It was previously reported that a fragment of Riz1 (aa 1514-1680)
was sufficient for the observed homo-dimeric binding to the PR/SET domain of Riz1
(Huang et al., 1998) suggesting that this region may non-selectively bind any SET
domain-containing protein. To test this possibility, a FLAG-Riz1C plasmid was co-
transfected into HeLa cells with either a DBD-PR-Set7 plasmid or a DBD-G9a plasmid
containing the SET domain. Western analysis following FLAG-immunoprecipitation
demonstrated that the Riz1 C-terminus bound PR-Set7 but did not bind the G9a SET
domain (Figure 11C). Importantly, a FLAG-Riz1ΔC plasmid (aa 1-1396) failed to
42
interact with PR-Set7, demonstrating that the C-terminus of Riz1 is required for
interaction with PR-Set7. These results indicate that the C-terminus of Riz1 directly and
selectively binds the C-terminus of PR-Set7 SET in vitro.
43
Figure 11. PR-Set7 and Riz1 interact via their C terminal domains
(A) Recombinant S-tag-PR-Set7 was expressed and purified from BL21 E. coli cells and
truncated FLAG-Riz1 proteins were expressed and purified from Sf9 insect cells. PR-
Set7 was incubated with Riz1 prior to immunoprecipitation with protein A dynabeads and
α-S-tag antibody. Bound material was subjected to western blot with a-FLAG antibody
and the results demonstrated that Riz1 C (aa 1397-1719) is capable of directly binding
PR-Set7. (B) Recombinant His or GST tagged proteins were expressed and purified from
BL21 E. coli cells. PR-Set7 and Riz1 proteins were incubated prior to
immunoprecipitation with glutathione sepharose beads. Bound material was subjected to
western blot with α-His antibody and it was observed that Riz1 C binds to the C terminal
domain of PR-Set7 (aa 129-352). (C) HeLa cells were co-transfected with FLAG-Riz1
plasmids and DBD-PR-Set7 or a DBD-G9a SET-domain containing plasmid. FLAG-
immunoprecipitation followed by western analysis demonstrated that Riz1 C binds PR-
Set7 but not the G9a SET domain. Additionally, FLAG-Riz1ΔC (aa 1-1396) failed to
interact with PR-Set7.
44
2.3.3. Riz1 predominantly functions as a histone H3K9 monomethyltransferase
It was previously reported that Riz1 catalyzes the methylation of histone H3K9 in
vitro, however, the product of Riz1 activity (mono-, di- or trimethyl H3K9) remained
unknown (Kim et al., 2003). To first address this, in vitro methyltransferase assays were
performed using a recombinant Riz1 protein fragment containing the catalytic PR
domain,
3
H-S-adenosyl-methionine as the methyl donor and histone H3 synthetic
peptides with different degrees of methylated H3K9 as substrates. Consistent with the
previous report, scintillation counting of the reactions demonstrated that Riz1 methylated
the unmodified H3 peptide (Figure 12A). Methylation of an H3K9me1 peptide was also
detected at about half the CPM of the unmodified H3 peptide, but methylation of the
H3K9me2 or negative control H3K9me3 peptides was not observed. Additional assays
using a recombinant G9a SET domain-containing protein revealed a similar trend for
methylation of the H3 peptide substrates. Since G9a is responsible for a large fraction of
cellular H3K9me1 and H3K9me2 (Shinkai and Tachibana, 2011), these findings indicate
that Riz1 is an H3K9 mono- and dimethyltransferase in vitro. The weaker
methyltransferase activity of Riz1 compared to G9a was not unexpected as a previous
structural study demonstrated that binding of the Riz1 PR/SET domain to a histone H3
peptide was significantly lower compared to other SET domain-containing proteins
(Briknarova et al., 2008).
Based on these findings, we hypothesized that depletion of Riz1 in cells would
reduce global levels of H3K9me1 and H3K9me2. To test this, HeLa cells were
transfected with a non-specific shRNA plasmid, a Riz1-specific shRNA plasmid or a
45
G9a-specific shRNA plasmid. Western analysis of whole cell lysates revealed that
depletion of G9a resulted in significant global decreases in all forms of H3K9
methylation compared to the non-specific shRNA control, as expected (Figure 12B).
Depletion of Riz1 also resulted in visible and consistent reductions in cellular H3K9me1
although less than depletion of G9a. In contrast to G9a, significant reductions in
H3K9me2 and H3K9me3 following Riz1 depletion were not observed. Similar decreases
in H3K9me1 were detected in both Riz1 and PR-Set7 depleted cells, however, changes in
H4K20me1 were not observed in Riz1 depleted cells compared to PR-Set7 depleted cells
(Figure 12C). These findings indicate that Riz1 is dispensable for PR-Set7-mediated
H4K20me1. Collectively, these results demonstrate that Riz1 possesses weak H3K9
mono- and dimethyltransferase activity in vitro, that Riz1 functions predominantly as an
H3K9 monomethyltransferase in cells and strongly suggests that Riz1 is largely
responsible for the previously observed PR-Set7-associated H3K9me1.
46
Figure 12. Riz1 possesses H3K9 monomethylation activity in vivo and in vitro
(A) In vitro histone methyltransferase assays were performed using recombinant Riz1 or
mouse G9a proteins. Briefly, 3 µg recombinant enzyme was incubated with 1 µg each of
the H3K9-methyl peptides and 1 µCi of S-adenosyl-L[methyl-3H]methionine in HMT
buffer (final concentration of 25 mM Tris-HCl pH 8.0, 5% glycerol) and incubated at
30°C for 90 minutes. The reaction was spotted on Whatman P-81 paper, washed and
counted by liquid scintillation. The indicated values were normalized to reactions lacking
peptide substrates. (B-C) HeLa cells were transfected with shRNA plasmids targeted to
G9a, Riz1, PR-Set7 or a non-specific sequence. Whole cell lysates were harvested and
western blot analysis was performed to examine global (B) H3K9 methylation and (C)
H4K20me1 levels.
47
Figure 13. The Riz1 C-terminal domain is necessary and sufficient for proper
cellular localization of Riz1
HeLa or U2OS cells were plated on glass coverslips and transfected with mCherry-tagged
Riz1 constructs for 24 hours. Cells were subsequently counter-stained with DAPI prior to
visualizing by fluorescence microscopy.
48
2.2.4. Interaction with PR-Set7 is required for Riz1 recruitment to chromatin
Taken together, the results above suggest that the direct binding of Riz1 to PR-
Set7 is required for Riz1 recruitment to chromatin. Therefore, we hypothesized that the
C-terminal domain of Riz1 would be sufficient for proper cellular localization. To test
this hypothesis, HeLa cells or U2OS cells were transfected with a plasmid encoding a
mCherry-Riz1 fusion protein. After 24 hours, cells were counterstained with DAPI and
visualized by fluorescence microscopy (Figure 13). Consistent with previous reports, we
observed that wild type full length Riz1 protein is predominantly localized in the nucleus
in both HeLa and U2OS cells (Liu et al., 1997)(Figure 13). Similarly, localization of the
mCherry-Riz1 C fusion protein was almost entirely nuclear in both cell types, indicating
that the C-terminal domain of Riz1 is sufficient for proper cellular localization.
Importantly, while the mCherry-Riz1ΔC mutant was also found in the nucleus, the lack
of the C-terminal domain correlated with a significant portion of the Riz1ΔC protein
remaining localized in the cytoplasm (Figure 13). Likewise, a mCherry-Riz1 N fusion
protein (aa 1-220), containing only the catalytic PR domain, was largely localized in both
the cytoplasm and nucleus of HeLa and U2OS cells, and was at times altogether excluded
from the nucleus. These results indicate that the PR domain alone is unable to properly
localize to the nucleus, and suggest that the PR-Set7 binding domain of Riz1 is required
for proper cellular localization.
These results found that a portion of the Riz1 ΔC mutant protein was found within
the nucleus. We hypothesized, however, that the Riz1 ΔC protein would display defective
localization within the nucleus and would be excluded from chromatin whereas the Riz1
49
C-terminus alone would be sufficient for recruitment to chromatin. To test this, HeLa
cells were transfected with a FLAG-Riz1 wild type (WT), FLAG-Riz1 ΔC or FLAG-Riz1
C plasmid. Nuclei were isolated and incubated with MNase at increasing times and the
various chromatin-associated fractions were collected. The soluble S1 fraction is mainly
composed of MNase-sensitive euchromatin, the S2 fraction is mainly composed of
MNase-resistant chromatin, such as heterochromatin, and the pellet (P) contains the
insoluble material associated with the nuclear matrix(Wu et al., 2010). Western analysis
revealed that FLAG-Riz1 WT was highly enriched in the P fraction but undetectable in
the S1 and S2 fractions even at extended MNase incubation times (Figure 14A). FLAG-
Riz1 C was also mainly enriched in the P fraction and extended MNase incubation times
did not shift the protein to the S1 or S2 fractions, as observed with HP1β. In contrast, the
majority of FLAG-Riz1 ΔC was enriched in the S1 fraction and undetectable in the S2
fraction. These findings indicate that the Riz1 C-terminus is required for Riz1
recruitment to chromatin in cells.
50
Figure 14. The Riz1 C-terminal domain is required for proper nuclear localization
of Riz1 and over-expression of this domain results in global reduction in H3K9me1
(A) HeLa cells were transfected with FLAG-Riz1 constructs for 24 hours. Nuclei were
isolated and digested with micrococcal nuclease (MNase) for the indicated times prior to
fractionation (see Chapter 5.8) and western analysis. (B) HeLa cells were transfected with
FLAG-Riz1 C or FLAG-GFP negative control plasmids. Western analysis of whole cell
lysates revealed a global reduction of H3K9me1, but no detectable changes in H3K9me2,
H3K9me3 or H4K20me1 in the FLAG-Riz1 C cells compared to FLAG-GFP negative
control cells.
2.2.5. Direct binding to PR-Set7 is required for Riz1-mediated H3K9me1
If Riz1 recruitment to chromatin is directly due to binding of PR-Set7, we
hypothesized that over-expression FLAG-Riz1 C would create a dominant negative
phenotype by preventing endogenous Riz1 binding to PR-Set7 thus resulting in reduced
levels of H3K9me1. To test this, FLAG-Riz1 C or FLAG-GFP negative control plasmids
were transfected into HeLa cells. Western analysis of whole cell lysates revealed a global
reduction of H3K9me1, but no detectable changes in H3K9me2, H3K9me3 or
H4K20me1 in the FLAG-Riz1 C cells compared to FLAG-GFP negative control cells
(Figure 14B). These findings indicate that direct binding to PR-Set7 is required for
51
recruitment of Riz1 to chromatin and that this interaction is necessary for a significant
amount of cellular Riz1-mediated H3K9me1.
These results imply that Riz1-mediated H3K9me1 is dependent on Riz1
interaction with PR-Set7, and suggest that PR-Set7 recruits Riz1 to chromatin. To test
this, a stable HEK 293 cell line containing a transgene consisting of 5 copies of the
GAL4 UAS in tandem with the a TK promoter was used. Cells were co-transfected with
a DBD-PR-Set7 or a control DBD-G9a SET-domain plasmid to target the fusion protein
to the transgene and either FLAG-Riz1 WT, FLAG-Riz1 N or FLAG-Riz1 C. Chromatin
immunoprecipitations (ChIPs) demonstrated that DBD-PR-Set7 and DBD-G9aC were
enriched near the TK promoter but not within the FKBP5 control gene (Figure 15).
Enrichment of DBD-PR-Set7 at the TK promoter was sufficient to promote recruitment
of FLAG Riz1 WT and Riz1 C. Importantly, recruitment of Riz1 C was not observed
when DBD-G9aC was targeted to the integrated transgene, demonstrating that Riz1
recruitment is selectively mediated by PR-Set7 (Figure 15). Conversely, Riz1 N,
containing the catalytic domain but lacking the PR-Set7 binding region, was not detected
at the TK promoter in the presence of either DBD-PR-Set7 or G9aC enrichment,
consistent with the findings above.
52
Figure 15. Riz1 recruitment to chromatin is selectively mediated by PR-Set7
HEK293-TK22 cells were co-transfected with a DBD-PR-Set7 or a control DBD-G9aC
SET-domain plasmid to target the fusion protein to the transgene and either FLAG-Riz1
WT, FLAG-Riz1 C or FLAG-Riz1 N. Chromatin immunoprecipitation was performed
using α-DBD, α-FLAG or IgG. 0.1 ng of ChIPed DNA was used as template in PCR
reactions with primers designed to amplify a region in the thymidine kinase promoter
(left) or a control region within FKBP5 (right).
53
2.2.6. Riz1 is dispensable for the transcription of PR-Set7 regulated genes
Similar to PR-Set7, it was previously reported that Riz1 functions as a
transcriptional repressor protein (Xie et al., 1997). The first three zinc finger motifs of
Riz1 (Figure 11A) can bind GC-rich Sp-1 elements and this interaction can induce the
transcriptional repression of both the HSV TK promoter and the SV40 early promoter
(Xie et al., 1997). These findings suggest that Riz1 functions in concert with PR-Set7 to
induce transcriptional repression of specific genes. Therefore, we hypothesized that
depletion of Riz1 would result in the de-repression of previously identified PR-Set7-
regulated genes (Table 3)(Spektor et al., 2011). To test this, quantitative RT-PCR was
performed on RNA isolated from HeLa cells transfected with a control shRNA plasmid, a
PR-Set7 shRNA plasmid or a Riz1 shRNA plasmid (Figure 12C). Consistent with the
previous results, depletion of PR-Set7 resulted in a significant de-repression of several
H4K20me1-associated genes but did not affect expression of H4K20me3-associated
genes (Figure 16). However, depletion of Riz1 and global reduction of H3K9me1 had no
effect on the expression of these same genes. These findings indicate that the PR-Set7-
mediated recruitment of Riz1 and H3K9me1 at specific genes is dispensable for
transcriptional regulatory effects mediated by PR-Set7.
54
Figure 16. Riz1 is dispensable for transcriptional regulation of PR-Set7-regulated
genes
Quantitative RT-PCR expression analysis of PR-Set7-regulated genes (Table 3) from
control shRNA (white), PR-Set7 shRNA (dark grey) or Riz1 shRNA (light grey)
transfected HeLa cells. Results are plotted relative to 18S expression and normalized to
control shRNA levels (y-axis). Error bars represent the standard error from three
independent biological replicates. The student t-test was used to determine statistically
significant changes at p<0.05(*).
55
2.2.7. PR-Set7 interaction with Riz1 may be critical for Riz1 tumor suppressor
activity
Besides a role in transcriptional regulation, Riz1 also functions as a tumor
suppressor and is commonly silenced or mutated in several different types of human
cancers (Table 4). A frequently occurring RIZ1 frameshift mutation in microsatellite
instable (MSI) human colorectal, gastric, endometrial and pancreatic cancers was
previously reported (Chadwick et al., 2000; Piao et al., 2000). The mutation is caused by
deletions in the poly-A tract of exon 8 resulting in a Riz1 protein lacking the wild type C-
terminus. These mutations were detected in 9 of 24 (37.5%) primary human MSI
colorectal tumors and in 6 of 11 (54.5%) cell lines, including HCT116 (Chadwick et al.,
2000). Since this region contains the majority of the PR-Set7 binding domain, we
predicted that Riz1 binding to PR-Set7 would be abolished in cancer cells carrying this
mutation. To test this, HeLa cells or HCT116 cells were transfected with either a FLAG-
PR-Set7 plasmid or a FLAG-GFP negative control plasmid. FLAG-immunoprecipitations
of nuclear lysates were performed prior to Western analysis. In HeLa cells, endogenous
wild type Riz1 selectively bound to FLAG-PR-Set7; binding was not observed to FLAG-
GFP (Figure 17). In contrast, binding of the endogenous truncated mutant Riz1 to FLAG-
PR-Set7 in HCT116 cells was not detected. Additionally, as predicted, a lower molecular
weight band was detected by the Riz1 antibody in the HCT116 nuclear lysates, consistent
with the reported C-terminal truncated mutant. These results demonstrate that a
frequently occurring frameshift mutation in cancer cells results in the expression of a
truncated Riz1 protein lacking the C-terminus that is unable to bind PR-Set7.
56
Cancer Poly(A) Frameshift
Frequency
Reference(s)
Gastric 36-48% (Piao et al., 2000; Sakurada et al., 2001)
Colorectal 25-38% (Chadwick et al., 2000; Piao et al., 2000;
Sakurada et al., 2001)
Endometrial 33% (Piao et al., 2000)
Melanoma 17% (Poetsch et al., 2002)
Pancreatic 10% (Sakurada et al., 2001)
Disease Deletion (LOH) Frequency Reference(s)
Pheochromocytoma 67% (Geli et al., 2005)
Paraganglioma 50% (Geli et al., 2005)
Liver 37-39% (Fang et al., 2001; Fang et al., 2000)
Colorectal 23% (Fang et al., 2001)
Breast 19 (Fang et al., 2001)
Gastric 12% (Fang et al., 2001)
Disease Promoter Hypermethylation
Frequency
Reference(s)
Gastric 69% (Oshimo et al., 2004)
Nasopharyngeal 60% (Chang et al., 2003)
Liver 45-62% (Du et al., 2001; Nomoto et al., 2007)
Breast 44% (Du et al., 2001)
Gallbladder 26% (Takahashi et al., 2004)
Ovarian 7% (Akahira et al., 2007)
Table 4. Summary of Riz1 mutations in human cancers
Reported mutations in the RIZ gene occurring by 1- or 2-bp frameshift deletion mutations
in two poly-adenosine tracks in exon 8 (top), loss of heterozygosity (middle), or gene
silencing by promoter hypermethylation (bottom). All mutations were observed in
primary human cancer specimens at the indicated frequencies.
57
Figure 17. HCT116 cells contain a C-terminally-truncated Riz1, which is unable to
interact with PR-Set7
HeLa or HCT116 cells were transfected with a FLAG-PR-Set7 or a FLAG-GFP control
plasmid. Nuclear lysates were isolated and incubated with FLAG-conjugated beads
followed by thorough washing and elution with FLAG-peptide. Eluted material was
analyzed by western blot using α-Riz1 and α-FLAG antibodies. 10% of HCT116 nuclear
lysates input material was also included in the western analysis to demonstrate the lower
molecular weight of the truncated Riz1 protein in HCT116 cells.
In several cancer cell lines in which Riz1 is silenced or mutated, it was reported
that ectopic expression of wild type Riz1 resulted in a G2/M cell cycle arrest and
apoptosis (Chadwick et al., 2000; He et al., 1998; Liu et al., 2012). Based on our findings,
we hypothesized that the observed cell cycle arrest and apoptosis was dependent on Riz1
binding to PR-Set7. We first sought to determine whether we could reproduce the
previously published results in the HCT116 cell line, which expresses a truncated Riz1
protein lacking its C-terminal domain. To do so, HCT116 cells were transfected with an
empty mCherry control plasmid or a plasmid encoding a mCherry-Riz1 WT fusion
58
protein (Figure 18). Four days post-transfection cells were harvested and stained with
Hoechst dye for DNA content analysis. Flow cytometry was performed to analyze the
DNA histogram in mCherry-positive cells. As predicted, ectopic expression of FLAG-
Riz1 WT resulted in a significant increase in cells arrested at G2/M as well as a
significant increase in cells with a sub-G1 content, indicative of apoptosis (Figure 18).
Experiments are currently underway to examine the cell cycle profile of cells ectopically
expressing a mCherry-Riz1 ΔC fusion protein. We predict that the ability of Riz1 to
induce cell cycle arrest and apoptosis in cell lines with silenced or mutated Riz1 is
dependent upon its binding with PR-Set7, and therefore predict that we will not observe
significant cell cycle changes in HCT116 cells expressing mCherry-Riz1ΔC.
Figure 18. Ectopic Riz1 expression in HCT116 cells induces G2/M arrest and
apoptosis
HCT116 cells were transfected with a mCherry control plasmid or a plasmid encoding a
mCherry-Riz1 WT fusion protein. Cells were harvested 96 hours after transfection and
stained for DNA content with Hoechst dye. Flow cytometry was performed to analyze the
DNA content of mCherry positive cells.
59
2.3. Discussion and future directions
We have identified a novel interaction between two histone methyltransferases,
PR-Set7 and Riz1. This interaction is required for Riz1 recruitment to chromatin and the
subsequent establishment of a trans-tail histone code consisting of monomethylated
H4K20 and H3K9. Through domain mapping in vitro binding assays, we determined that
Riz1 and PR-Set7 interact via their C-terminal domains. Importantly, a truncated Riz1
protein lacking the C-terminal PR-Set7 interacting region (Riz1 ΔC) is defective in
proper cellular localization and does not tightly bind insoluble chromatin like the wild-
type Riz1 protein. We used the PredictProtein software (Rost et al., 2004) to determine
whether the Riz1 C terminal may contain a nuclear localization signal that would account
for the loss of proper nuclear localization observed with the Riz1 ΔC mutant. This
software indicated that there is no nuclear localization signal present in the C-terminal
domain of Riz1, which further supports the model that the C-terminal is required for
Riz1’s PR-Set7-mediated recruitment to chromatin. Furthermore, we demonstrated that
targeting of PR-Set7 to a specific genomic region is sufficient to recruit Riz1, but not
Riz1 Ν.
Riz1 is frequently silenced or mutated in many human cancers, including breast,
liver, and colon cancers, and neuroblastoma, melanoma, osteosarcoma and malignant
meningiomas (Chadwick et al., 2000; He et al., 1998; Jiang et al., 1999; Liu et al., 2012;
Steele-Perkins et al., 2001). Two common mechanisms of Riz1 inactivation are gene
silencing by promoter hypermethylation and missense mutations targeting the catalytic
PR domain (Carling et al., 2003; Dong et al., 2012; Kim et al., 2003; Lal et al., 2006;
60
Oshimo et al., 2004; Steele-Perkins et al., 2001; Tokumaru et al., 2003). Of particular
interest to us in light of our discovery of Riz1 binding to PR-Set7, another frequent
mutation in the RIZ gene occurs through a frameshift mutation which results in a
truncated Riz1 protein lacking the majority of its C-terminal PR-Set7 interacting domain
(Chadwick et al., 2000; Piao et al., 2000). Our data suggests that the mutant truncated
Riz1 protein commonly found in microsatellite instable (MSI +) cancers is unable to
interact with PR-Set7 and therefore will not properly localize to chromatin to establish
H3K9me1. We would predict that this defect would have deleterious effects analogous to
Riz1 mutants containing inactivating PR-domain missense mutations.
In addition to an abundance of data demonstrating that Riz1 is silenced or mutated
in human cancers, Riz1 knockout mice develop a wide variety of tumors (Steele-Perkins
et al., 2001). Taken together, these data strongly suggest that Riz1 is a tumor suppressor
and link Riz1 inactivation to tumor formation in mammals. However, the exact
mechanism by which loss of Riz1 activity promotes cancer development is unclear. PR-
Set7 is required for proper cell cycle progression and DNA damage repair, both pathways
whose fidelity is critical to preventing oncogenesis. Future experiments will be performed
to determine whether Riz1 is also required for proper cell cycle progression and DNA
repair and, if so, if Riz1 and PR-Set7 interaction is required for proper function in these
pathways.
PR-Set7 and Riz1 have been reported to be involved in transcriptional repression
(Congdon et al., 2010; Xie et al., 1997), however, preliminary studies suggest that Riz1 is
dispensable for the transcriptional repression of PR-Set7 target genes (Figure 16). It has
61
additionally been reported that both Riz1 and PR-Set7 are important in estrogen-mediated
gene activation (Carling et al., 2004; Li et al., 2011a). Future experiments will be
performed to determine whether PR-Set7 and Riz1 function cooperatively to promoter
transcription of estrogen-responsive genes.
62
Chapter 3. PR-Set7 interacts with HDAC3 and this interaction may
promote transcriptional repression and epithelial to mesenchymal
transition
3.1. Introduction
The combinatorial nature of the histone code predicts that multiple histone
modifying enzymes may work together to establish various different modifications at the
same genomic locations. The presence of several different modifications may act as
‘layers’ to ‘lock’ chromatin in a particular state; for example, to compact chromatin in
regions that are transcriptionally repressed. One example of this is the direct interaction
between the maintenance DNA methyltransferase, DNMT1, and the HMT, G9a, which
coordinate during DNA synthesis to establish DNA and histone methylation at specific
genes and thus ensure faithful gene repression in daughter cells (Esteve et al., 2006).
Since we have established that PR-Set7 functions in the transcriptional repression of
human genes, we hypothesized that PR-Set7 might similarly interact with other enzymes
or factors known to repress transcription.
Acetylation of histone tails promotes transcription, in part by weakening histone-
DNA interactions and thus permitting chromatin to adopt a more open and accessible
state (Kurdistani and Grunstein, 2003). In this respect, histone acetyltransferases (HAT)
and deacetylases (HDAC) play important roles in transcriptional activation and
repression, respectively. Notably, recent analysis of ENCODE (Ernst et al., 2011) data by
Chendhore Veerappan revealed that enrichment of H4K20me1 is anti-correlative with
acetylation of H4K5, H4K8 and H4K16 (data not shown). Based on these results, we
predicted that PR-Set7 interacts with HDAC protein(s) to coordinate monomethylation of
63
H4K20 and concomitant removal of acetyl modifications from neighboring lysine
residues. To test this hypothesis, we isolated the PR-Set7 multi-protein complex from
cells by immunoprecipitation and evaluated whether the complex possessed HDAC
activity. As predicted, we found that PR-Set7 is in a complex with enzyme(s) possessing
HDAC activity. We further established that PR-Set7 interacts in vivo with Class I HDAC
proteins, and directly binds HDAC3 in vitro. Finally, we have confirmed published
reports that both PR-Set7 and HDAC3 promote epithelial-mesenchymal transition and
hypothesize that they function interdependently during that process.
3.2. Results
3.2.1. The PR-Set7 multi-protein complex contains histone deacetylase activity
To investigate whether PR-Set7 interacts with transcriptionally repressive
HDACs, we first utilized a HDAC activity kit containing an acetylated peptide substrate
(Active Motif, Cat. No. 56200). HeLa cells were transfected with either a FLAG-PR-Set7
or FLAG-GFP construct. FLAG immunoprecipitation was performed and the eluate was
used in the subsequent HDAC activity assay (Figure 19A). It was observed that the PR-
Set7 complex contained significantly more HDAC activity than the GFP control,
suggesting that PR-Set7 is indeed interacting with an enzyme that possesses HDAC
activity. Similar assays were performed that demonstrated that PR-Set7 does not
immunoprecipitate with enzymes possessing HAT activity (data not shown).
64
Figure 19. The PR-Set7 multi-protein complex contains HDAC activity
(A) HeLa cells were transfected with FLAG-PR-Set7 or FLAG-GFP plasmids.
Immunoprecipitations were performed using FLAG-conjugated beads and increasing
amounts of HeLa nuclear extract (HNE; positive control) or FLAG-IPed material was
incubated with acetylated substrate (Active Motif, Cat. No. 56200). (B) HeLa cells were
co-transfected with FLAG-HDAC plasmids and a HA-PR-Set7 plasmid (input; bottom).
Immunoprecipitation against PR-Set7 was performed using HA-conjugated beads
followed by western blot using α-FLAG and α-HA antibodies. These results indicate that
PR-Set7 selectively interacts with class I HDAC proteins in cells.
65
3.2.2. PR-Set7 specifically interacts with class I HDAC proteins
Next, in order to investigate which particular HDAC proteins may be interacting
with PR-Set7, we co-expressed FLAG-HDAC constructs (courtesy of Ed Seto, Moffitt
Cancer Center) with HA-PR-Set7 constructs. Immunoprecipitation against PR-Set7
followed by FLAG western blot revealed that PR-Set7 interacts in vivo with class I
HDAC proteins, most strongly with HDAC1 and HDAC3, but not with class II HDAC
proteins (Figure 19B).
3.2.3. PR-Set7 directly binds the class I HDAC, HDAC3
To investigate whether PR-Set7 can directly bind to class I HDAC proteins, GST-
tagged HDAC1, HDAC2, HDAC3 or HDAC10 were expressed and purified from BL21
E. coli cells and incubated with recombinant His-PR-Set7. GST pull-down followed by
western blot for His-PR-Set7 indicated that in vitro, PR-Set7 selectively binds to
HDAC3, but not to the other class I or II HDAC proteins examined (Figure 20A). To
further determine which domain of PR-Set7 is required for binding to HDAC3,
recombinant His-PR-Set7 N or C terminal truncated fusion proteins were incubated with
GST-HDAC proteins. GST pull-down followed by His western blot showed that the N-
terminal domain (aa 1-128) of PR-Set7 is responsible for the observed binding between
PR-Set7 and HDAC3 (Figure 20B).
66
Figure 20. PR-Set7 binds HDAC3 via its N-terminal domain in vitro
(A) Recombinant GST-HDAC proteins were incubated with recombinant His-PR-Set7
prior to immunoprecipitation against the HDAC proteins with glutathione sepharose
beads. Western analysis of the bound material using α-His antibody indicated that PR-
Set7 binds HDAC3 in vitro. (B) Recombinant His-PR-Set7 N- (aa 1-128) or C- (aa 129-
end) truncation proteins were incubated with GST-HDAC proteins prior to
immunoprecipitation against the HDAC proteins with glutathione sepharose beads.
Western analysis of the bound material using α-His antibody indicated that PR-Set7
binds HDAC3 via its N-terminal domain.
67
3.2.4. Selective inhibition of class I HDAC activity results in the de-repression of
PR-Set7-regulated genes
Since both PR-Set7 and HDAC proteins are involved in transcriptional repression
(Congdon et al., 2010; Ng and Bird, 2000) we hypothesized that PR-Set7 is interacting
with class I HDAC proteins in vivo to cooperatively facilitate transcriptional repression.
To test this hypothesis, we first utilized three common HDAC inhibitors: class I and II
inhibitors Trichostatin A (TSA) and Suberoylanilide Hydroxamic Acid (SAHA), and the
broad-range HDAC inhibitor sodium butyrate (NaBut). HeLa cells were treated for 24
hours with each of these HDAC inhibitors or DMSO vehicle control before harvesting
and extracting mRNA. Quantitative RT-PCR was performed to examine the expression of
PR-Set7 target genes identified from Illumina gene expression analysis (Table 3). The
results revealed that 4/7 (~57%) of PR-Set7 target genes examined displayed a 50% or
greater increase in expression after treatment with each of the three HDAC inhibitors
(Figure 21A). Furthermore, no changes in expression where observed for the H4K20me3-
associated gene TUG1. Previous studies have shown that inhibition of HDAC activity by
NaBut affects the expression of only 2% of mammalian genes (Davie, 2003). Taken
together, these results suggest that class I and/or II HDAC proteins also transcriptionally
regulate nearly half of genes that are transcriptional targets of PR-Set7. To determine
whether class I HDACs, class II HDACs or both were involved in the transcriptional
regulation of PR-Set7 target genes, we purchased inhibitors that can selectively inhibit
class I or II HDAC activity. HeLa cells were treated with 1 µM of HDAC1 specific (MS-
275), class I HDAC specific (MGCD0103), or class II HDAC specific inhibitor
(MC1568) for 24 hours. Quantitative RT-PCR analysis was performed and showed that 4
68
out of 6 (~66%) PR-Set7 target genes examined displayed a 2-fold or greater increase in
expression after treatment with either the class I HDAC specific or the HDAC1 specific
inhibitor (Figure 21B). Treatment with 1 µM of the class II HDAC specific inhibitor did
not affect the expression of the PR-Set7 target genes, and no significant changes in
expression of GAPDH or TUG1 were observed after treatment with any of the three
inhibitors. These data suggest that PR-Set7 is interacting with HDAC1, and possibly
other class I HDAC proteins, to repress the transcription of a subset of PR-Set7 target
genes in cells.
69
Figure 21. Inhibition of class I HDAC activity results in the de-repression of PR-
Set7 target genes
(A) HeLa cells were treated with 0.8 µM TSA (dark blue), 25 mM NaBut (purple), 2.5
µM SAHA (grey) or DMSO vehicle control for 24 hours prior to extracting RNA.
Quantitative RT-PCR analysis was performed to examine the expression of PR-Set7
target genes (Table 3). Results are plotted relative to 18S expression and normalized to
expression in DMSO-treated cells (dotted line), which are set at 1 (y-axis). Positive
control genes for TSA, NaBut and SAHA treatment are c-fos, IL-5 and p21, respectively.
(Figure 21, continued) Data points represent the cycle threshold average from 3 triplicate
PCR reactions, N=1. (B) HeLa cells were treated with 1 µM HDAC1-specific (blue),
class I HDAC (red) or class II HDAC (green) inhibitors for 24 hours prior to extracting
RNA. Quantitative RT-PCR analysis was performed to examine the expression of PR-
Set7 target genes (Table 3). Results are plotted relative to 18S expression and normalized
to expression in DMSO-treated cells (dotted line), which were set at 1 (y-axis). Error bars
for HDAC1 and class I HDAC inhibitor treatment represent the standard error from two
independent biological replicates, while class II HDAC inhibitor treatment is N=1.
70
UBE2L6
VAMP1
ARL14
REC8L1
EPSTI1
ADM
HES5
positive control
TUG1
0
5
10
Expression Fold Change (relative to 18S)
TSA
NaBut
SAHA
UBE2L6
VAMP1
ARL14
EPSTI1
HES5
NFKBIZ
p21
GAPDH
TUG1
0
2
4
6
8
10
Expression Fold Change (relative to 18S)
HDAC1 inhibitor
Class I HDAC inhibitor
Class II HDAC inhibitor
A
B
71
3.2.5. Ectopic expression of PR-Set7 or HDAC3 is sufficient to alter markers of
epithelial-mesenchymal transition
Interestingly, much like PR-Set7, HDAC3 is essential for the maintenance of
chromatin structure and genome stability (Bhaskara et al., 2010). Specifically, HDAC3
has critical roles in S-phase progression and DNA damage repair, and inhibition of
HDAC3 results in mitotic defects (Bhaskara et al., 2008; Warrener et al., 2010). In
addition, HDAC3 has also been implicated in transcriptional regulation and epithelial-
mesenchymal transition (EMT) (McQuown and Wood, 2011; Wu et al., 2011). Similarly,
PR-Set7 possesses dual transcriptional activities as a binding partner of the transcription
factor TWIST, a master regulator of EMT (Yang et al., 2011; Yang et al., 2004). These
reports suggest that there are numerous pathways in which the interaction between PR-
Set7 and HDAC3 may be biologically important. Since it has been demonstrated that
over-expression of either PR-Set7 or HDAC3 alone is sufficient to induce EMT, we first
hypothesized that PR-Set7 and HDAC3 function together in promoting EMT (Wu et al.,
2011; Yang et al., 2011). To begin, we sought to determine whether the previously
published results were reproducible in MCF7 cells. To do so, MCF7 cells were
transfected with FLAG-PR-Set7, FLAG-HDAC3 or FLAG-empty plasmids and
harvested after 48 hours. Western analysis using a FLAG antibody was performed to
confirm over-expression of PR-Set7 or HDAC3 (Figure 22). Western analysis using
antibodies against the epithelial marker, E-cadherin, or mesenchymal marker, N-
cadherin, demonstrated that over-expression of either PR-Set7 or HDAC3 results in an
up-regulation of the mesenchymal marker. Additionally, over-expression of PR-Set7, but
not HDAC3, led to a slight decrease in the epithelial marker, E-cadherin. These results
72
suggest that both PR-Set7 and HDAC3 do in fact play a role in promoting EMT, and
provide a solid foundation for future work to investigate whether there is a requirement
for the interaction between PR-Set7 and HDAC3 during EMT.
Figure 22. Over-expression of PR-Set7 or HDAC3 in MCF7 cells results in the up-
regulation of the mesenchymal marker N-cadherin
MCF7 cells were transfected with FLAG-PR-Set7, FLAG-HDAC3 or FLAG-empty
plasmids and harvested after 48 hours. Western analysis (left) was performed using α-
FLAG antibody or antibodies against the epithelial marker, E-cadherin, or mesenchymal
marker, N-cadherin. Densitometry (right) was performed on the western results and
plotted with error bars representing the standard error from three independent biological
replicates.
FLAG: Null PR-Set7 HDAC3
α-FLAG
α-H4
α-N-cadherin
α-E-cadherin
0
0.5
1
1.5
2
2.5
3
Control
PR-Set7
HDAC3
N-cadherin E-cadherin
Average Adjusted Density
73
3.3. Discussion and future directions
We discovered that the PR-Set7 multi-protein complex contains HDAC activity,
and further characterized that PR-Set7 interacts with class I HDAC proteins in vivo and
directly binds HDAC3 in vitro. While we found that PR-Set7 is in a complex with
HDAC1, HDAC2 and HDAC3 in cells, only HDAC3 was capable of interacting with PR-
Set7 in vitro. It is probable that the interaction between PR-Set7 and HDAC1 is mediated
by other protein(s). Additionally, the weak interaction between PR-Set7 and HDAC2 in
vivo may be reflective of the finding that HDAC1 and HDAC2 are in a complex together
(Johnson et al., 2002). Preliminary data using HDAC inhibitors suggests that class I
HDAC proteins are involved in the transcriptional repression of a subset of PR-Set7
target genes. It is unclear whether PR-Set7 is required for the recruitment of class I
HDACs to specific genes, or vice versa, and if these enzymes act in a synergistic manner
to repress gene expression. A combination of ChIP experiments and HDAC inhibitor
treatment in cells transfected with control or PR-Set7 shRNA plasmids will help clarify
the potentially cooperative function of PR-Set7 and class I HDAC proteins in
transcription. It is also unclear why the transcription of only a portion of the PR-Set7
target genes examined was affected by treatment with HDAC inhibitors. ChIP-
sequencing for PR-Set7 and the class I HDAC proteins could be informative in
identifying sequence motifs where they co-localize. Finally, through the use of shRNA
plasmids specifically targeted to HDAC1, HDAC2 or HDAC3, experiments are in
progress to distinguish which particular class I HDAC protein(s) is responsible for the
transcriptional de-repression observed upon treatment with class I HDAC inhibitor.
74
In vitro binding assays revealed that PR-Set7 directly binds HDAC3 via its N-
terminal domain. Following the discovery of this novel interaction, we sought to
determine which biological processes PR-Set7 and HDAC3 binding might function in.
Both enzymes contribute to maintaining chromatin structure and genome stability and
play key roles in proper cell cycle progression and DNA damage repair (Bhaskara et al.,
2008; Bhaskara and Hiebert, 2011; Houston et al., 2008; Warrener et al., 2010; Wu et al.,
2010). Furthermore, both PR-Set7 and HDAC3 were recently discovered to be central
players in epithelial-mesenchymal transition (EMT) (Wu et al., 2011; Yang et al., 2011).
In light of these recent reports we sought to investigate whether PR-Set7 and HDAC3
cooperate during EMT. It was reported that over-expression of PR-Set7 in MCF7 (breast
adenocarcinoma) or of HDAC3 in FaDu (hypopharyngeal carcinoma) epithelial cell lines
resulted in the down-regulation of epithelial genes and up-regulation of mesenchymal
genes. We sought to first determine whether these results were reproducible, and
importantly if over-expression of either PR-Set7 or HDAC3 could induce EMT
phenotypes in MCF7 cells. Preliminary experiments indicated that expressing either PR-
Set7 or HDAC3 in MCF7 cells led to a reproducible increase in the mesenchymal
marker, N-cadherin, protein levels but only a slight and somewhat inconsistent decrease
in the epithelial marker, E-cadherin, protein levels. It was reported that PR-Set7 works in
concert with the transcription factor TWIST to promote EMT. MCF7 cells have low
TWIST protein levels compared to MDA-MB-231 cells, a different breast cancer
epithelial cell line. Future experiments will be performed in the MDA-MB-231 cell line,
to see if more abundant levels of TWIST result in more pronounced down-regulation of
75
epithelial markers upon PR-Set7 or HDAC3 over-expression. If we find that PR-Set7 and
HDAC3 are able to significantly and consistently repress and activate epithelial and
mesenchymal genes, respectively, in MDA-MB-231 cells, we will next investigate
whether there is an interdependency for these enzymes to promote EMT. To do so,
MDA-MB-231 cells will be depleted of HDAC3 using shRNA prior to PR-Set7 over-
expression. We hypothesize that the lack of HDAC3 will inhibit the repression of
epithelial markers and activation of mesenchymal makers previously observed upon PR-
Set7 over-expression. Reverse experiments wherein PR-Set7 is first depleted followed by
HDAC3 over-expression may also be performed. ChIP analysis of EMT-associated gene
promoters in these experiments will be informative as to whether HDAC3 occupancy is
required for PR-Set7 recruitment, or vice versa. Finally, transcription reporter assays
along with invasion and metastasis assays will be employed to gain a more
comprehensive understanding of the potential cooperative role of PR-Set7 and HDAC3
binding during EMT. Dependent on the outcome of these studies it would be interesting
to collaborate with physicians and pathologists to determine whether expression of PR-
Set7 and HDAC3 correlate with each other and/or with prognosis in tumor tissue samples
representing varying degrees of metastasis.
76
Chapter 4. JNK-dependent phosphorylation of Serine 29 stabilizes PR-
Set7 against UV-dependent degradation
4.1. Introduction
PR-Set7 protein expression is tightly cell cycle regulated with peak expression
occurring during G2/M and undetectable levels in S phase (Oda et al., 2009; Rice et al.,
2002). Post-translational modification of PR-Set7, such as phosphorylation,
SUMOylation and ubiquitination are the primary mechanism by which proper PR-Set7
levels are regulated throughout the cell cycle (Abbas et al., 2010; Centore et al., 2010;
Oda et al., 2010; Spektor et al., 2011; Wu et al., 2010). Several independent groups have
recently demonstrated that PR-Set7 is a substrate of the E3 ubiquitin ligase complex
CRL4
Cdt2
(Abbas et al., 2010; Centore et al., 2010; Oda et al., 2010). PR-Set7 is targeted
for degradation by CRL4
Cdt2
in a PCNA-dependent manner during S phase and upon UV-
induced DNA damage. This degradation event is dependent on a PCNA interaction motif
present in PR-Set7 known as a PIP degron. Loss of this PIP degron, PCNA or the
CRL
Cdt2
complex all result in improperly high levels of PR-Set7 protein during S phase
or after DNA damage. Furthermore, inhibition of this degradation pathway leads to DNA
damage and checkpoint-mediated G2 arrest. PR-Set7 protein levels can also be regulated
by two other E3 ubuiquitin ligases, SCF/Skp2 and BBAP, however neither of these
ligases have been shown to directly ubiquinate PR-Set7 (Yan et al., 2009; Yin et al.,
2008).
In addition to ubiquitination, our lab has shown that phosphorylation is an
important event in regulating proper PR-Set7 protein levels. We demonstrated that PR-
77
Set7 is phosphorylated at serine 29 by Cyclin B/cdk1 during mitosis (Wu et al., 2010).
This post-translation modification causes PR-Set7 to disassociate from mitotic
chromosomes where it is subsequently dephosphorylated by the cdc14 phosphatase and
targeted for degradation via ubiquitination by APC/Cdh1. The dynamic regulation of PR-
Set7 protein levels by a variety of tightly coordinated post-translational modificaiton
events suggests that it is cruical to maintain precise levels of PR-Set7 and H4K20me1 for
proper cell cycle progression. Interestingly, the over-expression of a degradation-resistant
PR-Set7 led to the transcriptional repression of many genes (Abbas et al., 2010).
Importantly, it was shown that full gene repression was dependent on the catalytic
activity of PR-Set7. This report provides evidence suggesting that the proper CRL4
Cdt2
-
mediated degradation of PR-Set7 is critical for preventing the repression of genes during
the normal cell cycle, and further implicates PR-Set7 and H4K20me1 in transcriptional
repression.
Cdt1, an essential origin licensing protein, is another substrate of CRL4
Cdt2
, and is
also degraded upon UV irradiation. It was shown that pre-treatment with sorbitol, to
activate the stress kinase pathway by osmotic shock, blocks the UV-induced degradation
of Cdt1 (Chandrasekaran et al., 2011). Sorbitol-induced stabilization of Cdt1 is mediated
by p38 and JNK stress-activated MAP kinases and can be blocked by p38 and JNK
inhibitors. Since PR-Set7 and Cdt2 are tightly cell cycle regulated in a similar manner
and are two of only three known substrates of CRL4
Cdt2
, we predict that there will be
many similarities in the function and regulation of these two proteins. Consistent with
previous reports, we found that, like Cdt1, PR-Set7 is stabilized after osmotic stress by a
78
phosphorylation event. We have determined that the c-Jun N-terminal kinase (JNK)
phosphorylates serine 29 after osmotic stress, and that this modification stabilizes PR-
Set7 to prevent UV-induced degradation. Finally, we provide preliminary evidence that
phosphorylation of serine 29 blocks ubiquitination of PR-Set7, which we hypothesize is
due to inhibition of binding of the Cdt2 adaptor protein.
4.2. Results
4.2.1. Osmotic stress promotes JNK-mediated stabilization of PR-Set7 against UV-
induced degradation
We first sought to determine whether we could independently reproduce the
results that PR-Set7 is protected against UV-induced degradation by sorbitol pre-
treatment in a different cell line. To this end, the U2OS osteosarcoma cell line was
treated with or without 300 mM sorbitol for 15 minutes prior to UV-irradiation. As
expected, 60 minutes post-irradiation, PR-Set7 protein levels were significantly reduced
(Figure 23). Furthermore, as was reported in HeLa cells, pre-treatment of U2OS cells
with sorbitol protected PR-Set7 from degradation, and this protection was abolished
when cells were first treated with a combination of p38 and JNK MAP kinase inhibitors.
These results suggest that p38, JNK or both phosphorylate PR-Set7 in response to
osmotic stress, and this phosphorylation event is inhibitory to CRL4
Cdt2
ubiquitination of
PR-Set7. In addition to independently verifying the previous report, these results imply
that this pathway is not cell line specific, but rather is conserved across cell types.
79
Figure 23. Osmotic Stress Promotes MAPK-mediated Stabilization of PR-Set7
Against UV-induced Degradation
U2OS cells were subjected to UV irradiation and/or sorbitol treatment, in the presence
and absence of p38 and JNK MAPK inhibitors as indicated. 300 mM sorbitol and/or
MAPK inhibitors were added 15 min prior to UV irradiation and cells were harvested
after 1 hour. Western analysis was performed to examine PR-Set7, Cdt1 (positive
control)(Chandrasekaran et al., 2011), or histone H4 (loading control) levels.
Stress-induced phosphorylation of Cdt1 was completely blocked by pre-treatment
with a combination of p38 and JNK inhibitors, however pre-treatment with either
inhibitor alone had no detectable effect (Figure 23)(Chandrasekaran et al., 2011). To
determine whether the same was true for PR-Set7, U2OS cells were treated with the p38
inhibitor, JNK inhibitor or both inhibitors prior to sorbitol treatment and subsequent UV
irradiation. Treatment with the JNK inhibitor alone abolished the stress-induced PR-Set7-
stabilization, whereas treatment with the p38 inhibitor alone had no effect on the
stabilizing sorbitol treatment (Figure 24). These results indicate that, unlike what was
reported for Cdt1, JNK is the primary kinase responsible for phosphorylating PR-Set7
during osmotic stress, and that this phosphorylation can prevent degradation of PR-Set7
80
during DNA damage. To ensure that off-target effects of the JNK inhibitor do not
convolute these results, future experiments will employ shRNA to deplete cells of JNK
prior to sorbitol treatment and irradiation.
Figure 24. Osmotic Stress Promotes JNK-mediated Stabilization of PR-Set7 Against
UV-induced Degradation
U2OS cells were treated with 30 µM p38 inhibitor, 150 µM JNK inhibitor or DMSO
control for 15 minutes prior to a 15 minute treatment with 300 mM sorbitol or PBS. Cells
were subsequently UV-irradiated and harvested after 1 hour. Western analysis was
performed to examine PR-Set7 and tubulin (loading control) protein levels.
81
4.2.2. PR-Set7 is phosphorylated at Serine 29 after Osmotic Stress and this
modification may block UV-induced ubiquitination of PR-Set7
JNK, like other MAP kinases, has a lenient consensus sequence for substrates,
and can phosphorylate target serine or threonine residues that are followed by a small
amino acid, preferably proline (-S/T-P-). PR-Set7 only contains two amino acids that
satisfy this criteria: serine 29 (S29) and threonine 110 (T110). To investigate which, if
either, of these amino acids was being phosphorylated by JNK during osmotic stress, we
performed site-directed mutagenesis to mutate each site to alanine, thus eliminating the
target residue for phosphorylation. Plasmids encoding FLAG-PRSet7 WT, PR-Set7
S29A, PR-Set7 T110A or PR-Set7 S29A/T110A were transfected in to HeLa cells.
Twenty-four hours post-transfection, cells were treated with 300 mM sorbitol for 15
minutes prior to harvesting nuclear lysates and performing immunoprecipitation against
PR-Set7 using FLAG-conjugated beads. Western analysis of the FLAG-IPed material
using an antibody that specifically recognizes the phospho-Ser/Thr-Pro motif revealed
that, as predicted wild type PR-Set7 is phosphorylated during osmotic stress (Figure 25).
Mutation of threonine 110 to alanine had no detectable effect on PR-Set7
phosphorylation levels after sorbitol treatment, however, when serine 29 was mutated to
alanine, phosphorylation of PR-Set7 was no longer observed during the osmotic stress
response. These results suggest that S29 is the primary target for phosphorylation by
JNK, and is therefore a critical residue in regulating the stability of PR-Set7 under
conditions of cellular stress.
82
Figure 25. PR-Set7 is Phosphorylated at Serine 29 after Osmotic Stress
HeLa cells were transfected with a FLAG-PR-Set7 wild-type (WT) plasmid or FLAG-
PR-Set7 plasmids encoding mutations S29A, T110A or the double mutant S29A/T110A.
After 24 hours cells were treated with 300 mM sorbitol or PBS vehicle control for 15
minutes. Immunoprecipitations for PR-Set7 were performed using FLAG-conjugated
beads and bound material was eluted using FLAG peptide. Western analysis of the bound
material was performed using α-phospho-Ser/Thr-Pro antibody (Millipore, Cat. No. 05-
368).
Based on our results, we hypothesized that the PR-Set7 S29A mutant would be
more sensitive to UV-induced degradation whereas PR-Set7 S29D phospho-mimic
mutant would be resistant to degradation. To test this hypothesis, U2OS cells were
transfected with FLAG-PR-Set7 constructs encoding mutant proteins with alanine or
aspartic acid substitutions at S29 or T110 position. Cells were subsequently exposed to
UV irradiation followed by FLAG immunoprecipitation to isolate PR-Set7. Contrary to
our hypothesis, western analysis using a FLAG antibody did not reveal any detectable
differences in FLAG-PR-Set7 levels between any of the mutants (Figure 26). This is most
likely due to the high level of over-expression of the protein that would make it difficult
to accurately assess degradation levels. Interestingly however, in the UV-treated PR-Set7
WT sample we noticed several higher molecular weight bands. These bands disappeared
83
only when S29 was mutated to the phospho-mimicking aspartic acid (D) residue. Taken
together, we hypothesize that these higher molecular weight bands are ubiquitin
conjugates, and propose a model, similar to what was reported for Cdt1 regulation,
wherein phosphorylation of PR-Set7 at S29 by JNK stabilizes PR-Set7 against
degradation by blocking CRL4
Cdt2
ubiquitination. Further experimentation is required to
verify this model.
Figure 26. Phosphorylation at S29 May Block UV-induced Ubiquitination of PR-
Set7
U2OS cells were transfected with FLAG-PR-Set7 constructs encoding mutant proteins
with alanine or aspartic acid substitutions at S29 or T110. Cells were exposed to 800 J/m
2
UV irradiation and harvested after 1 hour. FLAG immunoprecipitation was performed to
isolate PR-Set7 followed by western analysis of the bound material.
800 J/m2 UV: - + - + - + - + - +
WT S29A S29D T110A T110D
pcDNA FLAG PR-Set7:
-FLAG
70
55
45
100
130
84
4.3. Discussion and future directions
It was recently reported that during S-phase or after UV-induced DNA damage,
PR-Set7 is degraded by CRL4
Cdt2
in a process that requires interaction with PCNA via a
conserved PIP domain in PR-Set7 (Jorgensen et al., 2011). It was also reported that
osmotic stress resulted in a MAP kinase-mediated stabilization of PR-Set7 thus
preventing degradation after UV irradiation (Chandrasekaran et al., 2011). We have been
successful in reproducing these results in a different cell line, U2OS, and have
additionally determined that osmotic stress leads to stabilization of PR-Set7 via JNK-
mediated phosphorylation of serine 29. Preliminary results for experiments using a PR-
Set7 phospho-mimic mutant (PR-Set7 S29D) suggest that S29 phosphorylation prevents
ubiquitination of PR-Set7. Immunoprecipitation against epitope-tagged ubiquitin will
help to determine whether PR-Set7 S29D is indeed unable to be ubiquitinated following
UV irradiation.
We have recently shown that another the cyclin dependent kinase 1
(cdk1)/cyclinB complex phosphorylates PR-Set7 at S29 during mitosis. This
phosphorylation event stabilizes PR-Set7 from degradation by another E3 ubiquitin
ligase, APC
Cdh1
, by directly inhibiting binding of the Cdh1 substrate adaptor protein. We
hypothesize that during other phases of the cell cycle or under conditions of stress, JNK
acts analogously to cdk1/cyclin B, to stabilize PR-Set7 by phosphorylating S29 and thus
blocking binding of the Cdt2 adaptor protein and subsequent ubiquitination by CRL4.
Furthermore, we predict that depletion of JNK will abolish osmotic stress-induced
stabilization of PR-Set7 after UV irradiation, and that immunoprecipitated PR-Set7 will
85
not be phosphorylated after sorbitol treatment in JNK depleted cells. Future co-
immunoprecipitation experiments will examine the effect of S29 phosphorylation on PR-
Set7 association with the Cdt2 adaptor protein and PCNA during S phase and after DNA
damage. Finally, we will examine what effect proper S29 phosphorylation has on the
precise cell cycle regulation of PR-Set7 protein expression. To do so we will utilize
lentivirus technology to knockout endogenous PR-Set7 with 3’UTR targeted shRNA and
subsequently replace with mutant PR-Set7. Cells will be synchronized in early S phase
and released and lysates will be collected at regular intervals for western blot analysis.
We predict that expression of the PR-Set7 S29D, phospho-mimic mutant, will remain
improperly high throughout S phase and will have deleterious effects on cell cycle
progression. Additionally, based on reports implicating PR-Set7 as a regulator of
replication origins, it is possible that lack of proper PR-Set7 degradation during S phase
by CRL4
Cdt2
could lead to origin licensing defects (Tardat et al., 2010) Taken together,
these results indicate that PR-Set7 protein levels are intricately regulated by different
kinases and ubiquitin ligases during every phase of the cell cycle and under conditions of
cellular stress and DNA damage.
86
Chapter 5. Methods
5.1. ChIP-Cloning
1. Fix HeLa cells in 1% formaldehyde for 10 minutes
2. Quench reaction with 0.125M glycine for 5 minutes
3. Isolate nuclei using nuclear isolation buffer (150 mM NaCl, 10 mM HEPES pH
7.5, 1.5 mM MgCl
2
, 10 mM KCl, 0.5% NP-40, 0.5 mM DTT, protease inhibitors)
4. Resuspend nuclear pellet in nuclear lysis buffer (50 mM Tris-HCl pH 8.1, 10 mM
EDTA, 1% SDS) at a concentration of 10
8
nuclei per ml
5. Sonicate nuclei to fragments of 200-600 bp
6. For each ChIP, sonicated nuclear material from 5 x 10
6
cells was
immunoprecipitated with either H4K20me1 (Active Motif, #39175), H4K20me3
(Active Motif, #39180) or FLAG (Sigma, #F7425) antibody.
7. Immune complexes were precipitated with Protein-A-conjugated sepharose
(Amersham, GE Biosciences) and washed with RIPA buffer (50 mM HEPES pH
7.4, 1 mM EDTA, 1% NP-40, 0.7% sodium deoxycholate, 500 mM LiCl)
8. DNA was eluted using 10% Chelex (Bio-Rad) solution
87
5.2. Immunoprecipitation
GST Pull-down
1. Incubate ‘bait’ GST fusion protein with other recombinant ‘prey’ protein in a final
volume of 400 µl Co-IP buffer (50 mM Tris pH7.4; 150 mM NaCl; 0.5% NP-40)
and rotate overnight at 4°C
2. Add 50 µl pre-equilibrated glutathione sepharose beads
3. Rotate for 1 hr at 4°C
4. Wash beads 3 times with 1 ml Co-IP buffer for 5 minutes on ice
5. Add 40 µl elution buffer (40 mM reduced glutathione, 50 mM Tris, pH 8.8) and
rotate for 10 min room temperature
6. Spin down and collect bound fraction
His Pull-down
1. Incubate ‘bait’ His fusion protein with 20 µl Protein A Dynabeads, 5 µg His
antibody (rabbit polyclonal) in a final volume of 400 µl Co-IP buffer (50 mM Tris
pH7.4; 150 mM NaCl; 0.5% NP-40) and rotate overnight at 4°C
2. Use magnet to pull Dynabeads to the side of tube and remove and save the
unbound fraction #1
3. Add other recombinant ‘prey’ protein and rotate at 4°C for 1 hour
4. Wash beads 3 times with 1 ml Co-IP for 5 minutes on ice
5. Add 40 µl 6X SDS loading dye (without DTT) and boil for 10 min at 70°C
6. Collect bound fraction
88
5.3. Plasmids and shRNA
PR-Set7 N corresponds to amino acids 1-128 and PR-Set7 C corresponds to
amino acids 129-352 of the human PR-Set7 protein. The sequences of PR-Set7 N and C
were cloned into the pET-45b(+) vector (Novagen) between BamHI and NotI for His-tag
fusion protein expression in E. coli and subsequent affinity purification.
The amino acids contained in each Riz1 truncation protein are as follows: ΔN is
221-1719; ΔC is 1-1396; N1 is 1-220; N2 is 1-504; M1 is 481-1142; M2 is 1136-1396; C
is 1397-1719. Riz1 amino acids N2, M1, M2 and C were cloned into the pFastBacHT-B
vector (Invitrogen) between StuI and NotI for baculovirus production and subsequent
protein expression in the Sf9 insect cell line and purification. Riz1 C was also cloned into
the pGEX-4T-1 vector (GE Healthcare Life Sciences) between BamHI and NotI for GST-
fusion protein expression in E. coli and subsequent affinity purification. Riz1 full length,
ΔC, N1 and C were cloned between the NotI and ApaI sites in a pcDNA 4/TO vector
containing a FLAG-HA sequence between BamHI and EcoRI. A pmCherry-C1 vector
was engineered (Creighton Tuzon) by replacing the EGFP sequence of pEGFP-C1 vector
with a mCherry sequence. Riz1 full length, ΔC, N1 and C were subsequently cloned into
pmCherry-C1 vector between the XhoI and BamHI sites to create N-terminally mCherry-
tagged Riz fusion constructs.
The pSUPER RNAi system (OligoEngine) was used to construct the pSuperior
Riz1 shRNA construct. The pSuperior.retro.puro vector (OligoEngine, Cat. No. VEC-
IND-0010) was used in concert with a pair of oligonucleotides containing the following
89
target sequence from the mRNA of human Riz1 (AACTGGCTGCGATATGTGAAT)
and following the manufacturer’s protocol to produce the shRNA containing construct.
5.4. Protein Expression and Purification
BL21 E. COLI
His-tagged Proteins
pET45b His Δ14 PR-Set7
1. Dilute 4mls o/n starter culture (from glycerol stock) into 300 ml LB/Amp
2. Grow 1-2 hrs until OD~0.8
3. Incubate at 4°C for 1 hr
4. Induce at room temperature with 0.5mM IPTG o/n (~16 hrs)
5. Spin at 6000rpm 15min 4°C
6. Resuspend in 20ml lysis buffer
7. Freeze/thaw one time in dry ice/ethanol
8. Sonicate: 10sec. pulses (3sec rest) output 2 for 8min total (Misonix Sonicator
3000)
9. Add 2mls 10% Triton-X-100 and rotate for 30min at 4°C
10. Spin at 14,000rpm for 10min at 4°C – save supernatant to load onto column (can
check for expression of His-PR-Set7 in the soluble fraction at this point)
11. Load 1.5ml Nickel beads onto 20ml drip column
12. Equilibrate beads with 15mls wash buffer 1
13. Load sample and rotate o/n at 4°C
14. Let sample flow through (collect for analysis)
90
15. Wash beads with 25ml wash buffer 1 (collect for analysis)
16. Wash beads with 25ml wash buffer 2 (collect for analysis)
17. Elute protein with 5mls elution buffer (collect 1ml fractions)
Lysis Buffer:
50mM Tris-HCl pH 8.0
150mM NaCl
1mM EDTA
1mM DTT
1mM PMSF
Protease Inhibitors
Wash Buffer 1:
50mM sodium phosphate pH 7.4
500mM NaCl
25mM Imidazole
Protease Inhibitors
Wash Buffer 2:
50mM sodium phosphate pH 7.4
500mM NaCl
Protease Inhibitors
Elution Buffer:
50mM sodium phosphate pH 7.4
500mM NaCl
300mM Imidazole
10% glycerol
Protease Inhibitors
GST-tagged Proteins
1. Dilute overnight starter culture in 500 ml LB and grow shaking at 37°C until
OD~0.6-0.8
2. Induce with 0.2 mM IPTG and grow for an additional 5 hours
3. Pellet cells by centrifugation for 15 min at 4°C at 6000rpm
91
4. Resuspend pellet in 25 ml NETN (100 mM NaCl, 1mM EDTA, 20 mM Tris-HCl
(pH 8.0), 0.5% NP-40, and protease inhibitors).
5. Lyse cells by mild sonication at 4°C [big tip, power setting 5, 1 minute total (1
second on, 0.5 seconds off)]
6. Centrifuge cell debris at 11,00 rpm at 4°C for 25 minutes
7. Collect supernatant and add 500 µl of pre-equilibrated glutathione sepharose
beads and rotate at 4°C overnight
8. Pellet beads by centrifugation at 1000 rpm for 5 min at 4°C
9. Wash once with NETN (0.5% NP-40) and twice with NETN (0.01% NP-40)
10. Elute fusion proteins by adding 150 µl of elution buffer (10 mM reduced
glutathione in 50 mM Tris-HCl (pH 8.0))
11. Repeat elution twice more and analyze fractions by SDS-PAGE electrophoresis
and coomassie staining.
SF9 INSECT CELLS
Recombinant bacmid containing the protein sequence of interest was produced
using the Bac-to-Bac Baculovirus Expression System from Invitrogen according to the
manufacturers protocol. Sf9 cells were transfected with recombinant bacmid using
Cellfectin II reagent (Invitrogen, Cat. No. 10362-100) and cells were incubated at 27°C
for 5 days before isolating virus from the supernatant by brief centrifugation. 2x10
9
Sf9
cells were transduced with 1ml supernatant and again incubated at 27°C for 5 days before
92
harvesting the P1 baculovirus stock. Virus was amplified one more time to obtain a high
titer P2 stock that was then used for large-scale transduction of Sf9 cells.
His-tagged Proteins
1. From approximately 40 ml of Sf9 cells, resuspend cell pellet in 10 ml lysis buffer
(50 mM sodium phosphate, pH 7.0, 500 mM NaCl, 20 mM imidazole, 10 mM b-
glycerophosphate, 15% glycerol, 0.01% NP-40, 0.2 mM PMSF, aprotinin,
leupeptin, pepstatin A)
2. Dounce thoroughly (35 strokes over a period of 30 minutes on ice)
3. Centrifuge at 11,000 rpm for 10 min at 4 deg.
4. Add 1 ml Ni beads (pre-equilibrated with lysis buffer) to the supernatant
5. Mix gently overnight
6. Wash beads twice with 7 ml lysis buffer
7. Wash beads twice with 4 ml wash buffer (50 mM sodium phosphate, pH 7.0, 100
mM NaCl, 20 mM imidazole, 10 mM b-glycerophosphate, 15% glycerol, 0.01%
NP-40, 0.2 mM PMSF, aprotinin, leupeptin, pepstatin A)
8. Elute protein with 4 successive cycles of addition and removal of 1 ml elution
buffer (Wash buffer + 480 mM imidazole). Allow 5 min elution time for each
step.
93
5.5. Histone Methyltransferase Assay
Methylation reactions (30-60 µl) contained HMT buffer, recombinant
methyltransferase, 10 µg free histones or peptides, and 3 µl [methyl-
3
H]adenosylmethionine. The mixture was incubated at 30°C prior to spotting on P-81
filter paper. Filer paper was washed 3 times with 50 mM sodium bicarbonate pH 9 for 15
minutes and then placed in scintillation tubes with 2 ml of scintillation fluid. Activity was
determined by scintillation counting using a (machine name).
Riz1 HMT Buffer
50 mM Tris-HCl, pH 9.0
5 mM MgCl
2
4 mM DTT
1 mM PMSF
PR-Set7 HMT Buffer
50 mM Tris-HCl, pH 8.0
10% glycerol
1 mM DTT
1 mM PMSF
94
5.6. Nuclear Fractionation
1. Harvest cells from a 10 cm plate that is ~80-90% confluent and wash with PBS.
2. Resuspend in 1ml NIB buffer and incubate on ice for 10 minutes.
3. Centrifuge at 600 x g for 5 minutes at 4°C to isolate nuclei.
4. Resuspend nuclear pellet in 600 µl nuclear buffer and aliquot 80 µl per time point.
5. Add 1 µl 6U/µl MNase to each reaction and incubate at 37°C for designated
times, e.g. 2, 4 and 8 minutes.
6. Terminate reactions by adding 1µl 0.5 M EDTA and 1µl 0.5M EGTA and
centrifuge at 14,000 rpm for 30 seconds. Collect supernatant, which is the S1
fraction.
7. Resuspend the pellet in 80µl 2mM EDTA and incubate on ice for 30 minutes.
8. Centrifuge at 14,000 rpm for 30 seconds. Collect supernatant, which is the S2
fraction.
9. Resuspend the pellet in 80µl lysis buffer. This is the P fraction.
Nuclear Isolation Buffer Nuclear Buffer Lysis Buffer
10mM Hepes, pH7.5 20mM Tris, pH7.5 50mM Tris, pH7.
1.5mM MgCl2 70mM NaCl 100mM NaCl
10mM KCl 5mM MgCl2 5mM EDTA
0.5% NP-40 20mM KCl 0.5% SDS
0.5mM DTT 3mM CaCl2
1mM PMSF 1mM PMSF 1mM PMSF
protease inhibitors protease inhibitors protease inhibitors
95
5.7. Cell Culture
Sf9 insect cells
Sf9 cells were grown in flasks in Grace’s Insect Medium, Supplemented plus 10% FBS
and 10 µg/ml gentamycin in a 28°C non-humidified incubator. To passage cells, fresh
medium was add and the cap of the flask was securely tightened before smacking hard
several times with hand until cells have become detached. Cells were then split into a
fresh flask at 1:10 ratio.
5.8. Stress Treatments
1. Treat U2OS cells with DMSO or 30 µM p38 inhibitor, SB203560, and/or 150 µM
JNK inhibitor, SP600125 for 15 minutes
2. Treat cells with 300 mM sorbitol for 15 minutes
3. Remove and save medium
4. Wash cells with PBS
5. Remove lid and expose cells to 800 J/m
2
UV
6. Add saved medium back to cells and incubate at 37 °C
7. Harvest after 30 minutes
96
Name Forward (5’-3’) Reverse (5’-3’)
General
GAPDH
(expression)
CAGCCGAGCCACATCGCTCAGACA TGAGGCTGTTGTCATACTTCTC
18S (expression) AACTTTCGATGGTAGTCGCCG CCTTGGATGTGGTAGCCGTTT
Lamin
(expression)
CAAGCTTGAGGCAGCCCTAG CTCACGCAGCTCCTCACTGTA
Cyclophilin
(expression)
TATTAGCCATGGTCAACCCCAC TCTGCTGTCTTTGGGACCTTGT
PR-Set7
(expression)
ATTGCCACCAAGCAGTTCTC CGATGAGGATGAGGTGAGGT
Riz1 (expression) AATTTGGGATGGATGTGCATTG GGCGCGATTGGCTTTAAAGT
OligoJW102 GCGGTGACCCGGGAGATCTGAATTC
OligoJW103 GAATTCAGATC
H4K20me1-genes (ChIP-chip)
PTTG1IP
(expression)
ACCGGCTTCCCTTTGTAAAT CTGGCCTTCTCCTCACTCCT
PFKL
(expression)
CAGAAAGCCATGGATGACAA TATACTGTGTGTCCATGGGAGAT
PDXK
(expression)
TGAATTCAGATCAGCTCCAG AAACTGGTTGGGCGTGATAA
LOC220686
(expression)
CTACAACTTCATCCGAAGCA CATCTCCATGAACCACTTGAA
BRD1
(expression)
ATGAAGGCTGCCAAAGAGAA TTTCCTCATTGTGGCAAAGTC
ADARB1
(expression)
TCGTGGATGGTCAGTTCTTT TTGGCATCTTTAACATCTGTGC
COL18A1
(expression)
TCAGTGCCACCACCATCTT GGATGTGGAACAGCAGTGAG
PTTG1IP
H4K20me1
positive 1 (ChIP)
ATCAGCAGGCACTCTTTTGG CAGGTTGGTACAGGGAGCAT
PTTG1IP
H4K20me1
positive 2 (ChIP)
CACACTGTCCACTCCACTCG TTTTGGCCCAGATTGAGTTC
PTTG1IP
H4K20me1
negative (ChIP)
GGATGAGCAGCAGGAGCA CTTTTTGAGGCTGCGCACT
BRD1
H4K20me1
positive 1 (ChIP)
TACCTGGTCCTATCGGATGC AAGACTGTCCCCTCAGACCA
BRD1
H4K20me1
positive 2 (ChIP)
CTCCAGGTACCCACTCAACC TAGGGCGACTCAGGACTTTG
Table 5 (Continued)
97
Name Forward (5’-3’) Reverse (5’-3’)
H4K20me3-genes (ChIP-chip)
CBR1
(expression)
TGATCCCACACCCTTTCATA AGCTTTTAAGGGCTCTGACG
TUG1
(expression)
ACCAAGGAGTCCCCTTACCT GCCTTTGGAAAACCAATGAT
MORC2
(expression)
AGCCCTCCACTTCCGAATG CCGTAAACAATTCCGGAGGAT
Pre-MORC2
(expression)
TGAATTTCCTTGGAGAGAGGTT AGTAGACTCAGGTGTTCGCTTG
CENPM
(expression)
CTGAAGGTCCACTTGGCAAAGT ACAAACACGATCAGGTCAATTCG
H4K20methyl-negative genes (ChIP-chip)
CYYR1
(expression)
TGCAAATCTTACTGCTGTGATG GATAGGAGGAGACGGTGTTGAT
MTMR3
(expression)
TCATGGAGAGAGCACAGAGTTT GATTGCGTTGTTCAGTCTCTTC
RBM9
(expression)
CTTTAGTTCCTGGCTTCCCTTA TATCCACCATAGAGGTCAGCAC
pre-IFNAR2
(expression)
TTGTTAGTGTCAGCTCCCAAAT ACCAAACACGAGGCTGATATAC
SYNJ1
(expression)
GCTGGATACAGTACAGCCAGAC AGTGGTTCAGGAAGGAAAGTTG
Pre-SYNJ1
(expression)
AACATCAGCTTCATTTGCTTTG CTCGAACATGAGACATTCTTCC
PR-Set7 target genes (Illumina)
NFKBIZ
(expression)
TGGTTGATACCATTAAGTGCCTA GTAAGCCTTTGCATTCACAAAA
ADM
(expression)
GGACGTCTGAGACTTTCTCCTT ACGACTCAGAGCCCACTTATTC
VAMP1
(expression)
AGCATCACAATTTGAGAGCAGT GTGTTGAGAGAGCAAACAGAGG
ARL14
(expression)
AGAACTGTTTGGGGCTGTTACT CACTGCAAAGCTTCTTCACTTT
HES5
(expression)
AGCTACCTGAAGCACAGCAA GAAGTGGTACAGCAGCTTCATC
REC8L1
(expression)
AATTCCAGGAACAACTGCAAA TTGGAACTTCAATCTTTCTCCTT
UBE2L6
(expression)
CCATGATCAAATTCACAACCA TTCGGCATTCTTTCTGAACA
CDA
(expression)
CAAGATGATTTTATCTCTCCATGTG GACATCTTTATGAAGTTCTCCAGGT
NFS1
(expression)
TCTATATGGATGTGCAAGCTACAA TTGCTATGTTGTTGGATTCAGTAG
CALCB
(expression)
CAGATGAATGACTCCAGGAAGA CTGTGATTCTGGCTTCTGGTAG
HERPUD1
(expression)
TAAATCGAGATTGGTTGGATTG GTTATTGTTGGGGTCCTGATTT
Table 5 (Continued)
98
Name Forward (5’-3’) Reverse (5’-3’)
H4K20me1-genes (ENCODE)
ZFAND5
(expression)
TAACTACCCCGAAAACAGAGGT CCACAAAACAAATTTCCACATC
Pre-ZFAND5
(expression)
TAACGGAATTTGGGCTTTTG CGGGGTCTGGTTAGTCTCCT
ASXL1
(expression)
GCCTGAAGTAGACAGACAGGTG CTTTTCCTTCTCCATTTCCTGT
Pre-ASXL1
(expression)
CCAGCGGTACCTCATAGCAT GTTTTGGTGTCATTGGAGCA
CD164
(expression)
TAGTGATTGTCAAGTGGGGAAC ATCAAAGGTAGACTTTCGCACA
Pre-CD164
(expression)
ATGGGTGGTTGATGCTGTTT GTCTGTCGTGTTCCCCACTT
HCFC1
(expression)
CACATCGACTACACCACCAAG GCCTTAGATTTCTTTGGAGCAG
Pre-HCFC1
(expression)
TTTGGGGAATGAGAGGAATG CGTCACACACGAAGCCATAG
GBA2
(expression)
TGGCCGCTATTACAACTATGAC TGGACGTTCAGCTCAAAGATAG
Pre-GBA2
(expression)
TGGGAGGTCACAGAGAGAGG GTCTTCCGGTACCACCACTG
RPL23
(expression)
GTCATTCGACAACGAAAGTCAT TTTGCTACTGGTCCTGTAATGG
Pre-RPL23
(expression)
TGCGGTGAATTACAGCTTGA CTCCTACCGGAAGACCCAAG
SAPS1
(expression)
ACCTCAGGAAGCCACAGAAG GACAGAGGTAGAGGGGTCTCTG
RPL4
(expression)
CTAAACCCATATGCAAAGACCA AGCAGCCTTCTTTCCTTTCTTA
Pre-ADRM1
(expression)
GCGTGTCTGAGTCTGCCTTA GAGGTCGTCATCCTGAAAGC
H4K20me1-negative genes (ENCODE)
NIT2
(expression)
GGGCTGTTGATAATCAGGTGTA TTCTGTCTAAAAACGGGGATTT
Pre-NIT2
(expression)
CTCTGTCTGAATCCAGCAGTTC AGAAACTATTTTGGCTCCTTGC
Pre-TBC1D23
(expression)
ATGCTGTGTATACCTTGGCATT AAACTTTGGCCCTCAGATCA
Pre-CEP97
(expression)
AGCCCTATGCTTAGCTGTTGTA TTAATCGTTTGCATTTCTCCAG
pre-CD47
(expression)
AACATTTTTCTTTTTCCTTTCCA ACTAAAGTCAGTGGGGACAGTG
Table 5. Complete list of all primers used
Sequences of forward and reverse primers used to perform quantitative PCR analysis of
DNA from chromatin immunoprecipitation (ChIP) or cDNA to examine RNA
expression.
99
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Abstract (if available)
Abstract
Within the eukaryotic nucleus, DNA is packaged via its interaction with histones and non-histone proteins into a structure known as chromatin. Chromatin is dynamic in nature, and can become more condensed or more accessible depending on, among other things, the post-translational modifications present on the histone proteins. Histone-modifying enzymes can alter the configuration of chromatin and are known to play essential roles in DNA-templated processes, including gene transcription, cell cycle progression and DNA damage repair pathways. This dissertation presents novel findings on the functions and binding partners of the PR-Set7 histone H4 lysine 20 monomethyltransferase. ❧ Several lines of evidence have been reported implicating PR-Set7 in both transcriptional repression and activation. Chapter 1 presents our efforts to more clearly elucidate the role of PR-Set7 and H4K20me1 in transcription. In short, we found that depletion of PR-Set7 or its catalytic activity resulted in the de-repression of newly identified PR-Set7 target genes, strongly suggesting that PR-Set7 and H4K20me1 function in the transcriptional repression of specific genes. ❧ A ‘histone code’ hypothesis has been proposed, in which specific combinations of post-translational modifications on histone tails may function coordinately to regulate distinct chromatin-templated processes. We previously reported a novel trans-tail histone code involving monomethylated H4K20 and H3K9. We found that global H3K9 monomethylation requires PR-Set7 but does not require its catalytic activity. We therefore predicted that PR-Set7 recruits an unidentified H3K9 methyltransferase to establish this novel histone code. In Chapter 2 we identify this H3K9 methyltransferase as Riz1 (PRDM2/KMT8). Riz1 is a tumor suppressor, and is frequently mutated in human cancers by a frameshift mutation resulting in the expression of a truncated protein lacking the C-terminal domain found to be required for PR-Set7 binding and proper localization. Finally, forced ectopic expression of wild type Riz1 in cancer cell lines carrying this Riz1 truncation mutation resulted in cell cycle arrest and apoptosis. These data indicate that Riz1 is an important tumor suppressor and imply that direct Riz1 binding to PR-Set7 is required for proper Riz1 localization and function. ❧ In Chapter 3 we demonstrate that PR-Set7 interacts with class I HDAC proteins in vivo, and that treatment with class I HDAC inhibitors results in the selective de-repression of PR-Set7 target genes. Furthermore, we found that PR-Set7 directly binds HDAC3 in vitro and that this interaction may be functionally significant in epithelial-mesenchymal transition. Together, these results suggest that PR-Set7 and class I HDACs cooperate to regulate transcription of specific human genes. ❧ Lastly, in Chapter 4 we present evidence that after osmotic stress, PR-Set7 is phosphorylated at serine 29 by JNK kinase, and that this modification protects PR-Set7 from degradation after UV irriadiation. Collectively, this dissertation presents novel findings regarding PR-Set7’s binding to other histone-modifying enzymes, Riz1 and HDAC3. These findings help to improve our understanding of PR-Set7 function during transcriptional repression and cell cycle progression, as well as provide insights into how perturbation of PR-Set7 may contribute to cancer development and progression.
Linked assets
University of Southern California Dissertations and Theses
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Asset Metadata
Creator
Congdon, Lauren Marie
(author)
Core Title
Multiple functions of the PR-Set7 histone methyltransferase: from transcription to the cell cycle
School
Keck School of Medicine
Degree
Doctor of Philosophy
Degree Program
Genetic, Molecular and Cellular Biology
Publication Date
11/15/2012
Defense Date
09/28/2012
Publisher
University of Southern California
(original),
University of Southern California. Libraries
(digital)
Tag
histone code,histone methylation,OAI-PMH Harvest,PR-Set7,Riz1,Transcription
Language
English
Contributor
Electronically uploaded by the author
(provenance)
Advisor
Rice, Judd C. (
committee chair
), An, Woojin (
committee member
), Goldkorn, Amir (
committee member
), Hsieh, Chih-Lin (
committee member
)
Creator Email
lauren.congdon@gmail.com,lcongdon@usc.edu
Permanent Link (DOI)
https://doi.org/10.25549/usctheses-c3-110854
Unique identifier
UC11288826
Identifier
usctheses-c3-110854 (legacy record id)
Legacy Identifier
etd-CongdonLau-1293.pdf
Dmrecord
110854
Document Type
Dissertation
Rights
Congdon, Lauren Marie
Type
texts
Source
University of Southern California
(contributing entity),
University of Southern California Dissertations and Theses
(collection)
Access Conditions
The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the a...
Repository Name
University of Southern California Digital Library
Repository Location
USC Digital Library, University of Southern California, University Park Campus MC 2810, 3434 South Grand Avenue, 2nd Floor, Los Angeles, California 90089-2810, USA
Tags
histone code
histone methylation
PR-Set7
Riz1