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Studies of the biological relevance of Histone H4 Lysine 20 monomethylation: discovery of its role in the cell cycle and localization within the human genome

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Studies of the biological relevance of Histone H4 Lysine 20 monomethylation: discovery of its role in the cell cycle and localization within the human genome

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Content STUDIES OF THE BIOLOGICAL RELEVANCE OF HISTONE H4 LYSINE
20 MONOMETHYLATION: DISCOVERY OF ITS ROLE IN THE CELL
CYCLE AND LOCALIZATION WITHIN THE HUMAN GENOME

by

Sabrina Ishimaru Houston

A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(BIOCHEMISTRY AND MOLECULAR BIOLOGY)

December 2007

Copyright 2007 Sabrina Ishimaru Houston
ii
Dedication
I dedicate this thesis to my mother, Setsuko Houston. Without her
support and friendship, I would have never been able to make it this far.
iii
Acknowledgements
I would like to acknowledge all the members of the Rice laboratory for
all the help and support they have given me. I would also like to thank them for
putting up with me for the last three years. I would also like to thank members
of the Lieber laboratory for all their help and guidance. In particular, I would
like to thank Dr. Sathees Raghavan for being patient beyond the call of duty
with a new graduate student who had much to learn. I would also like to thank
Ben Ng for generously teaching me how to use a microscope. Lastly, I would
like to thank my family, without whom I would be hopeless.

iv
Table of Contents
Dedication ii

Acknowledgements iii

List of Figures vi

Abstract viii

Introduction x

Chapter 1: Insights into H4K20 monomethylation in the genome 1
Standardization of novel panel of H4K20 methyl antibodies 1
Chromatin immunoprecipitation standardization 9
Discovery of H4K20 genomic localization 19
ChIP-chip 23
ChIP-chip statistical analysis 24
H4K20 monomethylation and transcription 34
Sites of H4K20 monomethylation have an inherent ability 41
to become condensed chromatin

Chapter 2: Role of PR-Set7 and H4K20 monomethylation in the cell cycle 45
H4K20 and H3K9 methylation during the cell cycle 45
PR-Set7 knockdown leads to growth defects 51
Knockdown of PR-Set7 in HEK 293T cells leads to G2 arrest 56
Loss of PR-Set7 leads to formation of multiple mitotic spindles 58
Loss of PR-Set7 leads to DNA damage 60
H4K20 monomethylation levels do not change in response 65
to genotoxic stress
Loss of PR-Set7 and H4K20 monomethylation leads to 68
decreased chromatin condensation
Loss of H4K20 monomethylation modification specifically 74
accounts for aberrant phenotypes
PR-Set7 and phosphorylation 79

Conclusions and future directions 88

References 102

v
Appendices
Appendix A (Tables of H4K20 monomethyl binding regions) 119
Appendix B (Schematic of PR-Set7 amino acid sequence) 129
vi
List of Figures
Figure 1: Characterization of a panel novel H4K20 methyl antibodies 5

Figure 2: Characterization of novel H4K20 methyl antibodies specificity 7

Figure 3: Standardization of MNase digestion 11

Figure 4: Preferential and selective enrichment of monomethylated 13
H4K20 and H3K9 within the same nucleosome in vivo
Figure 5: Preferential and selective enrichment of trimethylated H4K20 15

Figure 6: Localization of methylated H K20, methylated H3K9 17
and acetylated H4K16 in relation to level of MNase digestion

Figure 7: Visualization of H4K20 monomethylation patterns on 31
genes with high methylation density

Figure 8: Visualization of areas with high H4K20 trimethylation levels 33

Figure 9: H4K20 monomethylation occurs at specific sites in a 35
cell cycle regulated manner

Figure 10: H4K20 monomethylation has a repressive effect on 39
transcription

Figure 11: Sites of H4 K20 mono-methylation have an inherent 42
ability to repress transcription

Figure 12: Propidium iodide flow cytometry profiles for samples at 47
and following release from G1/S thymidine/mimosine block

Figure 13: H4K20 and H3K9 methylation throughout the cell cycle 50
Figure 14: Knockdown of PR-Set7 and H4K20 52
monomethylation in HCT116 cells leads to severe growth arrest

Figure 15: PR-Set7 knockdown in 293T cells leads to an 54
aberrant cell cycle profile
vii
Figure 16: 293T cells undergo an ATM-dependent G2 arrest 57
upon PR-Set7 knockdown.

Figure 17: PR-Set7 and H4K20 monomethylation knockdown 59
can lead to the development of multiple mitotic spindles

Figure 18: Knockdown of PR-Set7 and H4K20 monomethylation 62
leads to global increases in H2AX phosphorylation

Figure 19: H4K20 monomethylation levels do not change in 67
response to DNA damage

Figure 20: PR-Set7 knockdown leads to large nuclei phenotype 69

Figure 21: Loss of PR-Set7 and H4K20 monomethylation 72
leads to a global decrease in levels of chromatin condensation

Figure 22: Overexpression of catalytically dead, dominant-negative 76
PR-Set7 leads to growth arrest

Figure 23: Loss of H4K20 monomethylation, not PR-Set7 78
protein, leads to aberrant phenotypes

Figure 24: PR-Set7 is endogenously phosphorylated 80

Figure 25: PR-Set7 S29A lacks efficiency in nuclear localization 84

Figure 26: Standardization of an antibody raised against PR-Set7 87
phosphorylated at Serine 29

Figure 27: Model of H4K20 monomethylation function 92

viii
Abstract
The post-translational modification of histones is thought to play a
critical role in directing nuclear events involving chromatin. One such
modification, Histone H4 Lysine 20 (H4K20) methylation, a mark only seen in
multicellular eukaryotes, has been shown to be associated with inactive
chromatin. Here, we performed chromatin immunoprecipitation experiments on
genomic tiled arrays of human chromosomes 21 and 22 to discover the genomic
localization of H4K20 monomethylation. A novel statistical method was used to
cull genomic regions enriched for H4K20 monomethylation. It was found that
H4K20 monomethylation often occurs within genes, at minisatellite repeats, and
at sites of high conservation within higher eukaryotes. H4K20 monomethylation
was confirmed as being a transcriptionally repressive mark by analyzing the
expression analysis of highly H4K20 monomethylated genes in the presence and
absence of this modification. Sequences with a high degree of H4K20
monomethylation were found to have a negative effect on transcription, due to a
higher propensity for chromatinization. H4K20 monomethylation was also
found to have a cell cycle regulated profile, peaking at G2/M, while being
absent at the G1/S border. PR-Set7, a cell cycle regulated histone
methyltransferase, has been shown to be responsible for the bulk of H4K20
ix
mono-methylation. We found that knockdown of PR-Set7 and H4K20 mono-
methylation leads to severe growth arrest. Growth arrest was found to be
accompanied by the appearance of DNA damage, as measured by increased
levels of H2AX phosphorylation and comet assay. Multiple mitotic spindles,
another hallmark of DNA damage, were observed in cells lacking H4K20
monomethylation. These cells were also found to have high levels of enlarged
nuclei, whose chromatin appeared less densely packed than corresponding
normal sized nuclei. A global decrease in chromatin condensation upon PR-Set7
knockdown was confirmed with micrococcal nuclease assays. Together, these
studies indicate that while H4K20 monomethylation plays a role in
transcriptional repression, it also plays a major role in cell cycle progression,
chromatin condensation and protection from DNA damage.
x
Introduction
DNA contains the instructions necessary for the functioning of all living
organisms. DNA does not exist inside a cell’s nucleus alone, but as chromatin.
Chromatin is DNA and its associated proteins, the basic unit of which is a
nucleosome, a structure consisting of 147 basepairs of DNA wrapped around an
octamer of four core histone proteins. These four core histone proteins Histone
H2A (H2A), Histone H2B (H2B), Histone H3 (H3) and Histone H4 (H4), are
composed of an amino-terminal unstructured tail and a carboxyl-terminal
globular domain (Luger, Mader et al. 1997; Kornberg and Lorch 1999).
Genomic DNA in eukaryotes undergoes some 10,000-fold compaction to
be able to condense into a nucleus. This condensation presents a considerable
obstacle to nuclear processes involving DNA, such as transcription, replication
and DNA repair. One way that eukaryotic cells manage to overcome this
obstacle is through orchestrated, dynamic changes in their chromatin, most often
involving the histone cores of nucleosomes.
Covalent chemical modifications were first discovered on histones over
40 years ago when lysine methylation was reported on calf thymus histones
(Murray 1964). Subsequently, other histone modifications, such as lysine
acetylation and serine phosphorylation were discovered from a wide range of
sources (Kleinsmith, Allfrey et al. 1966; Ord and Stocken 1967; DeLange,
xi
Fambrough et al. 1968; Gershey, Vidali et al. 1968; Vidali, Gershey et al. 1968).
The next two histone modifications discovered were ubiquitylation and ADP-
ribosylation (Goldknopf, Taylor et al. 1975; Ueda, Omachi et al. 1975). Since
then, one other novel histone modification, sumoylation, has been found (Shiio
and Eisenman 2003).
Distinct histone modifications have been found to correspond with
whether chromatin is in a transcriptionally active or inactive state. This has led
to the proposal of a “histone code”. This code would be an “epigenetic marking
system (that) represents a fundamental regulatory mechanism that has an impact
on most, if not all, chromatin-templated processes, with far reaching
consequences for cell fate decision and both normal and pathological
development (Jenuwein and Allis 2001).” This histone code hypothesis carries
with it several predictions. One prediction is that histone modifications affect the
interaction between chromatin and its associated proteins. That chromatin
domains, such as euchromatin and heterochromatin are the results of histone
modifications is another prediction of the hypothesis. Lastly, the hypothesis
proposes that modifications on the same, or neighboring histones may depend on
each other.
For most of the time since the discovery of histone modifications, their
study was concentrated on those modifications that occur on the amino-terminal
tails of histones. One reason for this bias was that the main method for discovery
xii
of histone modifications was Edman degradation, a technique that favors
analysis of amino acids on the amino end of a protein. The application of mass
spectrometry has allowed the discovery of other post-translational modifications
on histones, beyond those on the amino tail. The first such modification to be
found in the globular domain of one of the histones, methylation of lysine 79 on
histone H3, was found in 2002 (Ng, Feng et al. 2002; van Leeuwen, Gafken et
al. 2002). The use of mass spectrometry has led to other newly identified histone
modifications localized to the nucleosomal cores, such as methylation of lysine
43 and 85 on Histone H2B, methylation of lysine 99 on histone H2A and
methylation of K59 on Histone H4 (Zhang, Tang et al. 2002; Cocklin and Wang
2003; Zhang, Eugeni et al. 2003). The importance of these newly identified core
histone modifications are just beginning to be realized. For instance, acetylation
of H3 Lysine 56 and methylation of H3 Lysine 79 have been found to play roles
in the DNA damage response (Huyen, Zgheib et al. 2004; Masumoto, Hawke et
al. 2005).
Although histone modifications other than acetylation and methylation
do not receive nearly as much press, they’re diverse functions are quite
remarkable. Summaries of the various known histone modifications follow.

xiii
Ribosylation
Poly ADP-ribose consists of a homopolymer of adenosine diphosphate ribose. It
is a transient modification that regulates the binding of nuclear proteins to DNA
, and is catalyzed primarily by polyADP-ribose-polymerase-1 (PARP 1) (Satoh,
Poirier et al. 1994; D'Amours, Desnoyers et al. 1999)
It is the most dramatic of posttranslational histone modifications, due to
its macromolecular nature. The size has led some to postulate that the function
of polyADP ribosylation is to interact with neighboring chromatin, as opposed
to directly playing a function on the nucleosome to which it’s attached (ref).
Although the exact nature of its biological function is still somewhat unclear,
polyADP-ribosylation has been implicated in playing a role in DNA repair and
in playing a role in long term memory in neurons (Boulikas 1989; Althaus 1992;
Cohen-Armon, Visochek et al. 2004).

Ubiquitylation
Ubiquitinated H2A and H2B are the most abundant ubiquitin conjugated
proteins in higher eukaryotes (Jason, Moore et al. 2002). Amino acid analysis
and peptide mapping led to the identification of histone H2A lysine 119 as the
first known site of histone ubiquitylation (Goldknopf and Busch 1975;
Goldknopf, Taylor et al. 1975; Olson, Goldknopf et al. 1976; Hunt and Dayhoff
xiv
1977). Subsequently, lysine 120 on Histone H2B was also found to be
ubiquitinated (Thorne, Sautiere et al. 1987).
Although ubiquitination of cellular proteins has long been associated with their
degradation by the 26S proteasome, two different studies have found that this
does not appear to be the function of histone ubiquitination (Seale 1981; Wu,
Kohn et al. 1981). The best-studied possible function of histone ubiquitination is
of its role in spermatogenesis. The levels of ubiquitinated histone have been
found to vary during spermatogenesis in several vertebrate species, but the
purpose of this variance has not been well established (Chen, Sun et al. 1998;
Baarends, Hoogerbrugge et al. 1999). Otherwise, histone ubiquitylation has also
been implicated in the cellular stress response. It has been found that levels of
ubiquitinated H2A are reduced following heat shock in several different cell
lines (Parag, Raboy et al. 1987). This has led to an interesting proposal that
ubiquitin released from H2A and H2B during the cellular stress response is
ligated to damaged proteins, and that ubiquitinated histone may serve as a
nuclear store for ubiquitin.

Sumoylation
Although sumoylation is similar to ubiquiylation, in that both represent
slightly bulky covalent additions (hence the moniker, small,ubiquitin-like,
modifier) its function as a histone modification appears to be highly dissimilar.
xv
The first instance in vitro and in vivo instance of histone sumoylation was found
on histone H4 (Shiio and Eisenman 2003). This study found that sumoylation of
histone H4 might particularly be associated with transcriptional repression, since
the mark appears to mediate gene silencing through recruitment of histone
deacetylases and heterochromatin protein 1 (HP1).
It has also been found in S. cerevisiae that sumoylation can participate in
a dynamic interplay with either acetylation or ubiquitylation, where SUMO
would act as a block to activating modifications (Nathan, Ingvarsdottir et al.
2006). In particular, this was the first negative histone modification to be found
in S. Cerevisiae, a model organism that lacks heterochromatin, and has a gene-
rich genome that seems “poised” for transcription.

Phosphorylation
Both Histone H3 and the linker Histone H1 have been found to be
hyperphosphorylated on condensed mitotic chromosomes. Both have been
postulated to be necessary for chromatin condensation. However, studies have
shown that histone H1 phosphorylation is not necessary for mitotic
chromosomes to condense (Ohsumi, Uchiyama et al. 1993; Shen, Yu et al.
1995).
Histone H3 phosphorylation is similarly contradictory in that it is highly
elevated on transcriptionally inactive mitotic chromosomes, yet it is also
xvi
correlated with some active chromatin. For instance, phosphorylated Serine 10
on histone H3 (the best studied of histone phosphorylation marks) has been
shown to have high levels on the hypertranscriped male X chromosome (Wang,
Zhang et al. 2001). The contradictory nature of this mark has yet to be clearly
delineated. The second known H3 phosphoryl mark on Serine 28 has similarly
been found at high levels on mitotic chromosomes. Its biological function is
similarly unclear.
However, the biological function of another histone phosphoryl
modification is somewhat more understood. The H2A variant H2AX has been
found to be phosphorylated on Serine 139 in response to DNA double-strand
breaks, and has been found to occur at the foci of these breaks (Rogakou, Pilch
et al. 1998; Sedelnikova, Rogakou et al. 2002). Cells lacking H2AX have been
found to be deficient in DNA repair, and it has been postulated that
phosphorylated H2AX may recruit DNA repair machinery to sites of DNA
damage (Vidanes, Bonilla et al. 2005).

Acetylation
In contrast to lysine methylation, only one acetyl group can be added to
the epsilon amino group of lysine. Acetylation of a lysine on a histone decreases
its overall positive charge. Since histones are basic proteins, it has been
postulated that acetylation of histones decreases their affinity for negatively
xvii
charged DNA, which allows transcriptional regulators greater access to their
chromatin templates (Norton, Imai et al. 1989; Hong, Schroth et al. 1993; Lee,
Hayes et al. 1993; Vettese-Dadey, Grant et al. 1996; Rice and Allis 2001).
Three decades of research has established that histone acetylation is
associated with gene activity, as opposed to inactivity. For instance, enrichment
of acetylated histones is often found on actively transcribed DNA sequences.
Originally, the pioneering use of chromatin immunoprecipitation assays using
antibodies against acetylated histones found that acetylated histones
preferentially localize to regions of general Dnase I sensitivity (Hebbes, Thorne
et al. 1988).
The first molecular evidence for a link between transcription and histone
acetylation was found when the transcriptional regulator Gsn5 was found to
have histone acetyltransferase (HAT) activity (Brownell, Zhou et al. 1996).
The first histone deacetylase (HDAC) was also discovered that same
year as the first nuclear HATs (Taunton, Hassig et al. 1996). This first HDAC
happened to be homologous to a known transcriptional corepressor in year
(Rpd3p). In the subsequent years, more transcriptional coactivators were
discovered to have HAT activity, and more corepressors were found to have
HDAC activity. This provided additional strong support for the idea that
hyperacetylated chromatin tends to be transcriptionally active, whereas
hypoacetylated chromatin tends to be transcriptionally repressed. HDAC activity
xviii
has also been implicated in the formation of heterochromatin-like domains.
Inhibition of HDAC activity has been shown to disrupt the formation of
heterochromatin regions in fission yeast (Ekwall, Olsson et al. 1997).
However, acetylation of Histone H4 lysine 12 has been implicated in
transcriptional silencing in yeast and Drosophila (Turner, Birley et al. 1992;
Braunstein, Sobel et al. 1996), and the HATs Sas3 and Hat1 have also been
implicated in transcriptional silencing in yeast (Reifsnyder, Lowell et al. 1996;
Kelly, Qin et al. 2000).
The bromodomain, a conserved protein motif, has been found to bind to
acetylated lysine residues. The first reported bromodomain was found in the
Drosophila Brahma protein (Tamkun, Deuring et al. 1992), and since,
bromodomains have been found in many other chromatin associated proteins.
For instance, bromodomains have been found in various HAT enzymes. The
presence of these domains has led to the postulation that this leads to a self-
perpetuation of acetylation at a certain locus (de la Cruz, Lois et al. 2005).
Interestingly, some histone methyltransferase enzymes have also been found to
contain bromodomains. Certain parts of chromatin remodeling machinery have
also been found to contain bromodomains. This has led to the hypothesis that
histone acetylation may precede recruitment of ATP-dependent chromatin
remodeling machinery (Agalioti, Lomvardas et al. 2000; Dilworth, Fromental-
Ramain et al. 2000; Syntichaki, Topalidou et al. 2000).
xix

Methylation
Other than the previously mentioned novel methylation marks
discovered on Histones H2A and H2B, the main histone methylation marks that
have been studied occur on H3 and H4. These methylation marks are catalyzed
by three different protein families. The PRMT family catalyzes methylation on
arginines (Zhang and Reinberg 2001; Bedford and Richard 2005). The SET
domain containing family (Kouzarides 2002; Lachner, O'Sullivan et al. 2003;
Martin and Zhang 2005) and Dot1 are responsible for methylating specific
lysines. Arginines can be either mono- or di-methylated, while lysines can be
either mono-, di- or tri-methylated (Bannister, Schneider et al. 2002).
Methylated lysines are recognized by at least four different protein domains- the
chromodomain, the tudor domain, the WD-40 repeat domain and the MBT
domain (Maurer-Stroh, Dickens et al. 2003; Wysocka, Swigut et al. 2005).
In contrast to histone arginine methylation, which has been linked to
transcriptional activation, histone lysine methylation can be associated with
either active or repressed chromatin. Chromatin immunoprecipitation (ChIP)
experiments have shown that active genes can be methylated at H3 Lysine 4
(H3K4), H3 Lysine 36 (H3K36) and H3 Lysine 79 (H3K79) (Santos-Rosa,
Schneider et al. 2002; Krogan, Kim et al. 2003; Schubeler, MacAlpine et al.
2004). These experiments have also shown methylation at H3 Lysine 9 (H3K9),
xx
H3 Lysine 27 (H3K27) and H4 Lysine 20 (H4K20) to be marks of inactive
genes (Cao, Wang et al. 2002; Peters, Kubicek et al. 2003; Rice, Briggs et al.
2003; Schotta, Lachner et al. 2004).
The best studied histone lysine methylation marks are H3K4, H3K9 and
H3K27. H3K4 methylation correlates with transcriptionally poised euchromatin.
Trimethylation of H3K4 accumulates upon activation of transcription at
promoter regions (Bernstein, Humphrey et al. 2002; Ng, Robert et al. 2003).
H3K4 monomethylation has in turn been found to occur at enhancers
(Heintzman, Stuart et al. 2007). Methylated H3K4 has also been found to create
a binding site for chromodomain containing protein Chd1p, which can recruit
acetyltransferase activity, which would in turn activate transcription (Pray-
Grant, Daniel et al. 2005). In addition, H3K4 methylation can also recruit
chromatin-remodelling activity that would contribute to nucleosomal
repositioning necessary for transcriptional activation (Santos-Rosa, Schneider et
al. 2003).
H3K9 methylation has been a cornerstone of the molecular definition of
heterochromatin in recent years. This advance originated from observations of
position effect variegation (PEV) in Drosophila, where the translocation of
normally active genes to areas proximal to heterochromatin led to the silencing
of those genes (Wakimoto 1998). Studies of PEV led to the identification of
PEV supressors including Su(var)3-9 (Tschiersch, Hofmann et al. 1994), and its
xxi
human homologue SUV39H1, which was later shown to be a histone lysine
methyltransferase that specifically methylated H3K9 (Rea, Eisenhaber et al.
2000). The role of H3K9 in heterochromatin later became clear when Suv39h1
was found to associate with heterochromatin protein HP1. This association was
cemented when it was found that HP1 itself contained a chromodomain that
could bind both di- and tri-methylated H3 K9 (Aagaard, Laible et al. 1999;
Bannister, Zegerman et al. 2001; Lachner, O'Carroll et al. 2001). H3K9
methylation was also implicated in genomic stability when Suv39h1/Suv39h2
knockout mice were found to have severe chromosomal segregation defects
(Peters, O'Carroll et al. 2001), and in developmental processes when it was
found that knocking out G9a, another H3K9 histone lysine methyltransferase,
resulted in early embryonic lethality attributed to severe differentiation defects
(Tachibana, Sugimoto et al. 2002).
H3K27 methylation has been implicated in homeotic (HOX) gene
silencing, X inactivation and genomic imprinting (Cao and Zhang 2004).
Members of the polycomb group (PcG) of proteins are the core of this silencing
system (Ringrose and Paro 2004). H3K27 is highly implicated as being an
underlying mechanism of developmental gene regulation as the homeotic genes
that H3K27 methylation silences encode transcription factors that specify
embryonic tissue identity (Ringrose and Paro 2004).
xxii
The dogma concerning histone methylation formerly placed it as being
potentially the most stable of the various histone modifications. This idea has
come undone with the recent proliferative discoveries of histone demethylases.
The first enzyme able to undo a histone methyl mark was peptidylarginine
deiminase 4 (PAD4), which converts monomethyl arginine from H3 and H4 into
citrulline by demethylimination (Cuthbert, Daujat et al. 2004; Wang, Wysocka
et al. 2004). Shortly thereafter, the first histone lysine demethylase LSD1 (lysine
specific demethylase 1) was identified. LSD1 specifically demethylates mono-
and dimethyl H3K4 (Shi, Lan et al. 2004), but when associated with the
androgen receptor LSD1 changes its substrate specificity to demethylate mono-
and dimethyl H3K9 (Metzger, Wissmann et al. 2005). The question remained as
to whether trimethylation of lysines was reversible, as LSD1 is an amine oxidase
that is chemically only able to reverse mono- and dimethylation. It was proposed
that proteins containing a JmjC domain could demethylate trimethylated lysine
residues in a manner similar to that by which DNA repair demethylase AlkB
hydroxylated the methyl groups of certain forms of DNA methylation damage
(Trewick, McLaughlin et al. 2005). This hypothesis was confirmed when the
first JmjC domain-containing histone demethylase JHDM1 was characterized to
specifically demethylate mono- and di-methyl H3K36 (Tsukada, Fang et al.
2006). JMJD2 was recently the first demethylase to be discovered to
demethylase a trimethyl residue, when its specificity was found to be for di- and
xxiii
trimethyl H3 K9 and H3 K36 (Whetstine, Nottke et al. 2006). New histone
demethylases are continually being discovered.
One mark of histone methylation, about which little has been
comparatively published is methylation of H4K20. H4K20 trimethylation has
been found to be a mark of heterochromatin, colocalizing with H3K9
trimethylation (Schotta, Lachner et al. 2004), although H4K20 mono- and
dimethylation have been found in non-heterochromatic transcriptionally-inactive
regions (Nishioka, Rice et al. 2002; Sims, Houston et al. 2006).
H4K20 methylation has also been implicated in DNA repair. Deletion of
the fission yeast H4K20 methylase Set9, against expectations, did not result in
defective heterochromatin formation, as assayed by measurements of
centromeric silencing, but instead showed a sensitivity to DNA double strand
breaks upon irradiation. It was hypothesized that the interaction of Crb2 (53BP1
orthologue in fission yeast) with methylated H4K20 was necessary for DNA
repair (Sanders, Portoso et al. 2004).
The bulk of H4K20 monomethylation has been found to be catalyzed by PR-
Set7 (Xiao, Jing et al. 2005). H4K20 monomethylation was also implicated in
playing a role in cell cycle progression when a loss of function mutant of the
PR-Set7 Drosophila homologue led to cell cycle arrest and second instar larval
death (Karachentsev, Sarma et al. 2005). Although, as describe above, H4K20
monomethylation has been found to be associated with inactive genes, other
xxiv
reports have the mark as being associated with active genes (Vakoc, Sachdeva et
al. 2006; Barski, Cuddapah et al. 2007), although these reports conflict on
whether the mark occurs mainly at promoters or well-within a gene.
Together, these previous studies imply that H4K20 monomethylation
may play some role in transcriptional regulation and also a role in progression
through the cell cycle.
Here, we present experiments that elucidate the function of H4K20
monomethylation through elucidating where this modification occurs within the
human genome, and whether this mark acts to counteract or promote local
transcription. We also present evidence as to what the overall function of this
mark may be in a context of cell biology, clarifying the relevance of this mark in
cell cycle progression.
1
Chapter 1. Insights into H4K20 monomethylation in the genome
H4K20 monomethylation has previously been found to be catalyzed by a
cell-cycle regulated histone methyltransferase, PR-Set7 (Rice, Nishioka et al.
2002; Xiao, Jing et al. 2005). A Drosophila mutant lacking PR-Set7 was found
to have developmental defects (first instar larval death) concomitant with cell
cycle defects (reduced imaginal disc cell numbers) (Karachentsev, Sarma et al.
2005).
As findings in Drosophila can often give insight into human
development and disease, we decided to gain comprehension of the biological
relevance of H4K20 monomethylation in human cells.
To gain insight into the function of this modification, an investigation
into the localization of this histone modification within the human genome was
performed by chromatin immunoprecipitation studies. Also, studies into the cell
cycle related functions of this modification were performed through knockdown
of PR-Set7 and analysis of resulting phenotypes.

Standardization of a novel panel of H4K20 methyl antibodies
The study of histone modifications has been able to expand in recent
years, due to the development of new technologies, such as mass spectrometry
and the development of highly specific antibodies. For many experiments
2
involving histone modifications, such as immunofluorescence, chromatin
immunoprecipitation and western analysis, the development of high titer, non-
cross reactive antibodies has been absolutely essential for attempting to
understand the biological relevance of a specific modification.
When the epsilon-amino group of a lysine is acetylated, it may only
accept one acetyl group at a time. On the other hand, the epsilon-amino group of
a lysine is capable of accepting up to three methyl groups, allowing a lysine to
be either mono-, di- or tri-methylated. Different histone methyltransferases will
bring an individual lysine to a specific methylation level. This implies that
different methylation levels have different biological functions. Therefore,
antibodies that can categorically discriminate between the three different methyl
states of a lysine are necessary to fully understand the biological function of
methylation on a distinct lysine residue. Experiments performed with antibodies
against a general “methylated” residue have become irrelevant in recent years.
We tested various commercial available antibodies raised against the
three methylation levels of Lysine 20 on Histone H4 (H4K20). Using ELISA
and peptide competition assays, none of these antibodies were found to be of
acceptable specificity, or titer, for our purposes. Branched peptides
encompassing two copies of the amino-terminal amino acid sequence of Histone
H4 mono-, di- or tri-methylated at H4K20 branched off of a central lysine
(Perez-Burgos, Peters et al. 2004) were synthesized for us by the Tufts
3
University Core Facility. These peptides were then used as immunogens in
rabbits (Figure 1A) by the Zymed Corporation, after which anti-serum was sent
to our lab for testing. The following antibody standardization experiments were
performed in conjunction with Tanya Magazinnik.
Once bleeds were received, they were tested for whether they would
recognize a non-modified version of H4, which had been expressed in bacteria,
as Histone proteins remain unmodified when expressed in bacteria.
Recombinant H4, along with histones acid-extracted from chicken blood, and
HeLa whole cell extract were separated on a SDS-PAGE gel, after which our
anti-H4K20 methyl bleeds were used as primary antibodies in Western blot
analysis.
For the purpose of characterizing our antibodies, 200 nanograms of
recombinant H4, 2 micrograms of acid-extracted chicken histones and whole
cell lysates from 10 ^5 HeLa cells were fractionated by SDS-PAGE and
transferred to polyvinylidene difluoride (PVDF). The PVDF membrane was then
blocked in 5% nonfat-milk in Tris-buffered saline before being incubated with
H4 K20 mono- (1:150,000), di- (1:100,000), or trimethyl (1:20,000) antibodies
diluted in 1% nonfat milk/Tris-buffered saline, rotating, for 1 hour at room
temperature. Primary antibody incubation was followed be three washes with
Tris-buffered saline containing 0.1% Tween 20. Blots were then incubated with
a horseradish peroxidase conjugated anti-rabbit secondary antibody (Jackson
4
Immunologicals) diluted in 1% nonfat milk/Tris-buffered saline (1:5000) in a
similar manner as the primary antibody. The blots were again washed three
times with Tris-buffered saline/ 0.1% Tween 20 before the addition of ECL Plus
(Amersham Biosciences). Results were visualized by exposing the blots to film
for 1 minute.
As expected, none of our H4K20 methyl bleeds recognized non-
modified H4, but all recognized modified H4 among both the HeLa acid-
extracted histone, and in the whole cell extracts (Figure 1B).
To test whether each individual bleed was specific for its respective
immunogen, and whether it would not recognize one of the other methyl levels
of H4K20, or unmodified H4K20, we performed peptide competition assays.
This was accomplished by taking linear peptides representing the H4 tail
(complementary to our immunogen peptides, but unbranched) that were either
unmodified or mono-, di- or trimethylated at H4K20, and pre-incubating varying
amounts of peptide with the bleed before using the bleed as a primary antibody
in western analysis. If an antibody binds to a peptide, it would make that
antibody molecule unavailable to bind to proteins bound on the western blotting
membrane. The minimum amount of immunogenic peptide necessary to make
an antibody unavailable for binding to a membrane containing whole cell extract
was determined (0.1 microgram for anti-H4K20 monomethyl, 0.5 micrograms
for anti-H4K20 trimethyl and 2 micrograms for anti-H4K20 dimethyl), and that
5
Figure 1.Characterization of a panel novel H4 K20 methyl antibodies
(Sims, Houston et al. 2006) A) Schematic of branched peptides, representing
amino acids 16-25 of histone H4, which were used as immunogens for
generating polyclonal antibody serum from rabbits. B) Western analysis with H4
Lys-20 methyl-specific antibodies using recombinant H4 (rH4), chicken
histones, and HeLa whole cell lysates (WCL). Each antibody only detects
modified H4, as recombinant H4 purified from bacteria are unmodified, whereas
histones from chickens or a human cell line would be modified in vivo.
6
amount of unmodified H4 peptide, and mono-, di- and tri-methyl K20 H4
peptide was incubated with each specific bleed, which was then used in Western
analysis of a blot containing HeLa whole cell extract. Each specific bleed was
only competed away from binding to the western blotting membrane by the
peptide against which it was raised, indicating that each antibody was highly
specific (Figure 2A).
Another problem that can be associated with histone modification-
specific antibodies is the phenomenon of “epitope masking,” where neighboring
modifications could potentially impair the ability of an antibody to bind its
target. The nearest potential modification site to H4K20 is Histone H4 Lysine
16 (H4K16), which is known to be acetylated. To test whether H4K16
acetylation could impair the ability of our H4K20 methyl antibodies to bind their
targets, we hyperacelated HeLa core histones with the NuA4 complex (Grant,
Duggan et al. 1997; Allard, Utley et al. 1999). After treatment with NuA4,
increased acetylation at H4K16 was readily detectable by western analysis. As
can be seen in Figure 2B, the efficiency of our H4K20 methyl antibodies to
recognize their targets were unaffected by this global increase in H4K16
acetylation (Figure 2B). We, therefore, had generated antibodies highly specific
for the mono-, di- and tri-methylated forms of H4K20, whose specificity were
unperturbed by known neighboring modifications. We were confident that these
7
Figure 2 Characterization of specificity of novel H4 K20 methyl antibodies. (Sims, Houston et al. 2006) A) Peptide
competition followed by Western analysis on HeLa whole cell lysates, using the different H4 K20 methyl-specific antibodies
with synthetic peptides that were unmodified or mono-, di-, or trimethylated at K20. Each antibody is highly specific as only
pre-incubation with the appropriate corresponding peptide was able to eliminate the signal for each of the antibodies. B)
HeLa core histones were hyperacetylated in vitro by incubation with the yeast NuA4 complex. Western analysis
demonstrated that the global increase in H4 K16 acetylation, the nearest known histone modification to H4 K20, did not
hinder the ability of the H4 K20 methyl-specific antibodies to detect their epitope. mock, minus NuA4
A B
9
antibodies could be used as reliable tools for understanding the biological
relevance of H4K20 monomethylation

Chromatin immunoprecipitation standardization
As scientific interest in histone modifications has increased in the last
decade, so has the interest in where these modifications are occurring within a
genome. A major tool used to answer this question is chromatin
immunoprecipitation (ChIP)
Standard ChIP involves treating cells with formaldehyde to cross-link
DNA and its bound proteins within a cell, in effect, freezing a nuclear portrait.
Formaldehyde treatment is followed by sonication to shear chromatin to useful
fragment sizes (usually less than 1000 base pairs.) The sonicated material is then
used as input in an immunoprecipitation (IP) reaction, where an antibody against
a DNA bound protein of interest is used to isolate the protein of interest, and the
specific sequences of DNA to which it is bound. A variation on this method
involves cutting chromatin with micrococcal nuclease (MNase), an
endonuclease that cuts at linker DNA between nucleosomes. This method is
only useful for IP of histones, or modified DNA itself (i.e. 5’ methyl-cytosine),
as transcription factors and other DNA binding elements would not, presumably,
reliably stay on their in vivo DNA targets (O'Neill and Turner 2003).
10
Most often, when these experiments are published, the experiments were
performed using antibodies that have been either commercially deemed “ChIP-
grade” or no experiments have been performed with the antibody to test if it is
specific for immunoprecipitation purposes. An antibody that may be very useful
for one experimental application may or may not be as useful to an experimental
application of a different nature. For our ChIP experiments, we decided to test if
our antibodies were specific in immunoprecipitation experiments with
nucleosomes, before we used them in ChIP experiments.
Nuclei from HeLa cells were isolated and subject to in nucleo MNase
digestion. Nuclei were resuspended in MNase digestion buffer (0.32 M sucrose,
50 mM Tris-HCl, pH 7.4, 4 mM MgCl2, 1mM CaCl2) to a concentration of 0.5
mg/ml as determined by A260. Micrococcal nuclease was added to the nuclei
(50 units/0.5 mg) and digestion occurred for 10 minutes at 37° C before the
reaction was quenched with the addition of EDTA to a final concentration of 10
mM. Mononucleosomes were then isolated by centrifugation, which removed
the nuclear pellet (Figure 3). One hundred micrograms of mono-nucleosomes
were incubated with 100 ul of ammonium sulfate precipitated H4K20 MonoMe
antibody. The immune complexes were precipitated using Protein A conjugated
Sepharose beads (Amersham Biosciences). Beads were washed sequentially in
phosphate buffered saline containing 50, 100 or 150 mM NaCl. The bound
material was eluted in TE (10mM Tris, pH 8.0, 1 mM EDTA pH 8.0) buffer
11
Figure 3. Standardization of MNase digestion. Ethidium bromide staining of
resulting DNA fragments after digestion with varying concentrations of MNase,
run along side a 100 basepair marker. Using various concentrations, we were
able to obtain mononucleosome only populations, or populations of
mononucleosomes with oligonucleosomes. Asterix represent the position of the
MNase digestion level at which we were obtaining pure mononucleosomes.

MNase
*
12
with 1% SDS by intermittent vortexing for 30 min. SDS-PAGE was then used to
fractionate 2% of the input and 5% of the bound fraction. The input and bound
fractions were then Western blotted with our H4K20 methyl antibodies to look
for enrichment of the mark of interest. As can be seen in Figure 4A, our IPs with
the H4K20 monomethyl antibody led to specific enrichment of H4K20
monomethylation, but not of di- or trimethylation.
Previously, experiments in the lab had shown that H4K20
monomethylation and Histone H3 Lysine 9 (H3K9) monomethylation
colocalized by indirect immunofluorescence (Sims, Houston et al. 2006). The
H4K20 monomethyl enriched IP bound fractions was then immunoblotted for
the presence of H3K9 methyl modifications. Antibody dilutions were used as
follows: H3K9 monomethyl (1:60,000; Upstate), H3 K9 dimethyl (1:80,0000;
Upstate), H3K9 trimethyl (1:10,000; Upstate). As can be seen in Figure 4B the
H4K20 monomethyl IP’d material also was enriched with a significant amount
of H3K9 monomethylation, to a similar enrichment level as that of H4K20
monomethylation. H3K9 di- and trimethylation levels in the bound fraction were
found to be insignificant in comparison. To us, this was a very significant
finding, showing that not only do H4K20 monomethylation and H3K9
monomethylation occur within the same regions on the genome, they even occur
within the same nucleosome
13
.
Figure 4. Preferential and selective enrichment of monomethylated H4 Lys-20 and H3 Lys-9 within the same nucleosome in
vivo. (Sims, Houston et al. 2006) A) mononucleosomes prepared from HeLa cells were immunoprecipitated (IP) with H4
Lys-20 monomethyl-specific antibody. Western analysis (WB) of 2% of the input material and 5% of the eluted bound
material indicates that the immunoprecipitated nucleosomes were specifically enriched for the monomethylated form of H4
Lys-20. NS represents nonspecific signal most likely generated by performing both the immunoprecipitation and the Western
analysis with rabbit polyclonal antibodies. B) Western analysis of the H4 Lys-20 monomethyl-enriched nucleosomes with H3
K9 methyl antibodies indicates that they are also selectively enriched for monomethylated H3 Lys-9.
14
We previously found in our lab that H4K20 mono- and tri-methylation
do not co-localize with each other by indirect immunofluorescence (Sims,
Houston et al. 2006). Since we wanted to have a negative control for our ChIP
experiments, we decided to test the specificity of our H4K20 trimethyl antibody
in IP experiments.
Immunoprecipitations with mononucleosomes were performed as above,
except with 180 microliters of ammonium sulfate precipitated H4K20
trimethylation antibody was used for pulldown. As can be seen in Figure 5A,
antibodies for H4K20 trimethylation enriched a significant amount of H4K20
trimethylation, but not a significant amount of H4K20 mono- or di-methylation.
This indicated that our H4K20 trimethyl antibody could be just as useful for IP
experiments as our H4K20 monomethyl antibody.
In the same vein as the experiments described above, we also blotted the
H4K20 trimethyl enriched fraction for the presence of H3K9 methyl
modifications. As can be seen in Figure 5B, a significant enrichment of H3K9
trimethylation was seen as being co-immunoprecipitated with H4K20
trimethylation, which was expected because previous studies have reported
similar results by immunofluorescence (Schotta, Lachner et al. 2004). Lesser
amounts of H3K9 mono- and dimethylation could also be seen, although the
significance of this finding is currently unknown to us.
15
Figure 5. Preferential and selective enrichment of trimethylated H4 K20. (Sims, Houston et al. 2006) A) Mononucleosomes
were immunoprecipitated with H4 Lys-20 trimethyl-specific antibody. Western analysis of 2% of the input material and 5%
of the eluted bound material indicated that the immunoprecipitated nucleosomes are specifically enriched for the
trimethylated form of H4 Lys-20. NS represents nonspecific signal most likely generated by performing both the
immunoprecipitation and the Western analysis with rabbit polyclonal antibodies. B) Western analysis of the H4 Lys-20
trimethyl-enriched nucleosomes indicates that there is a preferential enrichment for trimethylated H3 Lys-9 in vivo, although
the mono- and dimethylated forms of H3 Lys-9 are also detected to a lesser extent.
A
B
16
As part of standardization for MNase digestion, we had obtained
populations of oligonucleosomes of various sizes and we decided to test where
our histone modifications of interest occur in relation to levels of MNase
digestion. The dogma would be that euchromatin would be cut into
mononucleosomes, while heterochromatin would be represented by longer
oligonucleosomes, since more condensed chromatin would be more resistant to
cutting by MNase. Using this experiment, we would be able to obtain a rough
view on where certain modifications occur in relation to their level of chromatin
condensation.
To accomplish this, we performed an analytical MNase digest to
determine at what concentration, using the previously described MNase
digestion procedure, we would obtain the greatest range of oligonucleosomal
sizes. We then used this MNase concentration to perform a mass in nucleo
MNase digestion. The digested material was then placed on a 5-30% sucrose
gradient and centrifugation was performed at 80,000 g for 20 hours. Various
nucleosomal fractions from centrifugation of 5-30% sucrose gradient (as seen in
Figure 6A) were subjected to Western analysis for the presence of various
histone modifications. As can be seen in Figure 6B, there was not significant
bias for the localization of H4K20 methyl modifications in relation to the level
of MNase digestion, although a small enrichment for H4K20 di- and
17
Figure 6. Methylated H4 K20 and methylated H3 K9 occur in all levels of
condensed chromatin, while H4 K16 acetylation preferentially occurs in more highly
condensed chromatin. A) Fractions from a MNase digestion separated on 5-30%
sucrose gradient were run on a 1.2% agarose gel and stained with ethidium bromide.
Fractions chosen to represent mono-, mono- and di-, di- and tri-, di-, tri- and
quattronucleosomes are represented with red boxes. B) Western analysis of above
fractions. Methylated H4 K20 and methylated H3 K9 in all three methylation levels
show little preference for level of MNase digestion, whereas H4 K16 acetylation does
not occur in easily MNase digestible chromatin. An antibody against H3 general was
used as a loading control.
A
B
18
trimethylation could be seen in di- and trinucleosomes. This led to the
conclusion that H4K20 and H3K9 methylation can occur in both euchromatic
and heterochromatic regions.
Interestingly, the only modification that had preferential localization, in
terms of MNase digestion was H4 Lysine 16 (H4K16) acetylation. H4K16
acetylation had a tendency of being in fractions containing di- and tri-
nucleosomes, and did not often occur in fraction high in mono-nucleosomes
(Figure 6B). Since histone acetylation, H4K16 acetylation, in particular has been
found to antagonize chromatin folding in vitro (Hansen 2002; Shogren-Knaak,
Ishii et al. 2006). Our finding was almost counter-intuitive, as mono-
nucleosomes are often thought to be the least condensed transcriptionally active
regions, which is where you would expect histone acetylation to occur.
Although we had expected to find H4K16 acetylation almost exclusively
in the most easily MNase digestible regions of the genome, our results indicated
that H4K16 acetylation resides in areas of the genome that are less accessible to
MNase. From these results it became clear that the idea of repressed and active
chromatin being closed and open, respectively, is not quite as black and white as
it has been assumed to be.

19
Discovery of H4K20 monomethyl genomic localization
There are several different approaches that may be used to discover
novel genomic locations of DNA binding proteins. The first, and most simple,
approach is to perform ChIP for your protein, or modification, of interest and
sequence the immunoprecipitated DNA. Another method is ChIP display, where
duplicate DNA from duplicate ChIP experiments is PCR-amplified and analyzed
by restriction digest for the presence of non-background sequences (Barski and
Frenkel 2004). These two methods, while useful, are not considered optimal,
since they are low-thoroughput. Another method is ChIP followed by a modified
form of Serial Analysis of Gene Expression (ChIP-SAGE). In ChIP-SAGE,
ChIP’d DNA is treated with restriction enzymes and ligated with linkers in such
a way that you will end up being able to sequentially sequence long stretches of
ligated ChIP’d DNA sequences, usually 21 bp in length (Roh, Ngau et al. 2004).
The other high-thoroughput method is ChIP, followed by microarray analysis
(ChIP-chip). In these experiments, DNA ChIP’d for the protein, or modification,
of interest is labeled with a specific fluorophore, while total input DNA is
labeled with a different fluorophore. The ChIP’d DNA and the input DNA are
then denatured and allowed to hybridize with DNA on the microarray. At each
specific spot, if there is a higher abundance of a specific sequence in one DNA
population versus the other, there would be a greater amount of fluorescence
from a specific fluorophore to be measured. This allows us to measure at which
20
sequences ChIP’d DNA is enriched relative to total input DNA (Lee, Johnstone
et al. 2006).
Our first attempt to discover genomic locations of H4K20
monomethylation was to perform ChIP, and then directly sequence some of the
ChIP’d DNA to form a small ChIP library.
ChIP was performed as follows: 1 X 10^8 HeLa cells were fixed in
media (Dulbecco’s Modified Eagle Medium, with 10% Fetal Bovine Serum, L-
glutamine, Pennicilin and Streptomycin) containing 1 % formaldehyde for 10
minutes with agitation. This was followed by 5 min of quenching, where glycine
was added to a final concentration of 125 mM. Cells were washed twice in PBS
before being lysed in Nuclear Isolation Buffer. The nuclei were then washed
once more with PBS before being resuspended in Nuclear Lysis Buffer (150
mM NaCl, 10 mM HEPES, pH 7.4, 1.5 mM MgCl2, 1 mM KCl, 0.5% NP-40,
0.5 mM DTT) to a concentration of 1 x 10^8 nuclei/mL. Lysed nuclei were then
sonicated for a 30 second pulse, which resulted in chromatin fragments ranging
from 500-1000 basepairs.
The resulting sonicated chromatin from 1 x 10^7 cells was used for each
IP reaction. 8 microliters of H4K20 MonoMe antibody were used in the IP
reaction, and 10 ul of Protein A conjugated sepharose beads (Amersham
Biosciences) were used for precipitation of the immune complexes. The beads
were then washed 8 times in RIPA buffer (50 mM HEPES, pH 7.4, 1 mM
21
EDTA, 1% NP-40, 0.7% sodium deoxycholate, 500 mM LiCl) and once in TE
buffer (10 mM Tris pH 8.0, 1 mM EDTA pH 8.0). 100 ul of 10% Chelex
solution (Bio-Rad) was added to beads and this mixture was boiled for 10 min at
100° C to reverse crosslinks and denature the immunoprecipitated complexes
(Nelson, Denisenko et al. 2006). This first eluted fraction was then isolated
from the beads and Chelex. This was followed by a secondary elution with 100
ul of TE buffer, after which the pooled eluted fractions were treated with
Proteinase K for one hour at 55 ° C, before denaturing of the Proteinase K at
100° C for 10 min.
The eluate was then ethanol precipitated, resuspended, and blunted using
T4 DNA polymerase. Unidirectional linkers were made using the following
oligomers: oligoJW102 (5O gcggtgacccgggagatctgaattc 3O), and oligoJW103 (5O
gaattcagatc 3O). The blunted ChIP’d DNA was then blunt-end ligated with the
unidirectional linkers overnight. OligoJW102 was then used to non-specifically
PCR amplify the ChIP’d DNA (Oberley, Tsao et al. 2004). Since this PCR
amplification was accomplished using Taq polymerase, adenosine overhangs
were present on the 3’ ends of our ChIP’d DNA, which allowed us to clone the
DNA into vectors using “TA” cloning methodology. After cloning our ChIP’d
DNA into the pGEMT-EZ vector (Invitrogen), the resulting material was
transformed into bacteria, and colonies were screened for the presence of an
22
insert by lacZ blue-white screening. Plasmids from clones containing inserts
were then mini-prepped and sent for sequencing.
The resulting sequences were rather striking. Since ChIP is known as a
method that results in very high background, especially in human cells, due to
the overabundance of repeat DNA within the genome, we expected a large
proportion of sequences to be within repeat DNA.
Interestingly, 35 out of 86 (41%) sequences were found to be associated
with various repeat elements such as SINES, LINES, satellite repeats and LTRs.
Of the 35 sequences found within genes roughly half were found within known
genes and roughly half were found in unknown genes. No preferential
enrichment for any specific repeat sequence was observed, which is consistent
with a study which performed ChIP-chip for H4K20 monomethylation on an
array of mouse genomic repeats (Martens, O'Sullivan et al. 2005). This led us to
the conclusion that this modification is likely occurring in genes, to a large
extent.
While this data was compelling, we realized our need for a method that
would give high resolution insight into the genomic locations of H4K20
monomethylation as H4 this modification has been found occur throughout the
genome by immunofluorescence, and the possibility of gaining insight to which
genomic sequences are highly monomethylated by sequencing anything less
than several thousand ChIP’d sequences would be highly unlikely.
23
ChIP-chip
Of various methods available to discover novel genomic locations of
histone modifications, it was decided that a high-thoroughput approach would
be necessary to provide the high resolution necessary to discover sites of H4K20
monomethylation, and we chose to perform ChIP-microarray (ChIP-chip). Our
DNA microarray consisted of a tiled genomic array of human chromosomes 21
and 22. These arrays consisted of long oligomers (50-100 bp) of DNA
covalently attached to a specific microscopic spot.
As previously shown, our antibodies against monomethylated H4K20
and trimethylated H4K20 are highly specific in immunoprecipitation reactions
with nucleosomes and thus we used these antibodies in our ChIP-chip
experiments. For these experiments, we used the conventional ChIP technique,
followed by ligase-mediated PCR, as described for previous experiments. Three
independent experiments were performed, and for each, 4 micrograms of
ChIP’D DNA were obtained through PCR amplification with a primer specific
for the unidirectional linker. For each experiment 45-60 PCR cycles were
necessary to obtain the necessary amount of DNA.
For these experiments, we used arrays designed by the Nimblegen
Corporation. They designed genomic tiling arrays for our use, which
encompassed the majority of genomic sequence from human chromosomes 21
24
and 22. We chose to investigate H4K20 monomethylation on chromosomes 21
and 22 for two reasons. First, we would be able to achieve information on two
whole chromosomes, where we would reduce bias that might occur when
looking at only one chromosome. The second reason for choosing chromosomes
21 and 22 was that as their full sequences were published early, this resulted in a
wealth of information to be published about their various properties, including
the transcriptional status of all of their genes (Dunham, Shimizu et al. 1999;
Hattori, Fujiyama et al. 2001; Kapranov, Cawley et al. 2002)
We chose Nimblegen for our microarray purposes for several reasons.
First, Nimblegen was able to put 40 megabases worth of DNA on a single array.
Also, as opposed to the shorter DNA oligomers that the Affymetrix Corporation
used to construct their microarrays, Nimblegen used longer oligomers to achieve
less variation in hybridization due to melting temperature differences between
probes. Also, their arrays that could be stripped a reprobed, a great financial
advantage compared to Affymetrix’s one-time use arrays.

ChIP-chip statistical analysis
In collaboration with the lab of Ting Chen, a USC Associate Professor in
the Department of Molecular and Cellular Biology, we were able to assign
statistical significance to the enrichment of various genomic locations on
chromosomes 21 and 22 in our DNA ChIP’d for H4K20 monomethylation,
25
using a novel method developed by Xiting Yan, a graduate student in the Chen
Lab.
In the short history of analyzing ChIP-chip data, researchers have
struggled to overcome the problem of looking for statistically significant data
within the high levels of background intrinsic to ChIP experiments. Some
methods have detected peaks by directly using the log2 ratios of ChIP DNA
versus total input genomic DNA (Lieb, Liu et al. 2001) Since these methods
only looked at individual probes without taking neighboring effects into
account, these methods were not efficient for looking at the robustness of the
data of a specific area in relation to the background level noise of its
surroundings. To overcome this problem, other methods were developed that
take the neighboring effect into account, including PeakFinder (Glynn, Megee et
al. 2004) and ChIPOTle (Buck, Nobel et al. 2005). These methods all only
consider the log ratios themselves, as criteria for assigning statistical
significance. There are also other methods that consider the shapes (Kim,
Barrera et al. 2005) and lengths of peaks (Bieda, Xu et al. 2006).
To find statistically significant peaks within our H4K20 monomethyl
ChIP array data, we used a data analysis method based on the principle of
ChIPOTle. Average log2 ratios inside a moving window of pre-determined
length were calculated. A p-value was assigned to each window by comparison
of the average log ratios in the window with a reference normal distribution. In
26
comparison to ChIPOTle, which assumed the normal distribution to have a mean
value of 0, this method used permutation to estimate the mean and variance of
the normal distribution for each window. For a given probe i, the window
covered probes that were less than, or equal to, 600 basepairs away from i.
Within this window the Moving Average was defined as:

=
i
NP j
j
i
i
r
NP
M
#
1
,
where
i
NP was the set of probes that were within the window at probe i and
i
NP # was the size of this set. The p value for a specific probe i was calculated
as:
dz
z
M Z P p
i
M
i i

+

= > =
2
2
2
) ˆ , ˆ (
ˆ 2
) ˆ (
exp
ˆ 2
1
) (
2

µ

µ
,
where the sample mean and variance of these
*
i
M ’s were denoted by µ ˆ and
2
ˆ ,
respectively and
) ˆ , ˆ (
2
µ
Z is a random variable with the normal distribution ) ˆ , ˆ (
2
µ N .
Since there were more than 150,000 probes in our data sets, the method
proposed by Benjamini et al. (Benjamini, Drai et al. 2001) was used to control
for false discovery rates. The false recovery rate threshold in this data is 0.001.
If a specific probe is a true binding region, than some neighboring probes
would also have to appear to be true binding regions, due to the size of the
original sonicated chromatin fragments. Using this method, true potential
27
binding regions were defined as those that had at least 4 consecutive significant
probes within a 600 basepair window. The beginning of a binding region was
defined as the first probe found to be significant, while the ending position of
the regions was defined as the last consecutive probe found to be significant.
Using this method, we were able to find statistically significant areas of
H4K20 monomethylation at three different p values: <0.01, <0.005 and <0.001
(Appendix A). We were able to identify 116 peaks of p-value less than 0.001 on
human chromosomes 21 and 22. Out of these 116 peaks, 87 (75%) were found
within genes. This large percentage occurring in genes is consistent with our
sequencing of H4K20 monomethyl ChIP’d DNA. Of the peaks found within
genes, 15.6% were found to occur within exons, while 84.4% were found to
occur within intronic regions (Table 1).
When patterns of peaks were also examined at p values of less than 0.01
and 0.005, we were able to see that some characteristics of the peaks increased
along with the statistical significance of the p value. For instance, at a p < 0.01,
44.6% of peaks were found at minisatellite repeats, but at a p < 0.001, this
proportion increases to 52.5%. These minisatellite simple tandem repeats are not
to be confused with well known microsatellite repeats consisting of repeats of 2-
6 basepairs, which can result in tri-nucleotide expansion repeats, which occur in
several neurological disorders (Lenzmeier and Freudenreich 2003). The
minisatellite repeated where we were finding H4K20 monomethylation to occur
28
Table 1. Various visible trend of genomic locations of H4 K20 monomethylation on chromosomes 21 and 22
compared to p-value. Percentages and ratios of characteristics of statistically significant locations (peaks) of H4 K20
monomethylation at various p values are shown. Average size of peaks were found to increase with increasing statistical
stringency, although the range of sizes (below average size) remained the same for each p value. “Highly conserved site”
refers to a region containing at least one sequence of highest conservation, as measured by the UCSC genome browser using
phastCons system from Adam Siepel at Cornell University (http://compgen.bscb.cornell.edu/~acs/software.html).
Conservation was measured from humans down to zebrafish, xenopus and tetraodon. Peaks were considered to occur at
minisatellite repeats when there was at least one site deemed by the UCSC genome browser to have a “Simple” repeat, as
deemed by Arian Smit’s RepeatMasker program (http://www.repeatmasker.org). “% near or within genes” refers to peaks
within 1 kilobase of 5’ or 3’ ends of genes.
Average peak
size (base pairs)
% at highly
conserved site
% at mini-
satellite
repeat
% near or
within genes
% at exon
(of those
within genes)
% at intron
(of those
within genes)
P<0.01
(213 peaks)
650.8±586.5
(300-3600 bp)
33.8%
(72/213)
44.1%
(94/213)
78.4%
(167/213)
15.6%
(26/167)
84.4%
(141/167)
P<0.005
(177 peaks)
710±523.8
(300-3600 bp)
33.9%
(60/177)
46.9%
(83/177)
77.4%
(137/177)
15.3%
(21/137)
84.7%
(116/137)
P<0.001
(116 peaks)
840.9±586.5
(300-3600 bp)
38.8%
(45/116)
52.6%
(61/116)
75%
(87/116)
16.1%
(14/87)
83.9%
(73/87)
29
varied in their repeat length from 2 basepairs to 89, with the vast majority being
over 20 basepairs in length. Such minisatellites are generally thought to be GC
rich and have strong levels of strand asymmetry (Wyman and White 1980).
They have also been found to be associated with chromosome fragiles sites and
have been found near a number of recurring translocation breakpoints
(Sutherland, Baker et al. 1998). In addition they have been implicated as being
able to play a role in regulating gene expression (Kennedy, German et al. 1995)
Also, the percentage of peaks occurring at highly conserved genomic
areas increases from 33.8% to 38.8% when going from a p < 0.01 to p < 0.001.
“Highly conserved” refers to a region containing at least one sequence of highest
conservation, as measured by the UCSC genome browser using phastCons
system from Adam Siepel at Cornell University
(http://compgen.bscb.cornell.edu/~acs/software.html). Conservation was
measured from humans down to zebrafish, frogs and pufferfish.
Interestingly, the average size of each peak increases from 650.8±497.4
bp to 840.9±586.5 basepairs with increasing statistical significance. This data
suggests that H4K20 monomethylation prefers to occur in much larger genomic
stretches than of any known transcription factors. This could imply that H4K20
monomethylation functions to regulate in a more local manner, as opposed to
H3K9 methylation, which has previously been found to silence 20 kb of the
silent mating type region in S. Pombe (Noma, Allis et al. 2001).
30
We have also previously shown by immunofluorescence and
immunoprecipitation that H4K20 trimethylation does not colocalize with H4K20
monomethylation (Sims, Houston et al. 2006). As our H4K20 trimethyl antibody
has been shown to be suitable for immunoprecipitation experiments, it was
decided that a ChIP-chip for H4K20 trimethylation could serve as a suitable
negative control for ChIP-chip of H4K20 monomethylation. ChIP-chip for
H4K20 trimethylation was performed as previously described, except 10
microliters of anti-serum was used per immunoprecipitation reaction. We
compared the general patterns of the experimental to input fluorescence intensity
ratios for arrays hybridized with DNA ChIP’d for H4K20 monomethylation to
those of arrays hybridized with DNA ChIP’d for H4K20 trimethylation. We
found that areas of highly significant peaks of H4K20 monomethylation
(p0.001) had insignificant levels of H4K20 trimethylation (Figure 7) when
visualized side by side. Although it would appear that H4K20 trimethylation
was devoid of enrichment on chromosomes 21 and 22, this was not the case
(Figure 8).
To further our confidence in our ChIP-chip study, we confirmed a
population of our H4K20 monomethyl regions by conventional ChIP analysis.
As we will demonstrate in the following chapter, H4K20 monomethylation is a
cell cycle regulated mark, absent at G1 and peaking at G2/M. We performed
conventional ChIP on chromatin from HeLa cells either synchronized at the
31
32
Figure 7. Visualization of H4K20 monomethylation patterns on genes with high
methylation density. Graphs of log ratios of ChIP’d DNA to input were
generated using Nimblegen SignalMap software, where the y-axis indicates the
log2 ratio. For each gene, the top track represents DNA ChIP’d for H4K20
mono-methylation (H4K20 monomethyl) while the bottom track represents
DNA ChIP’d for H4K20 trimethylation (H4K20 trimethyl). Y axes are equal in
both H4K20 monomethyl and trimethyl ChIPs. Asterisks represent sites of
statistically significant H4K20 monomethylation (p<0.005). The gray boxes
represent the transcribed region of each gene. Gray boxes with a lower position
represent genes that are transcribed in the direction counter to chromosome
position numbering. Relative transcriptional start sites are indicated by arrows.

33
Figure 8. Visualization of areas with high H4K20 trimethylation density. Two areas with high levels of H4K20 trimethylation
are shown. The Y-axis depicts the log2 ratio of ChIP’d DNA to input DNA. The top track represents H4K20 monomethyl
ChIP’d DNA, whereas the lower track represents H4K20 trimethyl ChIP’d DNA. Y axes are equal for both H4K20
monomethylation and H4K20 trimethylation. These two sites are occurring at the genes USP41 (ubiquitin specific protease
41) and TUG1 (taurine upregulated gene 1), which are both located on human chromosome 22.
H4K20 1CH3 ChIP
H4K20 3CH3 ChIP
USP41
TUG1
MORC2
34
G1/S border, or at G2/M. This allowed us to ChIP both in the presence and
absence of H4K20 monomethylation, indicating the specificity of tested regions
as true genomic areas of H4 K20 monomethylation (Figure 9). This gave us high
confidence in the sequences we found enriched for H4K20 monomethylation
and allowed us to move forward with our investigations into the biological
relevance of this mark

H4K20 monomethylation and transcription
Once we were confident in our data for where H4K20 monomethylation
occurs within the genome, we took steps toward understanding the biological
relevance of the mark. Previously, other reports have described H4K20
methylation as a mark of transcriptional repression (Nishioka, Rice et al. 2002).
Other reports have the mark as associated with transcriptionally active genes,
although these reports conflict on whether the mark occurs mainly at promoters
or well within a gene (Vakoc, Sachdeva et al. 2006; Barski, Cuddapah et al.
2007). In our own lab, we have found the mark to be associated with
transcriptionally repressed chromatin (Sims, Houston et al. 2006). Expression
patterns of genes were tested in the presence and absence of H4K20
monomethylation in an attempt to gain insight into whether the mark is
repressive or activating.
35
36
Figure 9. H4 Lys20 mono-methylation occurs at specific sites in a cell cycle
regulated manner. A) Schematic of PTTG1IP gene and locations of regions for
where primers either positive or negative for H4K20 monomethylation (at
p<0.001) were designed. The open box represents the 5’ untranslated region,
while black boxes represent exons and shaded boxes represent introns. Scale bar
represents 5 kilobases of genomic sequence. B) ChIP assays on H4K20
monomethyl positive and negative regions on PTTG1IP using lysates from
HeLa cells either synchronized at G2/M or the G1/S border (H4 K20
monomethylation is absent ag G1/S, and peaks at G2/M) C) Schematic of BRD1
gene and locations of H4K20 monomethyl positive and negative regions for
which primers were designed.. Scale bar represents 10 kilobases of genomic
sequence. D) ChIP assays on H4K20 monomethyl positive and negative regions
on BRD1 primers using either G2/M or G1/S synchronized HeLa lysates.
37
The genes we tested were those with “high” degrees of H4K20 mono-
methylation. This “high” degree was awarded to the 7 genes, of those containing
three or more statistically significant H4K20 monomethylation sites
(peaks)(p<0.005), which had the most peaks per gene size (in kilobases). The
seven genes of highest ranking were subjected to expression analysis both in the
presence and absence of PR-Set7 and H4K20 monomethylation. The following
genes were tests: PTTG1IP, PFKL, PDXK, LOC220686, BRD1, ADARB1, and
COL18A1.
To obtain cells with an absence of H4K20 monomethylation, we
knocked down PR-Set7, the bulk H4K20 monomethylase, in 293T cells using
shRNA constructs that were either non-specific, or targeted to PR-Set7.
pSUPERIOR based shRNA containing vectors, also containing a puromycin
resistance gene as a selection marker, were transfected into HEK 293T cells
using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s
protocol. 24 hours post-transfection, 293T cells were subjected to puromycin
selection (4 ug/mL media). Six days post-transfection, reduced levels of both
PR-Set7 and H4K20 monomethylation were observed by Western analysis in
cells transfected with shRNA against PR-Set7 (Figure 10A). RNA from each
sample was purified using the Qiagen RNeasy Kit. RNA was converted to
cDNA using the ABI Reverse Transcription kit. Expression analysis on each
gene was accomplished by quantitative PCR on a Bio-Rad I-Cycler real-time
38
PCR machine, using SYBR Green to measure DNA levels. All genes were
normalized to GAPDH expression levels, which were not found to fluctuate due
to PR-Set7 knockdown.
Expression analysis of our highly H4 K20 monomethylated genes in
control vs. PR-Set7 and H4K20 monomethyl knockdown cells was striking
(Figure 10B). The expression of each gene was increased in the PR-Set7
knockdown sample. The change in expression levels was also rather consistent,
with the expression of each gene increased by an average of two-fold in the
knockdown sample, except for ADARB1, whose increase in expression upon
decrease of H4K20 monomethylation was more modest, which may have been
due to it being the largest gene tested. Regardless, all genes showed a
statistically significant increase in expression upon PR-Set7 shRNA treatment
(all p < 0.025 by student t test).
This data clearly indicated that H4K20 monomethylation is a repressive
mark. This suggests that while H4K20 monomethylation may have been found
to occur at active genes in certain studies, the modification occurred at these
genes for the purpose of reducing their transcription levels. While H4K20
monomethylation occurs near the 5’ regulatory regions of genes (see Figure 7),
it also occurs at regions within genes that are relatively far from the 5’
regulatory regions. This led to the hypothesis that regions where H4K20
monomethylation occurs may serve as aberrant enhancers, and that H4K20
39
Figure 10. H4 Lys 20 mono-methylation has a repressive effect on transcription.
A) Western analysis confirming reduced levels of H4K20 monomethylation
upon treatment with PR-Set7 shRNA. B) cDNA from HEK 293T cells
transfected with vectors containing either non-specific shRNA or shRNA
specific for PR-Set7 knockdown were subjected to expression analysis for
several highly H4 K20 monomethylated genes. The expression of each gene is
represented as its proportion of GAPDH expression. The expression of all tested
genes were found to increase in the absence of PR-Set7 and H4 K20
monomethylation (p values for all genes is less than 0.0005, except for BRD1,
which is less than 0.005, and ADARB1, which is less than 0.025).
0
0.0 2
0.0 4
0.0 6
0.0 8
0.1
0.1 2
0.1 4
0.1 6
0.1 8
P T T G 1I P P F K L P D X K L O C 2 20 6 86
0
0 .001
0 .002
0 .003
0 .004
0 .005
0 .006
BR D 1 A D AR B1
0
0.0 00 0 1
0.0 00 0 2
0.0 00 0 3
0.0 00 0 4
0.0 00 0 5
0.0 00 0 6
0.0 00 0 7
0.0 00 0 8
CO L 1 8 A 1
co ntro l shR N A
PR-Set7 sh RNA
A
B
-H4 general
-H4K20 monomethyl
control
shRNA
PR-Set7
shRNA
40
monomethylation at these regions would silence these enhancers and avoid
aberrant gene expression. To investigate this hypothesis, we introduced
genomic regions that by ChIP-chip were indicated to be rich in H4K20
monomethylation into a luciferase expression vector (TK-luc2) containing a
minimal thymidine kinase (TK) promoter. Sequences of statistically significant
H4K20 monomethylation (p<0.001) from the genes PTTG1IP, RUNX1 and
CECR5 were inserted just upstream of the TK promoter. Sequences from the
intergenic regions of chromosomes 21 and 22 (21R and 22R) that were found to
be H4K20 monomethylated were also used as inserts. If regions of H4K20
monomethylation do function as aberrant enhancers, increases in luciferase
expression would be observed for vectors containing H4K20 monomethyl
positive sequences compared to the basal luciferase expression level of the
empty vector. We also constructed an additional control vector containing a
region of the RUNX1 gene that was found to be H4K20 monomethyl negative
by ChIP-chip.
A consistent reduction in luciferase expression was observed in vectors
containing H4K20 monomethyl positive inserts compared to the basal level of
the vector. The vector containing the H4K20 monomethyl negative insert did not
have a similar repressive effect (Figure 11A), instead having expression
comparable to the empty vector.
41
This data negated our hypothesis that sites of H4K20 monomethylation
can act as enhancers. Rather, this data indicated that sites of H4K20
monomethylation have an inherent ability to repress transcription.

Sites of H4K20 monomethylation have an inherent ability to become
condensed chromatin
We have shown that the absence of H4K20 monomethylation results in
increased gene expression. We have also shown that introducing genomic sites
of H4K20 monomethylation into a vector upstream of a reporter gene will result
in transcriptional repression of that gene. These results led us to the hypothesis
that in the previously mentioned experiment, the vectors containing H4K20
monomethyl positive inserts were becoming chromatinized post-transfection,
due to the presence of the insert, resulting in decreased expression of the vector.
To examine this possibility, ChIP analysis was performed on the vectors
containing the H4K20 monomethyl positive insert with the most repressive
effect (22R) and the vector containing the H4K20 monomethyl negative.
Performing ChIP analysis on these vectors allowed us to test their in vivo
chromatinization status. Each vector was transfected into 293T cells using
Expressfect (Denville Scientific) according to the manufacturer’s protocol. The
cells were formaldehyde fixed and their nuclei were obtained one day post-
transfection, as previously described. Sonication was performed for four 20
42
Figure 5
43
Figure 11. Sites of H4K 20 monomethylation have an inherent ability to repress
transcription. A) Vectors containing a luciferase reporter gene and a basal TK
promoter and either no insert, or an insert either positive or negative for H4K20
monomethylation (by ChIP-chip data) were subjected to luciferase assays. All
data was normalized to renilla expression, which did not change appreciably
across samples. TK-luc2 refers to the empty vector. All inserts were found to
contain a statistically significant peak of H4K20 monomethylation by ChIP-chip
(p<0.001), except for the indicated H K20 monomethyl negative insert. 21R and
22R both refer to random genomic regions on chromosomes 21 and 22,
respectively, that were found to have high levels of H4K20 monomethylation,
but were not located within genes. All vectors containing an insert positive for
H4K20 mono-methylation showed repressed luciferase activity. The vector
containing and H4K20 monomethyl negative insert showed no repressive
activity. B) HEK 293T cells were transfected with either TK-luc2 containing H4
K20 monomethyl negative insert, or 22R insert. Sonicated, formaldehyde cross-
linked nuclear lysates from these transfected cells were immunoprecipitated
with either non-specific IgG or antibodies against Histone H3 or
monomethylated H4 K20. Samples were PCR amplified (0.01% of input, 1% of
immunoprecipitated samples) with a primer set spanning the TK promoter. The
TK promoter on the 22R containing vector contained much higher levels of
nucleosomal occupancy (By H3 levels) and H4 K20 monomethylation.
44
second pulses, with 10 second interventions. Immunoprecipitation, washing and
elution were performed as previously described. Primers in the region of the TK
promoter were used for PCR amplification.
From our ChIP analysis, it was observed that the vector containing the
H4K20 monomethyl positive insert with repressive activity (22R) showed a
much higher level of Histone H3 occupancy at the TK promoter than the vector
containing the H4K20 monomethyl negative insert (Figure 11B). This increase
in Histone H3 was accompanied by an increase in the levels of H4K20
monomethylation at the promoter. These data indicated that the decrease in
transcription resulting from the presence of genomic regions of H4K20
monomethylation was accompanied by an increased level of chromatinization.
These data led to the conclusion that genomic regions of H4K20
monomethylation have an inherent ability to recruit chromatinization factors and
PR-Set7.
45
Chapter 2. Role of PR-Set7 and H4K20 monomethylation in the cell cycle
H4K20 and H3K9 methylation during the cell cycle
As previously mentioned, PR-Set7, the bulk H4K20 monomethylase, is
cell cycle regulated. A Drosophila mutant lacking PR-Set7 was found to have
cell cycle defects and was unable to develop normally. These data indicated that
H4K20 monomethylation plays an important role in cell cycle progression in
Drosophila. This information led us to the hypothesis that H4K20
monomethylation is also critical for cell cycle progression within human cells.
The bulk of methylation on Histone H4 has been found to occur in a cell
cycle regulated manner, similar to the cell cycle regulated expression profile of
PR-Set7 (Rice, Nishioka et al. 2002). Only two methyl marks have been found
to occur on Histone H4 in vivo, Arginine 3 and Lysine 20 methylation (Zhang,
Eugeni et al. 2003). As H4 Arginine 3 methylation was not found to be cell
cycle regulated, that left methylation of H4K20 to be the cell cycle regulated
mark. This previous study had shown “methylated” H4K20 to have a debatable
cell cycle profile. We decided to test if H4K20 monomethylation was the cell
cycle regulated mark on Histone H4.
HeLa cells were synchronized at the G1/S border by a
thymidine/mimosine double block. To perform the first block, cells were placed
in media containing 2mM thymidine for 18 hours, followed by a fresh media
46
release for 6 hours. The second block, in media containing 450 µm L-mimosine
, which inhibits DNA synthesis by inhibiting deoxyribonucleotide metabolism
(Gilbert, Neilson et al. 1995), was for 18 hours, after which the cells were placed
in fresh media. Samples were taken at time 0, and every 2.5 hours for 20 hours.
The degree of cell synchronization at each time point was tested by propidium
iodide staining of ethanol-fixed cells. Propidium iodide is an intercalating DNA
dye, which, when measured by flow cytometry, measures the overall linear
DNA content profile of a population of cells. As seen in Figure 12, each sample
was highly synchronized compared to a propidium iodide profile of HeLa cells
in log phase.
Whole cell lysates from each sample were then subjected to Western
analysis for the presence of the H4K20 methyl modifications. An antibody for
general Histone H3 (1:200,000 dilution, from Abcam) was used as a loading
control, while an antibody against Histone H3 Serine 10 (H3S10)
phosphorylation (1:25,000 dilution, from Serotec) was used as a marker for
mitotic cells (Hendzel, Wei et al. 1997). Interestingly, H4 K20
monomethylation, but not di- or tri-methylation had a very specific cell cycle
regulated profile (Figure 13A). H4K20 di- and tri-methylation had a dip in
levels when cells were in S-phase, which would presumably be due to histone
deposition during DNA replication. H4K20 mono-methylation had an opposite.
47
G1
S G2/M
1n
48
Figure 12. Propidium iodide flow cytometry profiles for samples at, and
following release from G1/S thymidine/mimosine block. HeLa cells were
blocked at the G1/S border by a thymidine/mimosine double chemical block.
Samples were taken at time of release and every 2.5 hours for 20 hours after
release. Samples from each timepoint were ethanol fixed and stained with
propidium iodide. Graphs represent flow cytometry profiles of propidium iodide
staining. Samples are well synchronized at block, and at every timepoint
following release, compared to an asynchronous sample of HeLa cells in log
phase represented at the bottom of the figure.

49
profile, being absent at the G1/S border, and rising through S-phase, peaking at
G2/M, before levels declined as cells progressed back into G1
Since we had found that H4K20 monomethylation and H3K9
monomethylation colocalized, as previously mentioned, we also looked at the
levels of H3K9 methyl modifications over the course of the cell cycle. Although
they colocalize, H4K20 monomethylation and H3K9 monomethylation were not
found to share a similar cell cycle profile. All three H3K9 methyl modifications
had a cell cycle profile mirroring those of H4K20 di- and trimethylation, where
there was a slight dip during S-phase, but no other major cell cycle related
fluctuation in levels (Figure 13B). This data indicates that although H4K20
monomethylation and H3K9 monomethylation have a high degree of
colocalization, they are not superfluous. H4K20 monomethylation is a dynamic
modification, while H3K9 monomethylation is static. This would suggest that
these two modifications serve two very different biological functions.
These data also demonstrate the first instance of a truly non-epigenetic
histone-lysine methylation mark, as the hallmark of an epigenetic mark is its
ability to be transmitted to through rounds of cell division, and H4K20
monomethylation is turned over each round of the cell cycle.

50
A
B
A
Figure 13. H4 K20 and H3 K9 methylation throughout the cell cycle.
A)Western analysis of H4 K20 mono-, di- and tri-methylation levels in HeLa
whole cell extracts synchronized throughout the cell cycle. H4 K20
monomethylation is not present at the G1/S block, and increases through late S-
phase, peaking at G2/M, before levels decrease as cells go back into G1. H3
Ser10 phosphorylation is used as a positive mark of mitosis. H3 general levels
were used as a loading control. B) Western analysis of H3 K9 methyl levels
throughout the cell cycle.
A
B
51
PR-Set7 knockdown leads to growth defects
Since a Drosophila mutant lacking PR-Set7 displayed cell cycle defects,
we hypothesized that cell cycle related defects would occur in the absence of
PR-Set7 and H4K20 monomethylation. We knocked down PR-Set7 in HCT116
cells using the previously mentioned pSUPERIOR based shRNA containing
vectors. HCT116 cells were transfected with the vectors using Lipofectamine
2000 and the manufacturer’s protocol. The day after transfection, selection
commenced using puromycin (1.5 ug/ mL media). 48 hours after beginning
puromycin selection, a drastic slow-growth phenotype was observed in cells
transfected with the PR-Set7 specific shRNA vector, as compared to cells
transfected with the non-specific shRNA control vector. Growth curves over the
course of puromycin selection were made for both HCT116 cells, and their p53
null variant. The growth rate in the p53 null variant of the HCT116 cell line was
tested due to the fact that 293T cells, which we used for later experiments, are
p53 null, and we wanted to test if the presence or absence of p53 would hold any
significant bearing on the outcomes of these later experiments. Both HCT116
cell lines showed extreme growth retardation upon treatment with PR-Set7
shRNA (Figure 14). The implication was clear that H4K20 monomethylation
was required for cell cycle progression.
In order to further investigate the relationship of H4K20
monomethylation to cell cycle progression, we switched to 293T cells as our
52
Figure 14. Knockdown of PR-Set7 and H4 K20 MonoMe in HCT116 cells leads
to severe growth arrest. HCT116 cells, and a HCT116 p53 null variant cell lines,
were transfected with vectors containing non-specific shRNA or shRNA
targeted to PR-Set7. Puromycin selection for cells transfected with vector was
commenced one day post-transfection. Counting of cells was commenced 48
hours after puromycin selection (Day2) and performed each day for four days.
Both HCT116 cell lines transfected with vector containing PR-Set7 shRNA,
compared with cells transfected with the control shRNA vector, displayed
severe retardation in growth commencing 48 hours post-selection with
puromycin.

WT con tro l
p53 -/- contro l
W T PR -Se t7 shR N A
p53 -/- PR -Set7 shR N A
0
0.5
1
1.5
2
2.5
Da y2 Da y3 D a y 4 D a y 5
Proportion of
cell growth
relative to
Day 2
53
experimental cell line. In order to pursue further experiments, we needed a cell
line with higher transfection efficiency, as such a high degree of transfected
HCT116 cells died due to puromycin sensitivity, leaving us too few cells to
work with.
Once we transfected the 293T cell line with our shRNA vectors and
subjected the cells to puromycin selection, we also saw a severe growth defect in
cells transfected with the PR-Set7 specific shRNA vectors, although growth
curves were not generated.
Interestingly, when we looked at the propidium iodide staining profiles
of these cells, a very dramatic phenotype was observed. Compared to cells either
transfected with empty vector, or non-specific shRNA control vector, cells
transfected with PR-Set7 shRNA showed a dramatic shift. They had a
significantly lower percentage of cells in G1, concomitant with an increase in
cells at G2/M (Figure 15).
These cells also had a much higher “sub G1” population, indicating that
there were much higher numbers of apoptotic cells in the PR-Set7 shRNA
samples. Counts found by flow cytometry to have smaller amounts of DNA than
G1 nuclei are generally considered to be apoptotic, since part of the apoptotic
process is fragmentation of nuclei (Earnshaw 1995). The presence of cell “dirt”
in a sample can lead to increase sub G1 counts, but the possibility of the PR-
54
Null
Control
shRNA
PR-Set7
shRNA
A
B
C
55
Figure 15. PR-Set7 knockdown in 293T cells leads to aberrant cell cycle profile
A)Propidium iodide staining profiles of 293T cells transfected with empty
vector (Null), or a vector containing non-specific control shRNA or shRNA
specific for PR-Set7. B) Average percentages of populations of transfected 293T
cells in the stages of the cell cycle, from three independent experiments. PR-
Set7 knockdown leads to an increase in the percentage of cells at G2/M,
concomitant with a decrease in cells at G1. C) Western analysis of the
transfected cells confirming knockdown of both PR-Set7 and H4 K20
monomethylation in cells treated with PR-Set7 shRNA.

56
Set7 sample being “dirtier” than the control samples is unlikely, as all samples
were handled side by side.
These data indicate that loss of PR-Set7 and H4K20 monomethylation
leads to growth arrest due to perturbed cell cycle progression. This growth arrest
is also leading to increased rates of apoptosis in PR-Set7 knockdown cells,
indicating that many of these cells are unable to overcome the defect leading to
their growth arrests. This indicates that the defects experienced by PR-Set7
knockdown cells are of high severity.

Knockdown of PR-Set7 in HEK 293T cells leads to G2 arrest
Since we observed an increase percentage of cells in G2/M in PR-Set7
knockdown 293T cells compared to controls, we hypothesized that an arrest in
G2 or mitosis was occurring in these cells. We first performed Western analysis
on 293T PR-Set7 knockdown lysates to look for increased levels of mitotic
markers, H3S10 and histone H3 Serine 28 (H3S28) phosphorylation (Hans and
Dimitrov 2001), but actually found their levels to decrease. Cyclin B1 must be
degraded for cells to progress into mitosis (Smits and Medema 2001), and
increased levels of Cyclin B1 in PR-Set7 knockdown cells further confirmed
that these cells had a lower degree of mitotic cells (Figure 16). This decrease in
mitotic cells was later confirmed by immunofluorescence when DAPI staining
of control and PR-Set7 knockdown cells revealed a much lower percentage of
57
Figure 16. 293T cells undergo an ATM-dependent G2 arrest upon PR-Set7
knockdown. Histone H3 Serine 10 and Serine 28 phosphorylation, both marks of
mitotic cells, were found to be significantly reduced upon PR-Set7 knockdown.
Cyclin B1, which must be degraded for cells to progress into mitosis, was found
to be increased, again indicating a lower percentage of cells in mitosis. This
indicated that cells were arresting in G2. Activated p38 (pThr
180
/pTyr
182
), which
is involved in a cytoplasmically regulated G2 arrest, was not found to increase
upon PR-Set7 knockdown, although an increase in activated, phosphorylated
(S1981) ATM levels was observed in the PR-Set7 knockdown cells.

58
mitotic figures in the knockdown cells (13% in control cells versus 4.4% in
knockdown cells).
Since our PR-Set7 knockdown cells were not arresting in mitosis, this
indicated that a G2 arrest response was occurring. Recent evidence has shown
that two major signaling pathways can result in a G2 arrest. One pathway, which
takes place in the cytoplasm,involves the kinase p38 (Bulavin, Amundson et al.
2002). The other is nuclear, and involves ATM/ATR/DNA-PK pathway, which
is activated in response to DNA damage (Sancar, Lindsey-Boltz et al. 2004). We
looked at levels of activated p38 in control versus PR-Set7 knockdown cells and
found no change, although when we tested for levels of activated ATM a
dramatic increase was observed in PR-Set7 knockdown cells (Figure 16). These
data together indicate that PR-Set7 knockdown 293T cells were arresting at G2
due to an ATM dependent DNA damage response.

Loss of PR-Set7 leads to formation of multipolar mitotic spindles
After observing cell cycle arrest in cells with reduced levels of PR-Set7
and H4K20 monomethylation, we hypothesized that whatever defects were
leading to cell cycle arrest might lead to altered cell morphology. To test this
hypothesis, we stained for gamma-tubulin, to look at mitotic spindles, and
H4K20 monomethylation, and applied a DAPI (DNA dye) stain. To our
59
Figure 17. PR-Set7 and H4 K20 monomethylation knockdown can lead to the
development of multiple mitotic spindles. Indirect immunofluorescence in
shRNA treated 293T cells. Blue indicates DAPI staining. Red indicates staining
for H4 K20 monomethylation. Green indicates staining against gama-tubulin. In
control cells, high levels of H4 K20 monomethylation are seen in mitotic cells,
whereas in PR-Set7 knockdown cells, decreased levels of H4 K20 MonoMe are
seen in mitotic cells, compared to neighboring cells and some mitotic cells are
seen to have an aberrant numbers of mitotic spindles, an indicator of DNA
damage.

control shRNA
PR-Set7 shRNA
60
surprise, we found some mitotic cells with reduced levels of H4K20
monomethylation to have multipolar mitotic spindles (Figure 17). If a normal
mitotic spindle is defined as having a 2n mitotic pole content, a range from 1n to
8n mitotic pole content was seen in these cells. We were surprised to learn that
the appearance of multipolar mitotic spindles, due to centrosome
overamplification, is a hallmark of DNA damage (Tarapore and Fukasawa
2002). When cells accumulate DNA damage and linger longer in cell cycle
checkpoints, centrioles, which normally only replicate once per cell cycle, can
replicate two or more times, leading to multipolar spindles (Dodson, Bourke et
al. 2004; Srsen and Merdes 2006).
This data further indicated that loss of PR-Set7 and H4K20
monomethylation was leading to damage of DNA.

Loss of PR-Set7 leads to DNA Damage
A recent report of experiments performed in fission yeast has implicated
methylation of H4K20 in DNA damage repair. When the H4K20 methylase Set9
in fission yeast (which catalyzes all three methyl states) was deleted, cells
showed increases in H2AX phosphorylation(Sanders, Portoso et al. 2004).
H2AX contains an SQ motif on its C-terminal extension where this Serine is
phosphorylated in response to DNA damage (Serine 139 in human cells)
(Rogakou, Pilch et al. 1998). This phosphorylation of H2AX has been found to
61
be catalyzed by several DNA damage checkpoint protein kinases, HsATM, ATR
and DNA-PKcs (Burma, Chen et al. 2001; Ward and Chen 2001; Stiff,
O'Driscoll et al. 2004). This modification has been found to play an important
role in DNA damage repair, since it has been found to be essential for prolonged
recruitment of DNA damage repair proteins to DNA double strand breaks
(Redon, Pilch et al. 2003; Nakamura, Du et al. 2004).
Since PR-Set7 and H4K20 monomethylation knockdown resulted in an
ATM-dependent G2 arrest, we hypothesized that DNA damage was occurring at
an increased rate in cells with PR-Set7 knockdown. To test this hypothesis, we
took whole cell lysates from control and PR-Set7 knockdown 293T cells and
subjected them to Western analysis for levels of H2AX phosphorylation
(antibody from Upstate Biotechnology, used at 1:35,000). H2AX levels were
found to be significantly increased in PR-Set7 knockdown cells compared to
controls (Figure 17B). HCT116 control and PR-Set7 knockdown whole cell
lysates were also tested for levels of H2AX phosphorylation, and a significant
increase was observed in both the wild type and p53 null variant (Figure 18A).
We also looked at H2AX levels in 293T cells by immunofluorescence.
Five days post-transfection with shRNA-containing vectors, 293T cells were
split onto poly-lysine coated glass coverslips. The following day, the coverslips
were washed once with PBS before being fixed in 4% paraformaldehyde for 10
min. Cell were permeabilized and blocked simultaneously in 20% donkey serum
62
A
B
C
HEK 293T
0
2
4
6
8
10
co ntro l shR N A PR -Set7 shR N A
---- %positive
D
63
Figure 18. Knockdown of PR-Set7 and H4K20 monomethylation leads to global
increases in H2AX phosphorylation. A) HCT116 cells (wt and p53 null variant)
were transfected with vectors containing control shRNA or shRNA targeted to
PR-Set7, which led to a decrease in H4K20 monomethylation. This decrease in
H4 K20 monomethylation was met with an increase in H2AX phosphorylation.
B)HEK 293T cells were also transfected with a PR-Set7 shRNA containing
vector. Global increases in H2AX phosphorylation were also seen by western
analysis upon decrease in H4K20 monomethylation, and also in individual cells
by indirect immunofluorescence (C). D) Percentage of positive nuclei
(“comets”) from control and PR-Set7 shRNA treated 293T cells in a comet
assay (single cell gel electrophoresis), where migration of DNA was observed in
an electrophoretic field, indicative of DNA double strand breaks. Control
shRNA treated cells had less than 0.5% nuclei showing migration, whereas PR-
Set7 shRNA treated cells showed a positive migration rate of 9.6%, indicating
that DNA double strand breaks are occurring to a much greater extent in PR-
Set7 knockdown cells.

64
in PBS containing 0.5 % Triton for 2 hours at room temperature, with slight
agitation. Incubation with primary antibody was either performed overnight at
4° or at 37° C for 1 hour. All antibodies were diluted in 10 % donkey serum in
PBS. H2AX antibody was diluted to 1:1000 for immunofluorescence. All
secondary antibodies (either donkey anti-mouse or donkey anti-rabbit,
conjugated to Cy3 or FITC fluorophores, from Jackson Immunologicals) were
used at 1:200. After each antibody incubation, three washes were performed
with PBS containing 0.1% Triton. Coverslips were mounted using DAPI
containing mounting medium. Immunostaining was then visualized by
fluorescence microscopy, where exposure times for H2AX staining were equal
for all images. As expected, we found dramatic increases in H2AX
phosphorylation in individual cells by immunofluorescence (18C).
Another experiment performed to demonstrate that DNA damage was
indeed occurring in PR-Set7 knockdown cells was single cell gel electrophoresis
(Comet Assay), which allows for a measurement of the extent of DNA damage
in individual cells (Olive and Banath 2006). This experiment is based on the fact
that when cells are embedded in agarose and their nuclear membranes are lysed,
damaged DNA will have areas of negative supercoiling, which will allow DNA
to migrate if placed in an electrophoretic field, If DNA is stained and visualized
under a microscope, the DNA from a cell containing DNA damage will
65
resemble a comet. A comet assay was performed using a Trevigen CometAssay
kit, according to the manufacturer’s protocol.
This experiment was performed on a small scale, but the results were
still striking. Out of 194 nuclei visualized from cells transfected with control
shRNA, 0 (or <0.5%) were found to have DNA that had migrated in response to
the electrophoretic field. Out of nuclei visualized from cells transfected with PR-
Set7 shRNA, 13/135 (or 9.6%) were found to have DNA migration (Figure
18D).
Although the comet assay has a severe limitation, being unable to
distinguish between DNA double strand breaks, single strand breaks, and
apoptotic cells, these results further cemented our findings that PR-Set7
knockdown does indeed lead to increased levels of DNA damage.

H4 K20 monomethylation levels do not change in response to genotoxic
stress
These data led to the question of whether DNA damage is occurring in
PR-Set7 knockdown cells because PR-Set7 and H4K20 monomethylation play a
role in a cell’s DNA damage repair pathway. To test if H4K20 monomethylation
was induced in response to DNA damage, HeLa cells were treated with several
DNA damage inducing conditions. Cells were treated with UV light to induce
pyrimidine dimers (Whitmore, Potten et al. 2001) and hydrogen peroxide to
66
induce single strand lesions (Ananthaswamy and Eisenstark 1977).
Camptothecin, a topoisomerase I inhibitor, was used to induce double strand
breaks (Jacob, Aguado et al. 2001). For UV treatment, cells were placed in a
Stratagene Stratalinker and treated with either 18 or 48 joules of ultraviolet
radiation, and then allowed to recover for 1 hour at 37° C. Cells were also
treated with 1% hydrogen peroxide for either 30 minutes at 4° C of one hour at
37° C. Camptothecin treatment, either 1 micromolar or 5 micromolar, was
performed at 37° C for 1 hour. Etoposide, a topoisomerase II inhibitor that
induces DNA double strand breaks in a very potent manner, was also used
(Jacob, Aguado et al. 2001). Etoposide treatment was performed at 37° C for 1.5
hours.
Upon excessive treatment with UV light, there were no changes in
H4K20 monomethylation. As can be seen in Figure 19A, treatment with UV
light, hydrogen peroxide and camptothecin did not alter H4 K20
monomethylation levels. Camptothecin levels were further raised to 100
micromolar, but still no change in H4K20 monomethylation was seen (data not
shown). With etoposide treatment, a significant increase H2AX phosphorylation
was induced, indicating DNA double strand breaks did occur, but no changes in
H4K20 monomethylation were seen (Figure 19B). Two other studies have
subjected yeast to genotoxic stress and not observed changes in H4K20
methylation (Sanders, Portoso et al. 2004; Botuyan, Lee et al. 2006).
67
Figure 19. H4K20 monomethylation levels do not change in response to
DNA damage. A) HeLa cells were treated in three different manners to induce
DNA damage, by UV light, hydrogen peroxide (H2O2) and camptothecin. No
changes in H4K20 monomethylation were seen. B) HeLa and 293T cells were
treated with etoposide, a potent inducer of DNA double strand breaks. High
levels of DNA damage, measured by increased levels of H2AX phosphorylation,
did not induce changes in H4K20 monomethylation.
A
B
68
This led to the conclusion that although H4K20 methylation plays a role DNA
damage repair, it does not occur in reponse to DNA damage.

Loss of PR-Set7 and H4K20 monomethylation leads to decreased chromatin
condensation
Another phenotype observed by immunofluorescence of DAPI stained
PR-Set7 knockdown cells was the appearance of “large” nuclei (Figure 20). This
phenotype was also seen in the HCT116 cell line. Of PR-Set7 knockdown cells
counted, 49% were large nuclei compared to only 2.3% treated with the control
vector. Also, to figure out how much larger these nuclei could be, the volume of
the nuclei (by DAPI staining) were calculated. Using Axiovision software,
images were taken at every 0.8 microns of the nucleus and the area of DAPI
staining was measured at each plane. Random nuclear (i.e. random movement of
microscopic field and measurement of nuclei closest to center) volumes were
calculated for both the control and PR-Set7 knockdown cells and interestingly,
even with random analysis, the nuclear volume of PR-Set7 knockdown cells was
nearly twice that of control cells, with control cell nuclear volume averaging
16943±4664 cubic microns and PR-Set7 knockdown nuclear volume at
28079±7866 cubic microns. Also, when nuclear volume analysis of only large
69
HEK 293T HCT116
70
Figure 20. PR-Set7 knockdown leads to large nuclei phenotype. HEK 293T cells
and HCT116 cells treated with PR-Set7 specific or non-specific control shRNA
were stained with DAPI, a DNA stain. The appearance of much larger than
average nuclei appeared in large proportions in both HEK 293T and HCT116
cells. All images were taken at 40X magnification, with no manipulation.

71
nuclei was performed, they were found to be more than four times larger, at
70152±20804 cubic microns, than the average control nuclei.
Although quantitative experiments were not performed, by visual
examination these large nuclei had dim DAPI staining compared to neighboring
smaller nuclei. This led us to the possibility that these cells had chromatin that
was less condensed than that of control cells. To test this theory, we obtained
nuclei from both control and PR-Set7 knockdown cells and subjected them to
MNase digestion. MNase digestion was performed with 0.1 mg DNA in 0.5mL
MNase digestion buffer. Digestion was performed with equal amounts of
starting DNA, instead of with equal numbers of nuclei, since using equal
amounts of nuclei would have led to PR-Set7 knockdown cells having a larger
amount of DNA for the assay, since the cells had a much greater proportion of
cells at G2/M, and hence, a larger proportion of cells with a 2n DNA content.
MNase digestion was terminated after 1, 3 and 5 minutes using an excess
amount of EDTA. Nuclei were then pelleted, leaving digested chromatin in the
supernatant. 1% of the supernatant for each time point was subjected to
Proteinase K digestion before being electrophoresed on a 1.2% agarose gel and
being subjected to ethidium bromide staining. We found that the PR-Set7
knockdown nuclei were markedly more prone to MNase digestion than nuclei
from control cells (Figure 21) by the increased levels of mono- and
oligonucleosomes from PR-Set7 knockdown cells at each timepoint. This data
72
73
Figure 21 Loss of PR-Set7 and H4 K20 monomethylation leads to a global
decrease in levels of chromatin condensation
Nuclei isolated from shRNA treated 293T cells were subjected to Micrococcal
Nuclease (Mnase) digestion, based on DNA content. After a timecourse of
MNase digestion was performed (1, 3 and 5 minutes), nuclei were pelleted and a
fraction of the resulting supernatant was run out on an agarose gel for analysis.
No contamination of undigested DNA from the nuclear pellet can be seen in the
wells. Nuclei from PR-Set7 knockdown cells were more subject to Mnase
digestion, as indicated by the increased amount of mono- and oligo-nucleosomes
in those fractions. This indicated that these cells had lower global levels of
chromatin condensation. The position of mono-, di- and trinucleosomes are
indicated.

74
indicated that global levels of chromatin condensation were decreased upon PR-
Set7 knockdown.
It has been hypothesized that H4K20 methylation may recruit DNA
damage repair machinery, since the K20 position on the H4 tail would be buried
in the nucleosomal core; upon the occurrence of a DNA double strand break, the
H4K20 modification could become unburied and recruit members of the DNA
repair machinery (Sanders, Portoso et al. 2004). This hypothesis gained
credence when it was found that 53BP1, a DNA damage repair protein, can bind
to mono- and dimethylated H4K20 (Botuyan, Lee et al. 2006). Despite these
findings, since we saw no changes in H4K20 monomethylation upon genotoxic
stress, despite large increases in H2AX phosphorylation, and since we have
found that decreases in PR-Set7 and H4K20 monomethylation lead to global
decreases in chromatin condensation, we believe that this decrease in
condensation leads to chromatin instability, which in turn leads to increases in
DNA double strand breaks, implicating H4K20 monomethylation as playing a
major role in genomic stability.

Loss of H4K20 monomethyl modification specifically accounts for aberrant
phenotypes
Our previously described results indicated that PR-Set7 knockdown led
to aberrant phenotypes, illuminating the role of PR-Set7 and H4K20
75
monomethylation in chromatin integrity during the cell cycle. In these
experiments, reduced levels of PR-Set7 and H4K20 monomethylation were
present and the question remained of whether the absence of PR-Set7 or H4K20
monomethylation led to the described phenotypes. It is possible that PR-Set7 is
part of multi-protein complex of varied function that loses some, or all of its
function in the absence of PR-Set7. We hypothesized that the lack of the histone
modification itself (H4K20 monomethylation) and not lack of PR-Set7 protein
resulted in these aberrant phenotypes.
It has been previously shown that a point mutant of PR-Set7 (R265G, in
the SET domain) resulted in ablation of its histone-methyltransferase activity. It
was also found that when overexpressed, this point mutant acts as a dominant-
negative (Nishioka, Rice et al. 2002). We decided to take advantage of this point
mutant to knockdown H4K20 monomethylation, while still having both
endogenous and recombinant PR-Set7 physically available within cells.
HCT116 cells were transfected with pQCXIP vectors that overexpress
either green fluorescent protein (GFP) or the PR-Set7 catalytically dead
dominant negative mutant (PR-Set7 CD). The pQCXIP vector also contains a
puromycin resistance gene as a selection marker and transfections and
puromycin selection were performed as previously described. Interestingly, a
similar slow growth phenotype was observed with cells overexpressing PR-Set7
CD as that which was observed with PR-Set7 shRNA knockdown (Figure 22A).
76
Figure 22. Overexpression of catalytically dead, dominant-negative PR-Set7
leads to growth arrest. A) Growth curves of HCT116 and its p53 null variant
after transfection of cells with pQCXIP vectors expressing green fluorescent
protein (GFP) or PR-Set7 catalytically dead, dominant-negative point mutant
(PR-Set7 CD), which induces reductions in H4 K20 monomethylation. Cell
numbers were counted starting two days post puromycin selection and for three
days following. B) Propidium iodide profiles of HEK 293T cells overexpressing
GFP or PR-Set7 CD. Similar to PR-Set7 knockdown cells, am increase in the
proportion of cells at G2/M and a decrease of cells in G1 was seen upon
reduction of H4 K20 monomethylation (by PR-Set7 CD).
0
1
2
3
4
5
6
7
Day 2 Day 3 Day 4 Day 5
WT GFP
WT Set7 CD
p53-/- GFP
p53 -/- Set7 CD
A
B
77
This was also seen in the p53 null variant cell line. When 293T cells
overexpressed PR-Set7 CD, we also saw a phenotype almost exactly like that of
the PR-Set7 knockdown cells, where slow growth was observed, and propidium
iodide staining revealed cells with a lower proportion in G1 and a higher
proportion in G2/M, as compared to controls (Figure 22B). Upon specific
decrease of H4K20 monomethylation the same growth defects were observed as
upon PR-Set7 knockdown.
Since we observed the above growth defects upon H4K20
monomethylation by PR-Set7 CD, we next investigated whether DNA damage
would also occur in such conditions. Whole cell lysates from 293T cells
overexpressing either GFP or PR-Set7 CD were immunoblotted for
phosphorylated H2AX levels, and a significant increase was observed in cells
overexpressing PR-Set7 CD (Figure 23A). When these cells were subjected to
indirect immunofluorescence, the appearance of aberrant numbers of mitotic
spindles (Figure 23B) was observed in some mitotic cells lacking H4K20
monomethylation. These data indicate that phenotypes due to DNA damage seen
in PR-Set7 knockdown cells are also occurring in cells with H4K20
monomethylation reduced by PR-Set7 CD
These data made it absolutely clear that the lack of H4K20
monomethylation, not the lack of the PR-Set7 physical protein, is the cause of
the aberrant phenotypes that we previously observed. This is the first report to.
78
Figure 23. Loss of H4 K20 monomethylation, not PR-Set7 protein, leads to
aberrant phenotypes. A) Western analysis of HEK 293T cells overexpressing
either green fluorescent protein (GFP) or a catalytically dead, dominant-negative
point mutant or PR-Set 7 (PR-Set7 CD). Decreases in H4 K20 monomethylation
were observed in cells overexpressing PR-Set7 CD, concomitant with increases
in H2AX phosphorylation. B) Immunofluorescence of 293T cells
overexpressing PR-Set7 CD. Blue represents DAPI staining, green represent
staining for gamma-tubulin and red staining represent H4 K20
monomethylation. Multiple mitotic-spindles, similar to those seen in 293T PR-
Set7 knockdown cells, were observed.
A
B
79
demonstrate the biological relevance of a specific histone modification by
demonstrating that the lack of the specific histone modification itself, and not its
effector enzyme, leads to a specific phenotype

PR-Set7 and phosphorylation
A PR-Set7 homologue was previously found in a screen for mitotic
phosphoproteins in Xenopus (Stukenberg, Lustig et al. 1997).This led us to the
hypothesis that phosphorylation is a regulatory mechanism of PR-Set7 function.
To investigate this hypothesis, we first tested if PR-Set7 in a human cell
line is phosphorylated in vivo. This was accomplished by use of the Qiagen
Phosphoprotein Purification Kit, which purifies proteins phosphorylated a
serines, threonines and tyrosines. This kit contains columns that specifically
retain phosphorylated proteins. Although Qiagen did not divulge the nature of
the columns, they are likely to be immobilized metal cation columns (Ficarro,
McCleland et al. 2002). Untreated HeLa cell lysates were passed through the
columns and samples from the input, flowthrough and eluate fractions were
subject to Western analysis for the presence of PR-Set7. Using an antibody
specific for PR-Set7, it was observed that almost all of the endogenous PR-Set7
was retained on the column, while very little was found in the flow through
(Figure 24A), indicating that endogenous PR-Set7 is highly phosphorylated.
To test whether recombinant PR-Set7 would also be endogenously
phosphorylated, 293T cells were transfected with a pCMV-Tag3 vector that
80
Figure 24. PR-Set7 is endogenously phosphorylated. A)Untreated lysates from
HeLa cells were put though a Qiagen Phosphoprotein Purification Column. PR-
Set7 was almost entirely found in the bound fraction, indicating that is highly
phosphorylated in vivo. B) HEK 293T cells were transiently transfected with
vectors overexpressing FLAG-tagged PR-Set7, or Serine to Alanine point
mutant at Serine 29 (S29A) and put through phospho-column. FLAG-tagged
wild-type recombinant PR-Set7 was found to be highly phosphorylated in vivo,
but PR-Set7 S29A lost almost all ability to bind to the phospo-binding column.
C) Mutation of a cyclin dependant kinase binding motif on PR-Set7 (PR-Set7
RXL 149AAA) did not result in decrease of phosphorylation on PR-Set7.
A
B
C
81
contained PR-Set7 with an N-terminal myc tag. Lysates were taken 48 hours
post transfection and allowed to run through the phospho-column. Input,
flowthrough and eluate fractions were then subjected to western analysis for the
presence of the myc tag, so as to look at recombinant PR-Set7. Like the
endogenous protein, the vast majority of recombinant PR-Set7 was found in the
eluate fraction, indicating that the recombinant protein was phosphorylated in
vivo (Figure 24B).
After analyzing the PR-Set7 amino acid sequence for motifs that could
indicate potential phosphorylation sites, one site had the strongest consensus
sequence (Appendix B). This site, Serine 29, is a perfect consensus site for the
Cyclin dependent kinase2/Cyclin B1 complex, although it is also a consensus
site for Casein kinase 1 (Adams, Sellers et al. 1996; Watts, Hunt et al. 1999).
A serine to alanine point mutation (S29A) was made at this site using the
Stratagene Quik-change mutagenesis kit, according to the manufacturer’s
protocol. The above experiment was repeated with recombinant PR-Set7 S29A.
As can be seen in Figure 1B, compared to the wild type recombinant, PR-Set7
S29A had a dramatically lower proportion retained on the phospho-column,
indicating that the overall phosphorylation of the protein had been dramatically
reduced (Figure 24B). This indicated to us that Serine 29 is the main
phosphorylation site on PR-Set7, and that a cyclin dependent kinase may be
responsible for the bulk of PR-Set7 phosphorylation.
82
Studies found that a secondary motif (the RXL motif or hydrophobic
patch) other than a phosphorylation site consensus sequence, helps to stabilize
the binding of cyclin dependent kinases to their substrates (Schulman,
Lindstrom et al. 1998). PR-Set7 was found to contain such a motif, starting at
Arginine 149. To investigate whether this motif was important for
phosphorylation of PR-Set7 the possible RXL motif (amino acids 149, 150 and
151) were all mutated to alanines (RXL149AAA). The above experiment was
repeated with PR-Set7 RXL149AAA and interestingly, these mutations had no
effect on retention of recombinant PR-Set7 on the column, indicating that PR-
Set7 RXL149AAA was still phosphorylated to the same extent as the wild type
recombinant (Figure 24C). This indicates that while PR-Set7 may be
phosphorylated by a cyclin dependent kinase, the RXL motif is dispensible for
this to occur.
Although PR-Set7 is a nuclear protein, it only has a very weak nuclear
localization signal. The question of how it is so efficiently localized to the
nucleus has been a mystery. Since phosphorylation is known to affect cellular
localization of some proteins (Hagting, Jackman et al. 1999), we decided to test
if PR-Set7 phosphorylation may have any effect on its localization. 293T cells
were transfected with vector containing either FLAG-tagged wild type
recombinant PR-Set7 or PR-Set7 S29A. Cells were then harvested and
cytoplasmic and nuclear fractions were taken for both sets of transfections. The
83
cytoplasmic and nuclear fractions of PR-Set7 and PR-Set7 S29A were western
blotted for FLAG antibody, and interestingly, although it does not account for a
large fraction of the total, a significantly larger amount of PR-Set7 S29A was
found in the cytoplasmic fraction, compared to wild type (Figure 25A). These
transfected cells were also subjected to indirect immunofluorescence analysis
for the FLAG tag. In cells transfected with wild type recombinant PR-Set7, all
staining was found to exclusively reside in nuclei. Interestingly, in cells
transfected with PR-Set7 S29A, although the majority of FLAG staining was
seen in nuclei, there were still many cells where much of the FLAG tag was seen
in the cytoplasm (Figure 25B). Similar results were seen when these
experiments were repeated with myc-tagged recombinant PR-Set7 and PR-Set7
S29A. Although it is not the sole determinant of PR-Set7 localization,
phosphorylation of Serine 29 appears to play a significant role, in some cells, in
PR-Set7 nuclear localization.
Once we had discovered Serine 29 to be the major phosphorylation site
on PR-Set7, we decided to attempt a raise an antibody that would be specific for
this modification. Peptides containing amino acids 21-30 or PR-Set7,
phosphorylated at Serine 29, were used as immunogens in rabbits. To test the
anti-serum for specificity, a panel of lysates were blotted on a membrane
containing either lysates from cells overexpressing wild-type PR-Set7,
overexpressing PR-Set7 S29A, or lysates from cells overexpressing wild-type
84
B
A
85
Figure 25. PR-Set7 S29A lacks efficiency in nuclear localization. HEK 293T
cells were either transfected with FLAG tagged wild type PR-Set7 or FLAG
tagged PR-Set7 S29A. The day after transfection, cells were either plated on
coverslips for immunofluorescence analysis or harvested for western analysis.
A) Western analysis of cytoplasmic (cyto) and nuclear (nuc) fractions (4% of
each) of HEK293T cells transiently transfected with FLAG-PR-Set7 wt or
FLAG-PR-Set7 S29A. A higher proportion of PR-Set7 S29A is found in the
cytoplasmic fraction that that of PR-Set7 wt.
B) Immunofluorescence where blue represents DAPI staining. Red represents
staining for FLAG tag. FLAG-Pr-Set7 wt was always nuclear, while FLAG-PR-
Set7 S29A was often found outside of the nucleus. Experiments were performed
side by side.

86
PR-Set7 that have been treated with 10 units of calf intestinal alkaline
phosphatase (CIP) for 1 hour at 37° C. Our PR-Set7 phospho antibody
recognized PR-Set7 in lysates overexpressing wild-type PR-Set7, but not in the
corresponding lysates treated with CIP, and not in lysates overexpressing PR-
Set7 S29A (Figure 26). Since PR-Set7 is tightly cell-cycle regulated, only being
expressed near G2/M, we did some additional specificity testing on the
antibody. Lysates from HeLa cells that were synchronized at the G1/S border
and G2/M border were blotted, along with G1/S border lysates that had been
treated with CIP. Our antibody also showed specificity with this experiment, not
finding PR-Set7 at the G1/S border, but recognizing it at G2/M. The band seen
at G2/M disappeared upon treatment with CIP (Figure 26). These data indicate
that our antibody specifically recognizes PR-Set7 phosphorylated at Serine 29.

87
Figure 26. Standardization of an antibody raised against PR-Set7 phosphorylated
at Serine 29. Western analysis with polyclonal rabbit antibody raised against a
peptide representing PR-Set7 phosphorylated at Serine 29. The upper panel
represent whole cell lysates from 293T cells overexpressing recombinant PR-
Set7 wt, PR-Set7 S29A, or PR-Set7 wt that has been treated with calf intestinal
phosphatase (CIP). The lower panel represent lysates from HeLa cells that have
either been synchronized at the G1/S border (where PR-Set7 expression is
absent), at G2/M (where PR-Set7 expression is at its peak), or that have been
synchronized at G2/M and been treated with CIP
88
Conclusions and future directions
At the commencement of these experiments, H4K20 methylation was
considered to be a mark of repressive chromatin, about which little was known.
To further the understanding of the biological relevance of H4K20
monomethylation, we performed experiments to answer questions surrounding
the genomic localization of this modification, its nature relative to transcription
and its role in cell cycle progression.
The majority of described experiments relied on a novel panel of
antibodies, highly specific for the mono-, di- and trimethyl states of H4K20.
These antibodies were found to only recognize H4K20 methylated at a specific
state, and the ability of these antibodies to recognize their epitopes were
unhindered by the nearest neighboring histone modification. These antibodies
were standardized for immunoprecipitation (IP) with nucleosomes and it was
found that both our antibodies for H4K20 monomethylation and H4K20
trimethylation were suited for IP purposes.
As part of this IP standardization, we were able to show that H4K20
monomethylation and H3K9 monomethylation not only colocalize within the
genome, they also occur within the same nucleosome, to what may be to an even
greater extent than H4K20 trimethylation and H3K9 trimethylation.
89
Although active, acetylated chromatin is universally thought of as being
in a relaxed, open state, we found that chromatin acetylated at H4K16 was
actually more resistant to digestion by MNase than chromatin methylated for
H4K20 and H3K9, which are thought to occur at condensed, transcriptionally
repressed chromatin. Although one could argue that this resistance to digestion
at active, acetylated chromatin is due to the presence of various transcriptional
machinery, one would assume that since this chromatin is active, there would be
a certain amount of dynamism in that machinery. Presumably, that dynamism
would most likely allow MNase access to DNA.
Because of various reports regarding the biological relevance of H4K20
monomethylation, we made attempts to discover the genomic localizations of
this histone modification. We used two applications of chromatin
immunoprecipitation. The first application was to directly sequence DNA from
chromatin ChIP’d with our H4K20 monomethyl antibody. The second
application was to ChIP for H4K20 monomethylation, followed by microarray
analysis for enriched genomic regions.
Using novel statistical methodology, we were able to find genomic
regions on human chromosomes 21 and 22 that gave us information where
H4K20 monomethylation is occurring at p values of less than 0.01, 0.005, and
0.001. These data were confirmed by use of a H4K20 trimethylation, ChIP-chip
90
as a negative control, since we had found these two modifications to not
colocalize through immunoprecipitation and immunofluorescence analysis.
Our first method of direct sequencing of ChIP’d DNA was later found to
be much less useful, as sequenced areas found on chromosomes 21 or 22 were
not found to be statistically significant areas of H4K20 monomethylation in our
ChIP-chip data.
Since it had been previously reported that H4K20 monomethylation is
associated with transcriptionally repressed chromatin, we decided to test the
effect to expression of highly H4K20 monomethylated genes when PR-Set7 was
knocked down. The expression of all genes with a high level of H4K20
monomethylation increased in the absence of PR-Set7 and H4K20
monomethylation.
A hypothesis was formed that H4K20 monomethylation is occurring to
silence sequences that would otherwise function as aberrant enhancers. This
hypothesis was negated when H4K20 monomethylated sequences introduced
into a vector induced repression of a reporter gene. This led us to believe that
these vectors were becoming chromatinized in vivo.
To test this hypothesis, ChIP analysis was performed on the vector that
had the most repressed activity compared to empty vector and the vector
containing the insert that had no change in activity compared to empty vector.
When Histone H3 occupancy was tested at the TK promoter of both vectors, it
91
was found that the vector containing the insert positive for H4K20
monomethylation had a much higher level of nucleosome occupancy. We also
tested to see if H4K20 Monomethylation might be occurring at the TK
promoters of these vectors and it was found that the vector containing the insert
that is H4K20 monomethylated within the genome was in fact H4K20
monomethylated on this vector.
Collectively, these findings demonstrate that H4K20 monomethylation is
recruited to certain genomic regions in a sequence dependent manner, resulting
in chromatinization of that region, which results in the transcriptional repression
of a neighboring gene (Figure 27).
This then led to the question of what role H4K20 monomethylation plays
in cell cycle progression. By taking samples of synchronized cells over the
course of the cell cycle, we found H4K20 monomethylation to be cell cycle
regulated. This is one of the only histone lysine methyl marks known to be cell
cycle regulated. Another report has stated that H3K9 trimethylation is a cell-
cycle regulated mark, rising and falling during mitosis (McManus, Biron et al.
2006). Although in our experiments we saw no such mitotic fluctuation in global
H3 K9 trimethylation levels by Western analysis, it was found by quantitative
imaging microscopy (McManus and Hendzel 2005), that H3K9 trimethylation
increased by approximately 3 fold from interphase and early G2 to metaphase.
Although this may be somehow biologically interesting, H4K20
92
Figure 27. Model of H4K20 monomethylation function .
From our accumulated data, we have formulated the following model for the
biological function of H4K20 monomethylation: Genomic locations of H4K20
monomethylation recruit PR-Set7 in a sequence dependent manner, resulting in
site specific H4K20 monomethylation. The methyl mark then results in
condensation of adjacent chromatin, resulting in transcriptional repression of
neighboring genes.

93
monomethylation undergoes a much more dramatic cell-cycle regulated profile,
being virtually absent at G1 and rising a great deal more than 3 fold through S-
phase and G2/M, before levels fall as cell settled back into G1
We had previously found H4K20 monomethylation and H3K9
monomethylation to have a high degree of colocalization, such that they occur
on the same nucleosome. Surprisingly, H4K20 monomethylation and H3K9
monomethylation had very different cell-cycle profiles. This implies that these
modifications do not occur concurrently. Since H4K20 monomethylation is the
more dynamic of these two modifications, perhaps H3K9 monomethylation acts
as a marker for where H4K20 monomethylation should occur.
This could be tested by obtaining cells proficient and deficient in G9A,
the bulk H3K9 monomethylase (Tachibana, Sugimoto et al. 2002). ChIP-chip
for H4K20 monomethylation could be performed for H4K20 monomethylation
in the presence and absence of H3K9 monomethylation. If H4K20
monomethylation mapping changed dramatically in the absence of G9A, we
could conclude that H3K9 monomethylation does indeed act as a marker for
H4K20 monomethylation.
Also, although the majority of H4K20 monomethylation appears to
colocalize with H3K9 monomethylation by our IP experiments, not all of H3K9
monomethylation may colocalize with H4K20 monomethylation. Knockdown
experiments to reduce levels of H3K9 monomethylation could be performed to
94
see if similar aberrant phenotypes occur. If not, H3K9 monomethylation might
be recruit to similar domain as H4 K20 monomethylation, but the two marks
may actually have little to do with each other.
We also found that knockdown of PR-Set7 and reduction of global levels
of H4K20 monomethylation in human cell lines leads to severe reductions in
viability. In HEK 293T cells, a G2 arrest response was found to occur upon PR-
Set7 knockdown. This G2 arrest was found to be specifically regulated by the
DNA damage-dependent ATM/ATR/DNA-PKcs pathway.
The presence of DNA damage in the absence of PR-Set7 was
investigated and it was found that when H4K20 monomethylation levels are
decreased, they are accompanied by an increase in levels of phosphorylated
H2AX, an indicator of DNA double strand breaks.
While attempting to look at the morphology of PR-Set7 knockdown
cells, we discovered the appearance of multiple mitotic spindles in some
aberrant mitotic cells lacking H4K20 monomethylation. An aberrant number of
mitotic poles is considered a hallmark of DNA damage, and this further
cemented that PR-Set7 knockdown cells were prone to much higher levels of
DNA damage than controls.
To test if H4K20 monomethylation was performed in response to DNA
damage, we treated cells with DNA damaging reagents to induce genotoxic
95
stress. In all cases, no changes in H4K20 monomethylation were observed, even
when significant increases in H2AX phosphorylation were observed.
A large nuclei phenotype was also seen upon PR-Set7 knockdown. PR-
Set7 knockdown cells, as a population, have roughly twice the nuclear volume,
and that the large nuclei specifically had a volume, on average 4 times of that of
the average control cell nuclear volume.
As these large nuclei had dimmer DAPI staining compared to
neighboring nuclei of a normal size, we hypothesized that PR-Set7 knockdown
cells had a defect in chromatin condensation. We tested this by obtaining nuclei
from control and knockdown cells and found that PR-Set7 knockdown cells are
more sensitive to MNase digestion, confirming that chromatin was indeed more
relaxed in those cells. This was consistent with our previous findings that
H4K20 monomethylation leads to condensation.
Since we had knocked down both PR-Set7 and H4K20
monomethylation, we tested if phenotypes we were seeing were due to knocking
down the PR-Set7 protein, or due to loss of the histone modification. A
catalytically dead, dominant-negative point mutant of PR-Set7 was used to
knockdown H4K20 monomethylation and the same phenotypes were observed
as with PR-Set7 knockdown. This is the first time that loss of a histone
modification, and not the loss of the effector enzyme of that modification, has
been shown to be responsible for a resulting phenotype.
96
Based on a previous report from studies in Xenopus that PR-Set7 is a
mitotic phosphoprotein, we tested if PR-Set7 in a human cell line is
endogenously phosphorylated and found that to be the case. In addition, were
tested to see if recombinant PR-Set would be endogenously phosphorylated to
the same extent, which it was. We were then able to test if a recombinant PR-
Set7 point mutant at Serine 29 (a consensus site for phosphorylation by the
Cyclin B/cyclin dependent kinase 2 complex) would still be phosphorylated to
the same extent as wild type, and phosphorylation was all but abolished.
To further understand the biological relevance of PR-Set7
phosphorylation at Serine 29, one simple experiment performed was to test for
differential localization between wild-type and the phosphorylation mutant. By
immunofluorescence and Western analysis, it was found that recombinant PR-
Set7 S29A did not efficiently localize to the nucleus to the same extent as the
recombinant wild-type PR-Set7.
PR-Set7 only contains a very weak nuclear localization signal, and we
had questioned what the manner was of PR-Set7 being an entirely nuclear
protein in vivo. It appears that phosphorylation or PR-Set7 on Serine 29, while
not being the only variable, does play a role in nuclear localization of PR-Set7.
We were also able to generate an antibody that specifically recognizes
PR-Set7 phosphorylated at Serine 29. The use of this antibody could be helpful
97
in in vitro kinase assays, which could elucidate which kinase is performing
Serine 29 phosphorylation on PR-Set7.

These data together suggest that H4K20 monomethylation plays at least
two roles within the cell, one as a mechanism of transcriptional repression, the
other as a player in maintenance of genomic stability.
Two recent studies have revealed at least one methyl-binding protein that
specifically associated with mono- and dimethylated H4K20 (Kim, Daniel et al.
2006; Trojer, Li et al. 2007). This methyl-binding protein, L3MBTL1, was
found to bind to dimethylated H4K20 to a greater extent, but functional studies
were only performed in relation to its relationship with monomethylated H4K20.
It was revealed that upon siRNA knockdown of L3MBTL1, the expression of a
gene with H4K20 monomethylation was found to increase. L3MBTL1 was also
found to have the ability condense nucleosomal arrays, dependent on the activity
of PR-Set7.
These data are consistent with the results described here, implicating
L3MBTL1 as the key molecular translator for H4K20 monomethylation.
High-throughput ChIP analysis for L3MBTL1 occupancy throughout the
genome would be very insightful. If L3MBTL1 was found to have a high degree
of colocalization with H4K20 monomethylation, one would assume that its main
role is as effector of H4K20 monomethylation, although it may also colocalize
98
with areas of H4K20 dimethylation, serving a similar or alternate purpose. It
would also be use to knockdown L3MBTL1 to see if similar cell cycle defects
occur, as on PR-Set7 knockdown. To discover if L3MBTL1 has a cell cycle
regulated expression profile similar to that of PR-Set7 would also be insightful.
Although H4K20 monomethylation occurs at specific sequences,
L3MBTL1 was found to condense nucleosomal DNA regardless of sequence.
This suggests that the sequence specificity is on the side of PR-Set7. Since PR-
Set7 has an amino-terminal domain unique from other SET domain containing
proteins, perhaps this domain is responsible for sequence specific recruitment of
PR-Set7 to a region, upon which it performed monomethylation on an H4
molecule, although we could not find a consensus motif for sequences of H4K20
monomethylation among our ChIP-chip data.
As previously described, a considerable proportion of H4K20
monomethylation was found to occur at minisatellites. Satellite repeats have
been found to lead to altered DNA structure and fragility (Li, Korol et al. 2004).
Perhaps these regions where PR-Set7 is recruited undergoes a non-B DNA
conformation, which is a great possibility with the strand asymmetry observed in
the presence of minisatellite repeats (Buard, Bourdet et al. 1998). This non-B
structure could make these sequences recognizable by PR-Set7. Biochemical
experiments, such as gel shift analysis, could be performed with PR-Set7 and
99
DNA representing a genomic H4K20 monomethylated regions, to test for PR-
Set7 specificity, sequence or structure-wise.
One major point of interest would be the fate of H4K20
monomethylation at the end of a round of cell doubling, when the mark
disappears. One possibility is that a histone methyltransferase is increasing
H4K20 monomethylation to a di- or trimethyl state. Another possibility is that
H4K20 monomethylation is being removed at the end of the cell cycle by a
histone demethylase. The discovery of such an enzyme would be significant, as
it would presumably become the first cell cycle regulated histone demethylase to
be identified. It would also be the first histone lysine demethylase found to
completely turnover a modification. Lastly, it is possible that H4 molecules are
being replaced at the end of a round of mitosis.
Another unanswered question would be why the mark, if important for
genomic integrity, is dynamic. Other histone modification marks are known to
help cells condense their mitotic chromosomes, but H4K20 monomethylation
rises as cells proceed through S-phase, when most DNA double strand breaks
occur. If H4K20 monomethylation is involved in chromatin condensation that
protects from damage, why would it need to be removed in G1? Perhaps, since it
represses transcription, cells need to remove H4K20 monomethylation to have
adequate expression of genes necessary during G1.
100
A recent report found that H3K4 and H3K9 monomethylation, along
with H3K4 trimethylation can occur at chromosomal breakpoints (Barski,
Cuddapah et al. 2007). It was suggested that the open chromatin at these regions
makes them prone to breakage, leading to chromosomal translocation.
Interestingly, our ChIP-chip data also found H4K20 monomethylation to occur
near three sites of known chromosomal breakpoint: the chromosome 22 locus
where breakage leads to the bcr-abl fused protein (the Philadelphia
chromosome), the chromosome 21 locus, where breakage leads to the t(8;21)
translocation which can lead to acute myeloid leukemia, and the locus on
chromosome 22 where translocation leads to cat-eye syndrome. Perhaps,
condensation at these sites due to H4K20 monomethylation and L3MBTL1
protects from damage at these breakpoints. This could be tested by the
introduction of these sequences into an extrachromosomal recombination
substrate (Hesse, Lieber et al. 1987). If recombination was found to occur at
these sequences at a higher level than random controls, it could be concluded
that these sequences are broken, or cut, to a greater extent than most genomic
DNA. The catalytically dead PR-Set7 mutant could be co-transfected in, to see if
absence of H4K20 monomethylation would have an effect on the recombination
rates of these sequences.
101
Although this knowledge would not currently be able to keep these
biological phenomena from occurring, it would advance our overall
understanding of disease due to genomic instability.
Currently, there is one clear path that this data could take to help in the
fight against human disease. Since H4K20 monomethylation occurs in a cell
cycle regulated manner, and since disrupting this pathway leads to reduced
viability and cell death, perhaps blocking this pathway could be useful in
fighting proliferation of cancer cells. If an inhibitor of PR-Set7 catalytic activity
could be found, this compound could perhaps be a part of multi-drug
chemotherapy in cancer patients. Unfortunately, this form of therapy could
result in secondary cancers, due to aberrant chromosomal events that may occur
when, for instance, multipolar mitotic spindles occur. Such an event could lead
to secondary mutations, which could make a cancer cell even more virulent.
Hopefully, these data will eventually lead to greater understanding of
human disease in a chromatin-related context.

102
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119
Appendix A (Chromosome 21 H4K20 monomethyl regions, p<0.01)
Start End Start End
15325045
29593530
32594474
32880432
33775777
33844092
33845392
34739963
35042527
35104882
35107444
35133410
35138583
35157983
35171324
35182155
37367489
38050703
39101898
39113108
39208323
39227412
39419760
41473233
41959404
42045407
42049925
42123914
42137638
42393102
42415970
42521620
42552142
42587177
42663798
42704762
42956532
43134364
43141314
43466488
15326245
29594330
32595074
32881498
33776177
33844592
33847392
34740463
35042871
35105282
35107844
35133710
35139685
35160093
35171924
35182525
37367989
38051903
39102423
39113708
39208623
39227712
39420060
41473533
41959904
42045907
42050325
42124214
42138421
42393402
42416270
42522497
42553142
42587477
42664798
42705062
42956832
43134964
43142691
43467272
43576484
43622198
43636920
43663378
43738478
43762506
43826870
43848107
43964428
43970149
43971320
43994613
43997347
43999295
44011838
44030671
44038526
44096306
44124183
44128806
44155875
44183709
44203796
44494318
44550763
44555086
44561090
44660864
45008766
45106045
45107236
45109840
45115196
45137112
45155704
45164206
45192177
45198381
45287063
45325973
43577135
43622798
43637720
43664078
43738778
43762806
43828470
43848407
43966328
43970449
43971720
43994913
43997995
43999895
44012138
44031671
44039026
44096706
44124783
44129199
44156275
44184109
44204196
44494829
44551663
44555386
44561390
44662264
45009066
45106508
45107936
45110440
45116796
45137612
45156004
45164794
45192877
45198681
45287363
45326373
120
Start End
45339596
45371048
45373789
45377952
45381667
45385949
45393026
45396120
45401012
45446798
45500036
45552162
45624390
45663308
45728725
45757744
45881137
45990099
46142645
46168513
46303737
46329208
46364139
46413541
46445316
46464267
46508779
46597690
46626295
46643456
46647315
46742586
45340196
45372223
45373989
45379152
45382167
45386449
45393626
45396420
45401312
45447498
45500409
45552662
45625290
45663808
45729225
45758244
45881437
45991299
46142945
46169313
46304637
46330208
46364639
46415441
46446016
46464567
46509379
46598195
46629195
46643756
46648615
46742986
121
Chromosome 22 H4K20 MonoMe regions, p<0.01
Start End Start End
14609644
15959659
16001752
16805486
17333593
18242444
18269112
18272083
18346673
18484414
18783070
18800088
18836541
18964666
19099782
19211279
19233021
19247182
19615994
20035148
20061490
20097413
20113723
20114845
20172981
20174743
20176817
20193965
20230505
20251842
20322530
21609147
21848883
21874385
21886500
22763699
22874476
23887347
26525457
27931428
14610231
15960059
16003352
16805886
17334193
18242920
18270012
18272483
18347073
18484614
18784728
18801088
18837021
18964966
19100082
19211979
19233421
19248182
19616394
20035448
20061912
20098813
20114360
20115545
20173481
20175143
20178775
20195065
20230984
20252342
20322830
21609641
21849983
21874685
21887200
22764194
22874776
23887647
26526271
27931775
28973146
28999873
29804754
30351738
31050125
31540925
34033252
34062900
34103505
34136854
34299067
34322774
35049310
35084544
35558973
36380770
36925026
36950166
37016760
37035327
38004470
38032101
38034101
38241918
40164430
40206391
41657869
41667072
41734196
41799430
42001185
42556919
42758656
43027303
43754052
44684760
44803567
45250036
45322150
45331133
28973881
29000173
29805154
30352138
31051695
31541225
34033552
34063200
34103805
34137320
34299367
34323074
35050010
35085044
35559273
36381982
36925526
36950566
37017260
37036027
38004870
38032501
38034301
38242418
40164730
40206691
41658369
41667372
41734596
41799830
42001885
42557719
42758956
43027703
43754852
44685060
44803867
45250336
45323050
45334033
122
Start End
45355310
45451517
45452417
45523512
45546456
45786188
46019940
46968414
48496718
48537940
48539140
48540240
48739868
48761112
48769301
48780508
48974659
49032331
49202307
49403776
49408070
45355863
45451817
45452917
45523856
45546856
45786788
46020342
46969314
48497518
48538240
48539840
48540840
48740168
48763512
48770201
48781108
48976059
49032631
49202964
49404674
49411670
123
Chromosome 21 H4 K20 MonoMe regions, p<0.005
Start End Start End
15325045
29593530
32594474
32880432
33775777
33845392
34739963
35042527
35107444
35133410
35138583
35157983
35171324
37367489
38050703
39113108
39208323
39227412
39419760
41473233
41959404
42123914
42137638
42415970
42521620
42552142
42587177
42663798
42956532
43134364
43141314
43466488
43576484
43622198
43636920
43663378
43762506
43826870
43848107
43964428
15326245
29594330
32595074
32881498
33776177
33847392
34740463
35042871
35107844
35133710
35139685
35160093
35171924
37367989
38051903
39113708
39208623
39227712
39420060
41473533
41959904
42124214
42138421
42416270
42522497
42553142
42587477
42664798
42956832
43134964
43142691
43467272
43577135
43622798
43637720
43664078
43762806
43828470
43848407
43966328
43970149
43971320
43997347
43999295
44011838
44030671
44038526
44096306
44124183
44155875
44183709
44203796
44494318
44550763
44555086
44561090
44660864
45106045
45107236
45109840
45115196
45137112
45164206
45192177
45287063
45325973
45339596
45371048
45373789
45377952
45381667
45385949
45393026
45396120
45446798
45552162
45624390
45663308
45728725
45757744
43970449
43971720
43997995
43999895
44012138
44031671
44039026
44096706
44124783
44156275
44184109
44204196
44494829
44551663
44555386
44561390
44662264
45106508
45107936
45110440
45116796
45137612
45164794
45192877
45287363
45326373
45340196
45372223
45373989
45379152
45382167
45386449
45393626
45396420
45447498
45552662
45625290
45663808
45729225
45758244
124
Start End
45881137
45990099
46168513
46303737
46329208
46364139
46413541
46445316
46464267
46508779
46597690
46626295
46647315
46742586
45881437
45991299
46169313
46304637
46330208
46364639
46415441
46446016
46464567
46509379
46598195
46629195
46648615
46742986
125
Chromosome 22 H4 K20 MonoMe regions, p<0.005
Start End Start End
14609644
15959659
16001752
16805486
17333593
18242444
18269112
18272083
18346673
18783070
18800088
18836541
19099782
19211279
19233021
19247182
19615994
20061490
20097413
20113723
20172981
20174743
20176817
20193965
20230505
20251842
20322530
21609147
21848883
21886500
22763699
22874476
23887347
26525457
27931428
28973146
31050125
31540925
34103505
34136854
14610231
15960059
16003352
16805886
17334193
18242920
18270012
18272483
18347073
18784728
18801088
18837021
19100082
19211979
19233421
19248182
19616394
20061912
20098813
20114360
20173481
20175143
20178775
20195065
20230984
20252342
20322830
21609641
21849983
21887200
22764194
22874776
23887647
26526271
27931775
28973881
31051695
31541225
34103805
34137320
34299067
35049310
35084544
35558973
36380770
36925026
36950166
37016760
37035327
38032101
38241918
40164430
40206391
41657869
41667072
41734196
41799430
42556919
43027303
43754052
44684760
45250036
45322150
45331133
45355310
45451517
45452417
45523512
45546456
45786188
46968414
48496718
48537940
48539140
48540240
48761112
48769301
48780508
48974659
49032331
34299367
35050010
35085044
35559273
36381982
36925526
36950566
37017260
37036027
38032501
38242418
40164730
40206691
41658369
41667372
41734596
41799830
42557719
43027703
43754852
44685060
45250336
45323050
45334033
45355863
45451817
45452917
45523856
45546856
45786788
46969314
48497518
48538240
48539840
48540840
48763512
48770201
48781108
48976059
49032631
126
Start End
49202307
49403776
49408070
49202964
49404674
49411670
127
Chromosome 21 H4 K20 MonoMe regions, p<0.001
Start End Start End
15325045
29593530
32594474
32880432
33775777
33845392
34739963
35107444
35133410
35138583
35157983
38050703
39113108
39208323
41473233
42137638
42521620
42552142
42587177
42663798
43134364
43141314
43466488
43622198
43636920
43663378
43762506
43826870
43964428
43971320
43997347
44030671
44096306
44203796
44550763
44555086
44660864
45106045
45107236
45109840
15326245
29594330
32595074
32881498
33776177
33847392
34740463
35107844
35133710
35139685
35160093
38051903
39113708
39208623
41473533
42138421
42522497
42553142
42587477
42664798
43134964
43142691
43467272
43622798
43637720
43664078
43762806
43828470
43966328
43971720
43997995
44031671
44096706
44204196
44551663
44555386
44662264
45106508
45107936
45110440
45115196
45164206
45192177
45287063
45325973
45339596
45371048
45373789
45377952
45381667
45385949
45393026
45446798
45552162
45624390
45663308
45728725
45757744
45990099
46303737
46364139
46413541
46445316
46597690
46626295
46647315
45116796
45164794
45192877
45287363
45326373
45340196
45372223
45373989
45379152
45382167
45386449
45393626
45447498
45552662
45625290
45663808
45729225
45758244
45991299
46304637
46364639
46415441
46446016
46598195
46629195
46648615
128
Chromosome 22 H4 K20 MonoMe regions, p<0.001
Start End Start End
14609644
15959659
16001752
17333593
18242444
18269112
18272083
18346673
18800088
18836541
19099782
19211279
19247182
19615994
20061490
20097413
20113723
20176817
20193965
20230505
20251842
21848883
21886500
26525457
27931428
28973146
31050125
34103505
36380770
37035327
38032101
38241918
40206391
41657869
41667072
42556919
43754052
44684760
45322150
45331133
14610231
15960059
16003352
17334193
18242920
18270012
18272483
18347073
18801088
18837021
19100082
19211979
19248182
19616394
20061912
20098813
20114360
20178775
20195065
20230984
20252342
21849983
21887200
26526271
27931775
28973881
31051695
34103805
36381982
37036027
38032501
38242418
40206691
41658369
41667372
42557719
43754852
44685060
45323050
45334033
48537940
48539140
48540240
48761112
48769301
48780508
48974659
49202307
49403776
49408070
48538240
48539840
48540840
48763512
48770201
48781108
48976059
49202964
49404674
49411670
129
Appendix B

Abstract (if available)

Abstract The post-translational modification of histones is thought to play a critical role in directing nuclear events involving chromatin. One such modification, Histone H4 Lysine 20 (H4K20) methylation, a mark only seen in multicellular eukaryotes, has been shown to be associated with inactive chromatin. Here, we performed chromatin immunoprecipitation experiments on genomic tiled arrays of human chromosomes 21 and 22 to discover the genomic localization of H4K20 monomethylation. A novel statistical method was used to cull genomic regions enriched for H4K20 monomethylation. It was found that H4K20 monomethylation often occurs within genes, at minisatellite repeats, and at sites of high conservation within higher eukaryotes. H4K20 monomethylation was confirmed as being a transcriptionally repressive mark by analyzing the expression analysis of highly H4K20 monomethylated genes in the presence and absence of this modification. Sequences with a high degree of H4K20 monomethylation were found to have a negative effect on transcription, due to a higher propensity for chromatinization. H4K20 monomethylation was also found to have a cell cycle regulated profile, peaking at G2/M, while being absent at the G1/S border. PR-Set7, a cell cycle regulated histone methyltransferase, has been shown to be responsible for the bulk of H4K20 mono-methylation. We found that knockdown of PR-Set7 and H4K20 mono-methylation leads to severe growth arrest. Growth arrest was found to be accompanied by the appearance of DNA damage, as measured by increased levels of H2AX phosphorylation and comet assay. Multiple mitotic spindles, another hallmark of DNA damage, were observed in cells lacking H4K20 monomethylation. These cells were also found to have high levels of enlarged nuclei, whose chromatin appeared less densely packed than corresponding normal sized nuclei. A global decrease in chromatin condensation upon PR-Set7 knockdown was confirmed with micrococcal nuclease assays.

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Asset Metadata

Creator Houston, Sabrina Ishimaru (author)

Core Title Studies of the biological relevance of Histone H4 Lysine 20 monomethylation: discovery of its role in the cell cycle and localization within the human genome

School Keck School of Medicine

Degree Doctor of Philosophy

Degree Program Biochemistry and Molecular Biology

Degree Conferral Date 2007-12

Publication Date 09/19/2009

Defense Date 07/20/2007

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

Tag epigenetics,histone methylation,OAI-PMH Harvest

Language English

Advisor Rice, Judd C. (committee chair), [illegible], Ite A. (committee member), Frenkel, Baruch (committee member), Jadner, Robert D. (committee member)

Creator Email shouston@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m828

Unique identifier UC157971

Identifier etd-Houston-20070919 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-558150 (legacy record id),usctheses-m828 (legacy record id)

Legacy Identifier etd-Houston-20070919.pdf

Dmrecord 558150

Document Type Dissertation

Rights Houston, Sabrina Ishimaru

Type texts

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

Repository Name Libraries, University of Southern California

Repository Location Los Angeles, California

Repository Email cisadmin@lib.usc.edu