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Characterization and functional study of a novel human protein SFMBT, and PR-Set7 histone methyltransferase

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Characterization and functional study of a novel human protein SFMBT, and PR-Set7 histone methyltransferase

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CHARACTERIZATION AND FUNCTIONAL STUDY OF A NOVEL HUMAN
PROTEIN SFMBT, AND PR-SET7 HISTONE METHYLTRANSFERASE

by
Shumin Wu

__________________________________________________________________

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

December 2010

Copyright 2010 Shumin Wu
ii

Acknowledgements
To my family and parents. I could not have made it without your unconditional love
and support. Thanks to my husband Miou, for walking with me along the way, for all the
suggestions and inspirations; Thanks to my parents and my parents-in-law. I feel so
grateful to have such wonderful parents. You help taking care of my child so that I can
focus on my research; Thanks to my son Ethan for bringing so much joy and meaning to
my life.
To Dr. Judd Rice and all the members of the Rice lab. I would like to express my
special appreciation to my PI Judd Rice for his encouragements and guidances over these
years. His continued confidence in me has made my journey in science easier. Your
dedication and persistence for science is inspiring. Thanks to my lab mates, Creighton,
Lauren, Sai and Tanya. Each of you is so unique and wonderful. You guys make the lab
feel like a family. I learned a lot from you, scientifically and personally.
To my Committee members, Dr. Michael Stallcup, Dr. Debbie Johnson, Dr. Ite Laird
and Dr. Wei Li. Thanks for your encouragements and guidances over these years. Your
suggestions make my project progress much faster. I feel grateful to have you in my
Committee.
Last, but not the least. I would like to thank all my friends here at USC. Your
friendship and support have made my life richer and more meaningful. I feel lucky to
have you in my life.
iii

Table of Contents
Acknowledgements ii

List of Tables v

List of Figures vi

Abstract viii

Introduction 1
Chromatin and Its Regulation 1
Histone Methylation and Histone Methyltransferases (HMTs) 3
PR-Set7 and H4K20 monomethylation (H4K20me1) 4
Roles of PR-Set7 and H4K20me1 in Transcriptional Regulation 5
Roles of PR-Set7 and H4K20me1 in Cell Cycle Regulation 6
Roles of PR-Set7 and H4K20me1 in DNA Damage Responses 8
Malignant Brain Tumor (MBT) Proteins 10

Chapter 1: Characterization of a novel human protein SFMBT and its 12
function in transcriptional represssion
1.1. Introduction 12
1.2. Results 14
1.2.1. hSFMBT is a conserved Polycomb-like protein 14
1.2.2. hSFMBT is a cell type-specific nuclear protein 17
1.2.3. hSFMBT strongly associates with the nuclear matrix 19
1.2.4. The four MBT repreats of hSFMBT are necessary and sufficient for 22
potent transcriptional repression
1.2.5. hSFMBT binding to histone H3 and H4 is mediated by the four 27
MBT repreats
1.2.6. hSFMBT binds the N-terminal tail of histone H3 30
1.2.7. Identification of hSFMBT target genes in K562 cells 34
1.2.8. Identification of hSFMBT binding proteins 39
1.2.9. hSFMBT is associated with HDAC activity 43
1.3. Discussion and future directions 46

Chapter 2: Characterization and functional analysis of the phosphorylation 51
of PR-Set7 histone methyltransferase
2.1. Introduction 51
2.2. Results 53
2.2.1. Serine 29 is a major phosphorylated residue of human PR-Set7 53
2.2.2. Cdk1/cyclinB complex specifically and selectively phosphorylates 56
serine 29 of PR-Set7

iv

2.2.3. Phosphorylation of serine 29 does not affect PR-Set7 60
methyltransferase activity
2.2.4. Cdk1/cyclinB-mediated phosphorylation of PR-Set7 serine 29 occurs 64
from prophase to anaphase
2.2.5. PR-Set7 is removed from mitotic chromosomes following serine 29 68
phosphorylation
2.2.6. Phosphorylation of serine 29 prevents PR-Set7 degradation 71
2.2.7. Serine 29 phosphorylation directly inhibits APC
cdh1
-mediated 75
ubiquitination and degradation of PR-Set7
2.2.8. Cdc14 specifically and directly dephosphorylate PR-Set7 serine 29 81
2.2.9. Constitutive PR-Set7 S29 phosphorylation impedes early mitotic 85
progression
2.3. Discussion and future directions 91

Chapter 3: Elucidating the roles of PR-Set7 and H4K20me1 in DNA 96
damage responses
3.1. Introduction 96
3.2. Results 97
3.2.1. Global levels of PR-Set7 and H4K20me1 are elevated upon 97
IR induced DNA damage
3.2.2. PR-Set7 is targeted to DNA damage loci upon micro-irradiation 100
3.2.3. 53BP1 recruitment to DNA damage foci is impaired in the 104
absence of PR-Set7
3.3. Discussion and future directions 106

Chapter 4: Methods 108
4.1. Immunofluorescence studies on cells 108
4.2. GST pull-down assay 111
4.3. Western Blot analysis 111

4.4. Luciferase studies 113
4.5. Electroporation to K562 cells 114
4.6. Immunoprecipitation assay 115
4.7. Nuclear fractionation assay 117
4.8. HMTase assay 118
4.9. In vitro kinase assay 119
4. 10. APC ubiquitination and degradation asaay 119
4. 11. Cell cycle synchronization 120

References 122

v

List of Tables
Table 1. List of genes up-or down-regulated ≥2 fold in the absence of hSFMBT 38

Table 2. List of putative hSFMBT binding proteins from Y2H screening 42

Table 3. Dilutions of antibodies used for Western Blotting and 110
immunofluorescence experiments

vi

List of Figures
Figure 1. The MBT domains of hL(3)MBT, dSfmbt and hSFMBT are 16
conserved

Figure 2. Human SFMBT is a cell type-specific nuclear protein 18

Figure 3. The four tandem MBT repeats mediate the strong association 21
of hSFMBT with the nuclear matrix

Figure 4. All four MBT repeats of hSFMBT are required for potent 25
transcriptional repression

Figure 5. hSFMBT preferentially binds histone H3 and H4 29

Figure 6. The four MBT repeats of hSFMBT bind the N-terminal tail 32
of histone H3

Figure 7. Lack of hSFMBT leads to slow cell growth 36

Figure 8. hSFMBT functions in a multi-protein complex 40

Figure 9. hSFMBT recruits HDAC activity by interacting with HDAC1 45
and HDAC3

Figure 10. Serine 29 is the major phosphorylated residue of PR-Set7 55

Figure 11. Cdk1/cyclinB phosphorylates serine 29 of PR-Set7 58

Figure 12. PR-Set7 methyltransferase activity is not altered by serine 29 62
phosphorylation

Figure 13. Cdk1/cyclinB mediated PR-Set7 S29 phosphorylation occurs 65
at mitosis

Figure 14. PR-Set7 S29 phosphorylation occurs from prophase to anaphase 67

Figure 15. Phosphorylation of serine 29 results in removal of PR-Set7 70
from mitotic chromosomes to nuclear periphery

Figure 16. Phosphorylation of serine 29 prevents PR-Set7 degradation 72

Figure 17. S29A mutant undergoes rapid nuclear degradation 74
vii

Figure 18. PR-Set7 is ubiquitinated and degraded by APC
cdh1
E3 ligase 76

Figure 19. APC
cdh1
-mediated ubiquitination and degradation of PR-Set7 79
is directly inhibited by serine 29 phosphorylation

Figure 20. Cdc14 specifically dephosphorylate PR-Set7 serine 29 in vivo 82

Figure 21. Cdc14 specifically and directly dephosphorylate PR-Set7 84
serine 29 in vitro

Figure 22. Sustained PR-Set7 levels result in defective mitotic progression 86

Figure 23. Sustained PR-Set7 levels result in defective mitotic progression 89

Figure 24. Alterations in protein levels upon IR induced DNA damage 99

Figure 25. PR-Set7 and H4K20me1 is not detected at DNA damage foci 101
upon IR treatment

Figure 26. PR-Set7 is targeted to DNA damage foci upon micro-irradiation 103
treatment

Figure 27. 53BP1 recruitment to DNA damage foci is impaired in the 105
absence of PR-Set7

viii

Abstract
Eukaryotic chromatin, a dynamic structure composed of DNA and chromatin-
associated proteins, plays an essential role in key biological processes including gene
transcription, cell cycle progression and DNA repair. In this thesis, I wanted to gain
insights into the molecular mechanisms of these processes by focusing on: 1) a novel
human protein SFMBT-mediated gene silencing pathway; 2) PR-Set7 histone
methyltransferase-mediated cell cycle progression and 3) PR-Set7-mediated DNA
damage responses. First I present evidence here that human SFMBT (hSFMBT), a
putative Polycomb (PcG) protein, is a potent transcriptional repressor. hSFMBT
localizes to the nucleus where it associates with chromatin by directly binding to the N-
terminal tail of histone H3. Futhermore, the four tandem Malignant Brain Tumor (MBT)
repeats of hSFMBT are sufficient for nuclear matrix-association, N-terminal tail H3
binding, and required for transcriptional repression. These findings indicate that the
tandem MBT repeats form a functional structure required for biological activity of
hSFMBT. These repeats can be used to predict similar properties for other MBT domain-
containing proteins (Chapter 1). Second, the PR-Set7/Set8/KMT5a histone H4 lysine 20
monomethyltransferase is known to participate in gene silencing and to regulate
mammalian cell cycle progression, especially during G2/M. However it remained
unclear how PR-Set7 itself was regulated. I present here a novel mechanism that governs
PR-Set7 protein levels during mitosis. Perturbation of this regulation causes a defect in
mitotic progression. I found that PR-Set7 is phosphorylated at Serine 29 (S29) by the
cyclin dependent kinase 1 (cdk1)/cyclinB complex. S29 phosphorylation remains from
ix

prophase through anaphase subsequent to a global accumulation of the monomethylation
of histone H4 lysine 20 (H4K20me1). While S29 phosphorylation does not affect PR-
Set7 methyltransferase activity, it changes the location of PR-Set7 from mitotic
chromosomes to the nuclear periphery. S29 phosphorylation also stabilizes PR-Set7 by
directly inhibiting the anaphase promoting complex (APC)-mediated ubiquitination and
the subsequent degradation of PR-Set7. Furthermore, I found that Cdc14 phosphatases
dephosphorylates S29 resulting in PR-Set7 proteolysis. Since constitutive
phosphorylation of PR-Set7 resultes in delayed mitosis, the S29 dephosphorylation event
may play an essential role in mitotic progression. Collectively, I have elucidated the
molecular mechanisms that control PR-Set7 protein levels during mitosis and
demonstrated that this orchestrated regulation is important for normal mitotic progression
(Chapter 2). Finally I present evidence that PR-Set7 and its mediated H4K20me1 are
involved in ionizing irradiation (IR)-induced DNA damage responses. PR-Set7 protein
levels are elevated upon IR-induced DNA damage and it targets the damaged foci to
facilitate DNA damage foci formation for effective DNA repair (Chapter 3). Taken
together, I believe my thesis studies have provided new insights into the epigenetic
regulations of key cellular processes including gene silencing, mitotic progression and
DNA repair. These knowledges may have great impact in understanding human diseases
including cancer.

1

Introduction
Chromatin and Its Regulation
Eukaryotic genomes are assembled within the nucleus in the form of chromatin, a
structure that is composed of DNA and its associated proteins. The chromatin structure
creates a barrier to cellular machineries which need to gain access to the DNA in order
for DNA-templated processes to occur, such as gene transcription, DNA replication,
recombination or repair. The dynamic regulation of chromatin sets it into two
fundamental states, euchromatin and heterochromatin with quite different chromosomal
architecture. Euchromatin is less condensed and therefore more accessible to cellular
machineries such as transcriptional machinery, conferring its association with active
transcribed genomic regions. Heterochromatin, on the other hand, is more condensed and
therefore inaccessible to cellular machineries. Heterochromatin is typically associated
with transcriptionally silenced genes. The modulation of chromatin structure is critical
for numerous cellular processes. In unicellular organisms, most of the genes in the
genome are in a permanent active state with only a small fraction being specifically
recognized as targets for repression. By contrast, in mammals repression is a dominant
theme in regulation of gene expression with more than 50% of the genome being silenced
in any cell type throughout the development by epigenetic mechanisms (Lande-Diner and
Cedar, 2005). Normal cells have a highly coordinated epigenetic machinery to maintain
proper gene expression. For instance, DNA methylases can methylate DNA; chromatin-
modifying enzymes can modulate histones; effector proteins can bind modified DNA or
2

histones to trigger downstream events; nucleosome remodeling complexes can alter the
composition and spacing of nucleosomes.
The fundamental building block of chromatin is the nucleosome, which consists of
146 base pairs of DNA wrapping around an octamer with two copies of each core
histones H2A, H2B, H3 and H4. Each of the core histones contains two domains: a
globular domain and an unstructured N terminal tails. Numerous studies have shown that
specific amino acids on the histone tails undergo post-translational modifications
including acetylation, phosphorylation, ubiquitination, poly (ADP) ribosylation and
methylation. In addition, these histone modifications can be recognized by specific
protein modules or domains called “chromatin readers”. These proteins have binding
pockets that recognize specific histone marks and neighboring residues with high
accuracy (Taverna et al., 2007). These specific modified histone residues, together with
their binding proteins, play direct roles in essential cellular processes including gene
transcriptional regulation, DNA recombination and repair (Strahl and Allis, 2000). For
example, lysine residues on histone H4 tails can be acetylated and the acetylated lysines,
in turn, can bind the bromo domain of transcriptional co-activators to activate
transcription (Mujtaba et al., 2007). Similarly, methylation on lysine 27 of histone H3
allows the binding to the chromo domain of the polycomb protein to repress transcription
of developmentally related genes (Min et al., 2003). Also during DNA damage, the
histone variant H2AX is phosphorylated (γH2AX) and γH2AX binds the BRCT domain
of MDC1( mediator of DNA damage checkpoint protein 1) allowing effective DNA
repair (Stucki et al., 2005a). Together, these mechanisms contribute to the dynamic
3

regulation of chromatin structure and therefore are essential for cell growth, survival and
differentiation.

Histone Methylation and Histone Methyltransferase (HMTs)
Histone methylation which is mediated by histone methyltransferases (HMTs) occurs
on arginine and lysine residues of histone proteins. HMTs are a family of enzymes with
a conserved SET catalytic domain (Suppressor of variegation, Enhancer of Zeste,
Trithorax). The human genome encodes 48 SET domain-containing proteins with the
exception of DOT1L, which has HMT activity but does not contain a SET domain
(Albert and Helin, 2010). Histone lysine methylation can exist in a mono-, di- or
trimethylation state (me1, me2 and me3), catalyzed by the same or different HMTs. For
example, the mono- and dimethylation of histone H3 lysine 9 in human is mediated by
the G9a methyltransferase, whereas H3K9 trimethylation is mediated by the SUV39H1
enzyme (Peters et al., 2003; Rice et al., 2003). Similarly, monomethylation of histone H4
lysine 20 (H4K20me1) in mammals is mediated only by the PR-Set7 methyltransferase
(Couture et al., 2005; Fang et al., 2002; Nishioka et al., 2002; Xiao et al., 2005), whereas
the di- and trimethylated histone H4 lysine 20 (H4K20me2/3) is mediated by SUV4-
20H1/H2 enzymes (Schotta et al., 2004; Yang et al., 2008). In addition, different states
of lysine methylation at the same residue can be involved in distinct biological processes.
For instance, H4K20me3 is associated with repressed chromatin due to its targeting to
constitutive heterochromatin, repetitive elements and imprinting control regions (Delaval
et al., 2007; Martens et al., 2005; Schotta et al., 2004) whereas H4K20me2 is distributed
4

evenly in euchromatic regions with a potential function in 53BP1-mediated DNA repair
(Botuyan et al., 2006c; Sanders et al., 2004; Yang et al., 2008). The exact function of
H4K20me1 in transcription regulation is still controversial. In addition to regulating
transcription, several lines of evidence suggest that histone lysine methylation also
functions to maintain genome integrity and cellular identity. Recent studies from our lab
demonstrated that PR-Set7 mediated H4K20me1 is essential for mitotic entry and
genomic stability (Houston et al., 2008). Others also showed that PR-Set7 dependent
H4K20me1 is required for genome replication and S phase progression (Jorgensen et al.,
2007; Tardat et al., 2007; Yin et al., 2008).
With increasing numbers of histone demethylases (HDMs) identified, our initial view
of histone methylation as a permanent mark has changed (Yin et al., 2008; Klose et al.,
2006). Recent studies demonstrated that PHF8 is a bona fide histone H4 lysine 20
demethylase (Liu et al., 2010; Qi et al., 2010). Therefore, a tight regulation of HMTs and
HDMs is required for appropriate gene expression and other chromatin related processes.
Numerous mouse HMT knockout studies have demonstrated the importance of HMT in
embryonic development. In addition, deregulation of at least 22 out of the 49 human
HMTs have be linked to cancer occurance and other human diseases (Albert and Helin,
2010).

PR-Set7 and H4K20 Monomethylation (H4K20me1)
One of the first identified histone modifications was the methylation of histone H4
lysine 20 (H4K20) (Murray, 1964). This lysine residue can accept one, two or three
5

methyl group(s) (Thomas et al., 1975). As described above, the three states of H4K20
methylation are mediated by specific HMTs and have distinct biological consequences.
PR-Set7 is the only mammalian methyltransferase that is responsible for
monomethylation of H4K20 (Couture et al., 2005; Fang et al., 2002; Nishioka et al.,
2002; Xiao et al., 2005). PR-Set7 belongs to the SET domain histone methyltransferase
(HMTase) family. Structural studies have demonstrated that PR-Set7, through its SET
domain, specifically recognizes a stretch of amino acid sequence surrounding lysine 20 of
histone H4 (RHRK
20
VLRDN) and that this sequence is absolutely required for lysine 20
methylation (Yin et al., 2005). Besides histone substrate, PR-Set7 has been shown to
methylate p53 at lysine 382 to modulte its function (Shi et al., 2007).

Roles of PR-Set7 and H4K20me1 in Transcriptional Regulation
PR-Set7 and H4K20me1 were originally identified as being associated with silenced
chromatin. Subsequent studies demonstrated that PR-Set7-mediated H4K20me1 is
required for chromatin compaction and transcriptional repression of specific genes
involved in mammalian differentiation (Biron et al., 2004; Kalakonda et al., 2008; Sims
and Rice, 2008; Trojer et al., 2007). For instance, PR-Set7 mediated H4K20me1 can
recruit a “chromatin modifier” protein L3MBTL1 to repress the expression of specific
genes including Runx1, a critical regulator of hematopoietic differentiation (Sims and
Rice, 2008). Moreover, using Illumina expression array, out lab demonstrated that the
down-regulation of PR-Set7 and H4K20me1 by RNAi caused an approximately 2-fold
increase in all the analyzed H4K20me1-associated genes, confirming its role in gene
6

silencing (Congdon et al., 2010). However, several genome-wide analyses demonstrated
that H4K20me1 is enriched within actively transcribed genes suggesting an association
of PR-Set7 mediated H4K20me1 with actively transcribed genes (Barski et al., 2007;
Talasz et al., 2005; Vakoc et al., 2006). This discrepancy of PR-Set7 functions in
transcriptional regulation awaits future studies.

Roles of PR-Set7 and H4K20me1 in Cell Cycle Regulation
The eukaryotic cell cycle is composed of interphase including G1, S and G2, and
mitosis. During S (DNA sysnthesis) phase the DNA are replicated, and during M
(mitosis) phase the sister chromatids segregate into two identical daughter cells. The S
phase and M phase are separated by two gap phases, G1 and G2, which govern the
readiness of the cells to enter S or M phase respectively. G1 phase cells can, before
commitment to S phase, enter a resting stage called G
0.
Cyclin-dependent kinases
(CDKs), the key cell cycle regulators, are a family of serine/threonine proteain kinases
that are activated at specific stage during the cell cycle by specific cyclins. CDK protein
levels remain stable during the cell cycle whereas cyclin protein levels fluctuate to
periodically activate CDKs (Evans et al., 1983; Pines, 1991). CyclinD binds to CDK4 or
CDK6, and the CDK-cylcinD complexes are essential for G1 entry (Sherr, 1994).
CyclinE associates with CDC2 to regulate progression from G1 into S phase (Ohtsubo et
al., 1995). CyclinA binds to CDK2, and this complex is required for S phase progression
(Walker and Maller, 1991; Girard et al., 1991). In addition, CyclinA also associates with
7

CDK1 to regulate progression from G2 into M phase. Mitosis is further regulated by
cyclinB in complex with CDK1 (King et al., 1994; Arellano and Moreno, 1997).
Mitosis can be further divided into discrete stages including prophase, prometaphase,
metaphase, anaphase, telophase and cytokinesis (Pines and Rieder, 2001). Two types of
enzymes play essential roles in controlling mitotic progression: mitotic CDKs which
drive cells into metaphase and an ubiquitin ligase called anaphase promoting complex
(APC) which triggers sister chromatids segregation and mitotic exit. These CDKs
mediated phosphorylation events lead to a profound reorganization of the cell
architecture including nuclear envelope breakdown, centrosome separation, spindle
assembly and chromosome condensation (McIntosh and Koonce, 1989; Nigg et al.,
1996). These phosphorylation events are reversed by certain phosphatases, and the
delicate interplay between them determines the timely execution of mitotic events. The
APC functions as an E3 ubiquitin ligase to control the timely destruction of key cell cycle
regulators involved in anaphase onset, mitotic exit, and G1 events (Page and Hieter,
1999; Peters, 2002; Harper et al., 2002; Zachariae and Nasmyth, 1999). APC is activated
by two distinct activating proteins, Cdc20 and Cdh1. Association with Cdc20 activates
APC during metaphase to anaphase transition, while association with Cdh1 activates
APC during mitotic exit and the ensuing G1 phase. Many substrates for APC share but
are not limited to a consensus sequence motif known as Destruction box (D-box) (Fang et
al., 1998).
Studies from our lab demonstrated that PR-Set7 and its mediated H4K20me1 has a
distinct cell cycle profile in mammalian cells: low at G1, increased during the late S
8

phase and G2, and maximal from prometaphase to anaphase, suggesting a role of PR-
Set7 in cell cycle regulation (Houston et al., 2008). Indeed, several recent studies
indicated that depletion of PR-Set7 and H4K20me1 results in defective S phase
progression or G2/M progression. For example, the loss of PR-Set7 and H4K20me1
leads to defective replication fork activity and delayed S phase progression (Jorgensen et
al., 2007; Tardat et al., 2007). Our lab found that the loss of PR-Set7 and H4K20me1
causes defective mitotic entry and genomic instability with striking phenotypes including
global chromosome condensation failure, aberrant centrosome amplification and
substantial DNA damage (Houston et al., 2008). These findings provide an explanation
for the observation that gene disruption of PR-Set7 leads to embryonic lethality in both
Drosophilia and mice (Fang et al., 2002; Karachentsev et al., 2005; Nishioka et al., 2002;
Huen et al., 2008).

Roles of PR-Set7 and H4K20me1 in DNA Damage Responses
Genomic instability, a hallmark of human aging and cancer, is largely caused by
DNA damage in the form of double strand breaks (DSBs). Both exogenous DNA
damaging agents and endogenous sources including metabolites and naturally occurring
processes (DNA recombination and DNA replication) could cause DSBs. Since DSBs
are potentially lethal, eukaryotic cells have developed two major repair mechanisms to
counteract them, the homologous recombination (HR) and the nonhomologous end
joining (NHEJ). HR typically occurs during S and G2/M phases where a sister chromatid
is utilized a as the homologous template for DNA copy and restoration. This process is
9

mediated by two factors, Rad5 and RPA, which catalyze the invasion of a single strand
DNA into the homologous template for effective repair (Baumann et al., 1996). By
contrast, NHEJ repairs DSBs in cell cycle independent manner by joining two ends of
DSB via a homology-independent mechanism (Delacote and Lopez, 2008). NHEJ is an
intrinsically error-prone pathway while HR results in comparatively more accurate repair.
These two pathways, although distinct, share some commonality whereby they respond to
DSBs through quickly detecting the damage, recruiting proteins to the site, activating
checkpoints to stall cell cycle progression and ultimately fixing the damaged DNA. Since
DSBs occurs in the context of chromatin, a dynamic structure composed of DNA and its
associated histone proteins, the execution of these repair steps is orchestrated, at least in
part, by modifications of histone proteins.
Previous studies from our lab and others have demonstrated that PR-Set7 and
H4K20me1 are required to suppress aberrant DNA damage and genomic instability
(Houston et al., 2008; Huen et al., 2008; Jorgensen et al., 2007; Tardat et al., 2007).
Recent studies demonstrated that PR-Set7 is rapidly degraded upon UV-induced DNA
damage, suggesting a role of PR-Set7 in DNA damage responses (Centore et al., 2010;
Abbas et al., 2010). A very recent study further demonstrated that PR-Set7 is quickly
recruited to the laser-induced DNA damage site to monomentylate H4K20 which, in turn,
recruits 53BP1 for DNA repair (Oda, H., et al. in press). Taken together, these studies
strongly suggest that PR-Set7 and its mediated H4K20me1 play critical roles in DNA
damage responses.

10

Malignant Brain Tumor (MBT) Proteins
As mentioned above, histone modifications can be recognized with high accuracy by
specific protein modules or domains called “chromatin readers”. Among these domains,
chromo-, bromo-, WD40 and PHD fingers have been intensively studied (Ruthenburg et
al., 2007a; Sims, III and Reinberg, 2006; Taverna et al., 2007). By comparison,
Malignant Brain Tumor (MBT) domain, despite its structural similarity to chromo-,
PWWP- and Tudor domains, is less studied and poorly understood (Maurer-Stroh et al.,
2003).
The MBT domain is invariably found in tandem arrays of two to four repeats. Each
MBT repeat is composed of an N-terminal arm and a C-terminal core, with each arm
packed against the core of the adjacent repeat. Two MBT repeats form a saddle-like
interlocked structure (Sathyamurthy et al., 2003); three MBT repeats form a clover-like,
three-leaved propeller structure (Wang et al., 2003); four MBT repeats form a three-
leaved propeller with the extra MBT repeat grafted asymmetrically onto one side
(Ruthenburg et al., 2007b; Grimm et al., 2009). Although in most cases only one of the
MBT repeats of the same protein is required for binding to mono- and di-methylated
lysines, the other MBT repeats may provide structural support or recognize other ligands.
Functionally, MBT proteins can be classified into three families: 1) Proteins that are
implicated in transcriptional repression of developmental genes and maintenance of cell
identity. One example is the Drosophila SCM protein that contains two MBT repeats, a
SPM (also known as SAM, sterile alpha motif) domain and zinc fingers. 2) Proteins that
are involved in cell cycle regulation and associated with E2F/Rb complexes, such as
11

Drosophila L3MBT that contains three MBT repeats. 3) Proteins that share functions
described in both 1) and 2), such as Drosophila Sfmbt (dSfmbt) that functions as a PcG
protein and regulates the function of E2F/Rb complexes as well(Bonasio et al., 2010).

12

Chapter 1: Characterization of a novel human protein SFMBT and its
function in transcriptional repression

1.1.Introduction
The ability to regulate transcription of specific sets of genes is critical in determining
cell fate. Importantly, these transcription patterns are propagated to progeny by
epigenetic mechanisms that ensure the maintenance of cellular identity. The expression
of developmental-associated genes is largely regulated by multi-protein complexes that
either activate or repress gene transcription, which must operate in concert for proper
differentiation. The best studied regulatory complexes are Polycomb group (PcG) or
Trithorax (TRX) proteins (Ringrose and Paro, 2004). The PcG proteins, which were first
discovered in Drosophila, function to specify positional identity by creating a repressive
chromatin structure at homeotic (Hox) genes resulting in their transcriptional silencing
(Bantignies and Cavalli, 2006; Lewis, 1978). Currently there are three known PcG
complexes: PRC1, PRC2 and PhoRC (Schwartz and Pirrotta, 2007). The main
component of the PhoRC complex is Pleiohomeotic (Pho), a sequence-specific DNA-
binding protein that targets Polycomb response elements (PREs) in the genome (Brown et
al., 1998). Drosophila Pho was recently shown to heterodimerize with a novel PcG
protein, known as SFMBT (Scm-related gene containing four mbt domains); which is
required for Hox gene silencing (Klymenko et al., 2006).
13

The mammalian SFMBT was cloned seven years ago, and yet, little is known about
its biological function (Usui et al., 2000). Structurally, SFMBT protein contains four
tandem malignant brain tumor (MBT) domains and a conserved protein-interacting sterile
alpha motif (SAM), which was first identified in the PcG gene Scm and also found in
both ph and l(3)mbt (Bornemann et al., 1996). To gain new insights into the function of
human SFMBT (hSFMBT), I found that hSFMBT specifically partitions to the nucleus as
a potent transcription repressor, consistent with its putative role as a PcG protein.
hSFMBT strongly interacts with the nuclear matrix and it selectively binds to histones H3
and H4, both in vitro and in vivo. The binding occurs at the N-terminal histone tail,
suggesting that hSFMBT sequesters transcriptionally inert chromatin at the nuclear
periphery. Interestingly, I observed that all four MBT repeats were necessary and
sufficient for nuclear matrix attachment, transcriptional repression and histone binding.
In addition, the findings that all four MBT domains are required for repressor activity
indicate that the higher-order structure formed by the four MBT repeats is essential for
biological function. This is consistent with the structural characterization of the MBT
repeats in human SCML2 and L(3)MBT, where the MBT domains fold cooperatively
through interdigitation to form unique higher-order structures (Montini et al., 1999;
Sathyamurthy et al., 2003). Lastly, I found that hSFMBT is preferentially expressed in
certain cell types, suggesting that it is an important regulator of transcriptional programs
during developmental and differentiation processes (Wu et al., 2007).
Subsequent studies demonstrate that hSFMBT acts as part of a multi-protein complex
with a molecular weight around 440KDa ~ 700KDa. Yeast-Two-Hybrid experiment
14

enables the identification of putative hSFMBT binding proteins which are related to
protein translation and catalytic functions of certain enzymes. hSFMBT also recruits
HDAC activity by interacting specifically with HDAC1 and HDAC3, supporting its
involvement in transcriptional repression. By depleting hSFMBT in K562 cells, I
identified some putative target genes of hSFMBT. These genes are highly related to cell
growth and apoptosis, consistent with the finding that cells lacking hSFMBT display a
slow growth phenotype. Further investigations are needed to validate these findings and
to elucidate the molecular mechanisms underlying hSFMBT-mediated gene silencing.

1.2. Results
1.2.1. hSFMBT is a conserved Polycomb-like protein
In order to gain insights into the possible biological functions of hSFMBT, we
compared its amino acid composition and structure to other metazoan MBT-containing
proteins. The human, mouse and rat SFMBT proteins are structurally similar, where each
contains four N-terminal tandem MBT repeats and a SAM domain near the C-terminus.
While Drosophila SFMBT (dSfmbt) retains the C-terminal SAM (sterile alpha motif)
domain, its four tandem MBT repeats are located towards the C-terminus and dSfmbt
contains a zinc finger motif which is lacking in mammals (not shown). Despite these
structural differences, sequence alignments of the MBT domains of hL(3)MBT, dSfmbt
and hSFMBT revealed a substantial degree of homology within their respective repeats
(Fig. 1). A pairwise amino acide alignments of these individual repeats from the fly and
human SFMBT proteins revealed a 48% sequence homology and 34% sequence identity
15

between the two. This high degree of conservation strongly suggests that hSFMBT
functions as a Polycomb-group protein, similar to dSfmbt (Klymenko et al., 2006) .

16

Figure 1. The MBT domains of hL(3)MBT, dSfmbt and hSFMBT are conserved.

Sequences comprising the MBT repeats of each protein were obtained from the SMART
Domain Database (Schultz et al., 1998). Sequences were aligned using ClustalX
(Thompson et al., 1997) and rendered using CHROMA (Goodstadt and Ponting, 2001).
Residues sharing sequence identity within the MBT repeats are denoted with a gray
background, while conserved hydrophobic and hydrophilic residues are illustrated with
yellow and cyan backgrounds, respectively. Conserved acidic residues are red. The MBT
repeats of fly and human SFMBT are 34% identical and 48% homologous.

17

1.2.2. hSFMBT is a cell type-specific nuclear protein

Since hSFMBT is a putative PcG protein, I hypothesized that it functions as a nuclear
protein. To determine this, a GFP-tagged wild type hSFMBT was created and transiently
transfected into HeLa cells. Fluorescence microscopy of the cells revealed that GFP–
SFMBT was enriched within nuclei as demonstrated by its co-localization with nuclear
DAPI staining (Fig. 2A). In contrast, GFP protein by itself was evenly dispersed
throughout the HeLa cells. The transfected HeLa cells were also fractionated into nuclear
and cytoplasmic components, which were then subjected to Western analysis using a
GFP antibody. The results confirmed that the GFP–SFMBT fusion protein was
specifically enriched within the nuclear compartment (Fig. 2B).
To verify that endogenous hSFMBT partitioned to the nucleus as well, a polyclonal
SFMBT- specific antibody was created, characterized (Fig. 2C) and used in Western
analysis of whole cell lysates from several commonly used human cell lines.
Interestingly, hSFMBT was only detected in cettain cell types, mainly those of
hematological origin (Fig. 2D). While the highest levels of hSFMBT were detected in the
erythroblastic K562 and myeloblastic HL-60 cells, hSFMBT was also detected in the B-
cell lymphoblastic Daudi cells. In contrast, hSFMBT was barely detected in epithelial cell
lines derived from uterine (HeLa), breast (MCF7) and kidney (HEK-293) tissues.
Furthermore, K562 cells were fractionated into nuclear and cytoplasmic components for
Western analysis using the hSFMBT antibody. Similar to the GFP–SFMBT, I found that
endogenous hSFMBT was also specifically enriched within the nucleus (Fig. 2E).
18

Figure 2. Human SFMBT is a cell type-specific nuclear protein.

(A) Fluorescent microscopy of HeLa cells expressing GFP-SFMBT fusion protein or
GFP-null. GFP-SFMBT (green) specifically co-localizes with nuclear DAPI staining
(blue) whereas GFP-null is dispersed evenly through the cell. (B) Western analysis of the
nuclear (N) and cytoplasmic (C) fractions of HeLa cells expressing GFP-null or GFP-
SFMBT fusion proteins. GFP-SFMBT selectively partitions to the nuclear compartment
compared to GFP-null. (C) Peptide competition assay to determine specificity of a novel
polyclonal SFMBT antibody. Antibody was incubated with increasing amount of
immunogenic peptide (amino acids 767-780 of SFMBT; 2.5 μg, 10 μg or 25 μg; triangle)
or 25μg of a same length, non-specific peptide for 2 hours at room temperature before
probing nuclear extract from K562 cells. (D) Western analysis of whole cell lysates from
several common cell lines using the hSFMBT-specific antibody. (E) Western analysis of
the nuclear (N) and cytoplasmic (C) fractions of K562 cells using hSFMBT antibody.

19

1.2.3. hSFMBT strongly associates with the nuclear matrix

To further dissect the sub-nuclear localization of hSFMBT, nuclei were isolated from
K562 cells, as depicted in Fig. 3A, and were partially digested with micrococcal nuclease
(MNase) for 1, 4 or 16 minutes before isolating the various chromatin components by
centrifugation (Huang and Garrard, 1989; Rose and Garrard, 1984). DNA analysis
demonstrates that the MNase-sensitive soluble S1 fraction is mainly composed of mono-
and dinucleosomal sized DNA fragments, typically associated with euchromatin (Fig.
3B). In contrast, the insoluble S2 fraction is composed of MNase-resistant
oligonucleosomes, typically associated with heterochromatin, as observed by the higher
molecular weight laddering. The P fraction represents the nuclear material that remains
bound to the nuclear matrix. With increasing time of MNase digestion, more of the S2
and P fractions become soluble and shift into the S1 and S2 fractions, respectively.
Western analysis of these fractions using the hSFMBT antibody revealed that
endogenous hSFMBT selectively and strongly associates with the nuclear matrix as it
failed to shift from the P fraction even at extended MNase digestion times (Fig. 3C). As a
marker for proper fractionation, RNAP II was found to preferentially associate with the
nuclear matrix, as previously reported (Jackson and Cook, 1985) whereas the β isoform
of heterochromatin protein 1 (HP1β) was enriched in the S2 fraction but was liberated to
the S1 fraction upon extended digestion with MNase (Thiru et al., 2004). These findings
demonstrate that hSFMBT strongly interacts with the nuclear matrix.
Based on the evolutionary conservation of the four tandem MBT domains (Fig. 1), I
hypothesized that the MBT repeats were sufficient for nuclear matrix attachment. To test
20

this, a Gal4–DBD fusion construct with an hSFMBT truncation mutant containing only
four MBT repeats (4×MBT) was transfected into HEK-293 cells for fractionation
analysis (Fig. 3A). Similar to the findings for endogenous hSFMBT, the Gal4–DBD-4×
MBT fusion protein was preferentially bound to the nuclear matrix while the Gal4–DBD-
null plasmid was ubiquitously distributed amongst the fractions (Fig. 3D). Therefore,
these findings indicate that the four MBT repeats of hSFMBT are sufficient for nuclear
matrix attachment.

21

Figure 3. The four tandem MBT repeats mediate the strong association of hSFMBT
with the nuclear matrix.

(A) Flowchart of nuclear fractionation assay. Nuclei from the indicated cells were
subjected to partial micrococcal nuclease (MNase) digestion for 1, 4 or 16 minutes.
Following digestion, the soluble euchromatic fraction (S1), insoluble heterochromatic
fraction (S2) and matrix-associated fraction (P) were collected for analysis. (B) DNA
from each fraction and a 100 bp ladder (L) were electrophoresed on a 2% agarose gel.
Bands corresponding to the expected sizes of mono-, di-, tri- and oligonucleosomes are
indicated. (C) Equal volumes of each fraction were analyzed by Western blotting for
hSFMBT, RNA polymerase II (RNAP II) or heterochromatin protein 1 (HP1β). (D)
Nuclear fractionation assay was performed with HEK-293 cells transfected with either a
Gal4-DBD-4×MBT or Gal4-DBD-null plasmid. Western analysis using a Gal4-DBD
antibody indicates that the four MBT repeats are sufficient for nuclear matrix attachment.

22

1.2.4. The four MBT repreats of hSFMBT are necessary and sufficient for
transcriptional repression
Since SFMBT associates with the nuclear matrix, similar to RNAP II, I hypothesized
that SFMBT could function as a transcription co-activator. To test this, the pGK1-luc
reporter construct containing five tandem repeats of the Gal4 upstream activating
sequence (5×UAS) followed by a TATA box and luciferase reporter gene was employed
as previously described (Fig. 4A) (Lee et al., 2006). HEK-293 cells were co-transfected
with pGK1-luc and Gal4–DBD fusion constructs containing either the CARM1 co-
activator as the positive control (Chen et al., 1999), full length hSFMBT or 4×MBT; a
Gal4–DBD-null vector served as the negative control. In addition, cells were co-
transfected with the pRL reporter vector to normalize for transfection efficiency (Rice et
al., 1998). As predicted, the CARM1 co-activator increased luciferase gene expression by
sevenfold compared to control (Fig. 4A). In contrast, both Gal4–DBD-SFMBT and Gal4–
DBD-4×MBT failed to activate transcription but, instead, greatly reduced even basal
levels of luciferase expression when compared to the Gal4–DBD-null negative control.
These findings demonstrate that hSFMBT does not act as a transcription co-activator and,
instead, they suggest that hSFMBT functions as a transcriptional repressor protein.
To test if hSFMBT acts as a transcriptional repressor, the pSV40-luc reporter
construct containing five tandem repeats of the Gal4 UAS (5×UAS) followed by an SV40
promoter that constitutively activates the transcription of a luciferase reporter gene was
employed as previously described (Fig. 4B) (Ishizuka and Lazar, 2003). HEK-293 cells
were co-transfected with the pSV40-luc reporter and the Gal4–DBD fusion constructs
23

generated above. The Gal4–DBD-SMRT repressor protein served as the positive control
for repression (Zamir et al., 1997) and the pRL reporter vector was also used to normalize
for transfection efficiency. Consistent with previous reports, the SMRT repressor protein
produced a fivefold decrease in transcription of the luciferase gene compared to the
negative control (Fig. 4B). Interestingly, both Gal4–DBD-SFMBT and Gal4–DBD-
4×MBT caused a more than sevenfold and ninefold decrease in luciferase gene
expression, respectively. The failure of a FLAG-tagged full length SFMBT construct to
reduce luciferase activity in these assays confirmed that the observed repressive effects
were not indirectly due to ectopically expressed hSFMBT. These data indicate that
hSFMBT is a potent transcription repressor and that the four MBT repeats of SFMBT are
sufficient to induce repression.
To further define which of the MBT repeats is required for the observed repressive
effects of hSFMBT, truncation mutants of the Gal4–DBD-4×MBT fusion construct were
created, as depicted in Fig. 4C, and used in the repression assays. Initial studies of
constructs lacking the two MBT repeats closest to the N-terminal (ΔMBT1,2) or C-
terminal (ΔMBT3,4) resulted in a complete loss of repression. Therefore, I speculated
that the two central MBT repeats were required for repression. However, these two
repeats (ΔMBT1,4) alone were not sufficient to restore repression, suggesting that an
additional MBT repeat flanking either the N-terminal (ΔMBT4) or C-terminal (ΔMBT1)
was also required. Interestingly, the lack of a single N- or C-terminal MBT domain also
resulted in a complete loss of repression. The lack of repression could not be due to
differences in the expression levels of the fusion proteins (Fig. 4C bottom). Collectively,
24

these findings indicate that all four MBT repeats of hSFMBT are required for potent
transcriptional repression.

25

Figure 4. All four MBT repeats of hSFMBT are required for potent transcriptional
repression.
(A) In co-transfected HEK-293 cells, the Gal4-DBD fusion protein binds the Gal4
Upstream Activating Sequence (UAS) of the pGK1-luc reporter construct and promotes
luciferase transcription if it functions as a co-activator, such as CARM1. Comparative
quantitative measurements were made by normalizing to renilla activity and plotting the
fold change in luciferase activation relative to the Gal4-DBD-null negative control. (B) In
the repression assay, the Gal4-DBD fusion protein binds the Gal4 UAS of the pSV40-luc
reporter construct, which constitutively drives expression of luciferase, and decreases
luciferase transcription if it functions as a repressor, such as SMRT. Comparative
quantitative measurements were made as described above and the fold decrease in
luciferase activity was plotted relative to the Gal4-DBD-null negative control. (C)
Deletion of any single MBT repeat restores luciferase transcription indicating that all four
MBT repeats of hSFMBT are required to induce repression. Comparable protein levels of
all the truncation mutants are shown at the bottom.

26

Figure 4: Continued

27

1.2.5. hSFMBT binding to histone H3 and H4 is mediated by the four MBT
repreats

Recent structure studies suggested that the MBT domain closely resembles the
chromodomain of HP1 and Pc (Sathyamurthy et al., 2003; Wang et al., 2003), which
selectively bind methylated histone H3 lysine 9 and 27, respectively (Fischle et al., 2003;
Jacobs and Khorasanizadeh, 2002; Min et al., 2003). Consistent with this, Drosophila
SFMBT was shown to bind to the mono- and dimethylated forms of histones H3 lysine 9
and H4 lysine 20 in vitro (Klymenko et al., 2006). To explore the possibility that human
SFMBT could bind histones in vivo, the Gal4–DBD-4×MBT or Gal4–DBD-null
constructs were transfected into HEK-293 cells and immunoprecipitated with a Gal4–
DBD antibody (Fig. 5A). Western analysis of the bound material indicates that the four
MBT repeats of SFMBT could specifically bind endogenous histones H3 and H4 with a
preference for H3. This was a specific interaction as Gal4–DBD-4×MBT failed to
immunoprecipitate UBC9, an ubiquitously expressed protein (Wilson and Rangasamy,
2001).
To determine if hSFMBT directly binds histones, GST pull-down assays were
performed using either purified recombinant GST, GST-SFMBT or GST-4×MBT and
acid-extracted histones from HEK-293 cells. While GST alone failed to pull-down
histones, both GST-SFMBT and GST-4×MBT bound core histones with higher
selectivity for H3 compared to H4, consistent with the in vivo findings (Fig. 5B). Both
constructs also bound histones H2A and H2B, but to varying degrees and with far less
affinity compared to H3 and H4. Histone H1 was not detected in any of the pull-down
experiments. To confirm that the observed in vitro interaction was specific to histones,
28

the experiments were repeated using GST-4×MBT with either myelin basic protein
(MBP) or bovine serum albumin (BSA) – two highly positively charged proteins that
mimic histones (Farbiszewski and Rzeczycki, 1975; Moskaitis et al., 1987). In both
cases, GST-4×MBT failed to pull-down MBP or BSA (Fig. 5C). Collectively, these
findings indicate that the four MBT repeats of hSFMBT directly and specifically bind
histones H3 and H4 in vivo and in vitro.

29

Figure 5. hSFMBT preferentially binds histone H3 and H4.

(A) The Gal4-DBD immunoprecipitated material from HEK-293 cells transfected with a
Gal4-DBD-null or Gal4-DBD-4×MBT plasmid was analyzed by Western blotting. (B)
GST pull-down experiments with 100pmol of recombinant GST, GST-SFMBT or GST-
4×MBT incubated with 50μg of HEK-293 acid-extracted histones. Coomassie staining
following SDS-PAGE demonstrates that the four MBT repeats are sufficient for direct
binding to histones H3 and H4. (C) GST pull-down experiments of recombinant GST-
4×MBT with either myelin basic protein (MBP) or bovine serum albumin (BSA).

30

1.2.6. hSFMBT binds the N-terminal tail of histone H3

To determine the region of histone H3 responsible for interacting with the MBT
repeats of hSFMBT, GST pull-down experiments were performed using purified
recombinant GST or GST-4×MBT on beads with either acid-extracted histones or “tail-
less” histones isolated from trypsinized HEK-293 oligonucleosomes (Fletcher and
Hansen, 1995). Western analysis of the bound fractions using a general H3 antibody
revealed that the four MBT repeats of hSFMBT failed to bind the H3 histone-fold region.
This finding suggests that the N-terminal tail is required for the interaction (Fig. 6A).
To confirm this observation, co-immunoprecipitations were performed in HEK-293
cells expressing the full length FLAG-SFMBT construct with an N-terminal GST fusion
construct containing the N-terminal tail of human H3 ( the first 41 amino acids; H3 1–
41aa) (An and Roeder, 2003). Western analysis of the FLAG-SFMBT
immunoprecipitats demonstrated that the GST–H3 1–41 fusion protein, but not GST
alone, bound to hSFMBT (Fig. 6B top and middle). As negative controls for this
experiment, a FLAG-null immunoprecipitates failed to bind GST–H3 1–41, and the
GST–H3 1–41 fusion protein did not bind the FLAG beads (Fig. 6B bottom). This data
indicated that SFMBT specifically interacts with the N-terminal tail of H3. To confirm
these observations conversely, the experiment was repeated with glutathione-sepharose
beads instead of FLAG beads on the same lysates. Western analysis of the GST–H3 1–41
immunoprecipitated material verified binding to FLAG-SFMBT (Fig. 6C). This was a
specific interaction as the GST–H3 1–41 failed to immunoprecipitate UBC9.
31

Collectively, these finding demonstrate that the four MBT repeats of hSFMBT
specifically bind the N-terminal tail of histone H3.

32

Figure 6. The four MBT repeats of hSFMBT bind the N-terminal tail of histone H3.

(A) Acid-extracted histones or “tail-less” histones prepared from trypsinized
nucleosomes were used as substrates for GST-null and GST-4×MBT pull-down
experiments. Western analysis of the bound material using a general histone H3 antibody
demonstrates that 4×MBT binding requires the N-terminal tail of H3. (B) HEK-293 cells
expressing FLAG-SFMBT with either GST or GST-H3 1−41 were immunoprecipitated
with anti-FLAG agarose beads. The input and bound materials were analyzed by Western
blotting. (C) Immunoprecipitations for GST were performed on the cell lysates in (B).
Western analysis was performed to confirm that the first 41 amino acids of H3 bind
hSFMBT.

33

To further explore whether the interaction between hSFMBT and histone H3 tail is
mediated by specific modifications on histone H3 tail, we applied two approaches: 1)
Fluorescence Anisotropy (FA). This assay can measure the kinetic interactions between
the protein of interest and fluorescently labeled modified histone peptides in solution
(Fischle et al., 2003). In collaboration with Dr. Wolfgang Fischle, we explored the
interactions of 4×MBT with a panel of differentially modified histone peptides; 2)
Peptide Microarray. This assay can measure the binding affinity between protein of
interest coated on slide and biotinylated modified histone peptides in an ELISA-like
format (Kim et al., 2006). This experiment was performed in collaboration with Dr. Mark
Bedford. Unfortunately, initial studies using both methods failed to display significant
discriminations of hSFMBT in recognizing modified peptides (data not shown),
suggesting that hSFMBT binds histone H3 tails in a modification-independent manner.

34

1.2.7. Identification of hSFMBT target genes in K562 cells
Our findings suggested that hSFMBT is recruited to specific genomic regions to
repress transcription. Since hSFMBT is abundant in K562 cells, I decided to define
hSFMBT target genes in K562 cells by RNAi technology. pGIPZ lentiviral vectors
containing GFP and shRNA bi-cistronic transcript were electroporated into K562 cells.
GFP positive cells were selected by Fluorescence Activated Cell Soring (FACS) and
analyzed for the down-regulation of hSFMBT. Results of a representative experiment are
shown in Figure 7A. In comparison to the Non-Silencing shRNA control (NS), two
hSFMBT-specific shRNAs (shRNA3 and shRNA6) decreased mRNA levels of hSFMBT
by four fold and six fold, respectively. Importantly, I observed decreased hSFMBT
protein levels in these cells. Interestingly, the cells lacking hSFMBT grew more slowly
than control cells. To confirm this observation, K562 cells electroporated with NS
shRNA or shRNA6 were FACS sorted and kept growing for 2, 3 or 4 days. Cells were
trypan blue stained and the unstained live cells were counted. As shown in Figure 7B, NS
shRNA cells showed a normal proliferation rate, whereas shRNA6 cells displayed a
significantly slower growth. The decreased cell numbers suggestse that, instead of
growing, the cells underwent cell death. To verify this result using a different cell line,
HeLa cells were transfected with NS shRNA or shRNA6, and the cell numbers were
counted 1 or 2 days after transfection. Consistent with the observation in K562 cells,
shRNA6 treated HeLa cells displayed a slower proliferation rate as compared to NS
shRNA cells. Since the transfection efficiency was about 70% in HeLa cell, we argued
that the sustained growth of shRNA6 cells was attributed to the non-transfected cells.
35

Taken together, these data provide evidence that hSFMBT plays a positive role in
controlling cell growth, although the mechanism remains to be investigated.

36

Figure 7. Lack of hSFMBT leads to slow cell growth.

(A) Representative graph showing that both mRNA and protein levels of hSFMBT
decreased in K562 cells after shRNA treatment. K562 cells were electroporated with
Non-silencing shRNA (NS) or two hSFMBT-specific shRNA (shRNA3 and shRNA6)
constructs. On the next day, GFP positive cells were sorted by FACS and kept growing in
media. Samples were collected at different days for analysis. Arrow indicates the band
for hSFMBT. (B) Cell growth of shRNA treated K562 cells was monitored at 2, 3 or 4
days after electroporation. The error bars represent standard deviation from three
independent replicates. (C) Cell growth was measured in HeLa cells transfected with NS
shRNA or shRNA6 constructs.

37

HumanRef-8 Expression Beadchip (Illumina) which contains eight arrays of > 24,000
probes derived from RefSeq database was applied to identify hSFMBT target genes in
K562 cells. Total RNA was extracted from either untreated, NS shRNA-treated or
hSFMBT shRNA treated K562 cells (as described above). However, the integrity of the
RNA extracted from NS shRNA-treated cells failed to reach the standards and therefore
could not be used. In the eight-array Beadchip, three arrays were used for detecting
RNAs from untreated K562 cells; another three arrays were used for detecting RNAs
from shRNA3-treated cells of independent triplicate experiments, and the rest two arrays
were used for detecting RNAs from shRNA6-treated cells of independent duplicate
experiments. The results indicated that in three out of the five experimental arrays,
hSFMBT was down-regulated by 40%. Therefore, these three arrays, referred to as A, B
and C, were used for final data analysis. Compared to controls, expression of 114 probes
changed in A, B and C with p<0.001. Surprisingly, among these genes only 38% were
up-regulated in the absence of hSFMBT, contradictory to our findings that hSFMBT
functions as a transcriptional repressor. Table 1 displays all the genes which are up-
regulated or down-regulated by ≥2 fold in the absence of hSFMBT. Real-Time PCR
needs to be conducted to further verify these putative target genes.

38

Table 1. List of genes up- or down-regulated ≥ 2 fold in the absence of hSFMBT.
RNAs extracted from hSFMBT shRNA K562 cells or untreated K562 cells (control) were
applied to a HumanRef-8 Expression Beadchip (Illumina). Shown above is a list of
genes that were up-regulated or down-regulated ≥2 fold in the absence of hSFMBT.

39

1.2.8. Identification of hSFMBT interacting proteins
My previous study suggested that hSFMBT functions as a potential PcG protein. PcG
proteins are known to bind to other proteins to form multi-protein complexes and their
binding proteins regulate the function of the PcG proteins. Therefore, it is important to
identify binding proteins in order to understand how hSFMBT functions in vivo. To this
end, I first applied conventional chromatography using a Superdex 200 column to
determine the molecular weight of the endogenous hSFMBT complex from K562 nuclear
extract. As shown in Figure 8A, hSFMBT was eluted in the fractions between the size of
440 KDa and 700 KDa, indicating that hSFMBT is part of a multi-protein complex as
monomer hSFMBT is around 130KDa.
Two approaches were applied to define hSFMBT binding proteins: 1) Tandem Tag
Immunoaffinity Purification (TAP). By creating a stable cell line that contains a tandem
tagged FLAG-HA-hSFMBT fusion protein, I can purify hSFMBT complex by epitope-
mediated two-step purifications. The purified proteins can be identified by mass
spectrometry; 2) Yeast-Two-Hybrid (Y2H). Full length hSFMBT can be used as bait
protein to “fish out” potential binding proteins from a mammary epithelial derived cDNA
library. To generate a FLAG-HA-hSFMBT containing stable cell line for TAP assay, a
lentivirus plasmid containing FLAG-HA-hSFMBT was created and used to infect K562
cells followed by puromycin drug selection. Unfortunately, multiple experiment trials
failed due to the lack of hSFMBT expression in the drug-selected cells. Besides technical
problems, we argued that over-expression of hSFMBT could be lethal to cells. Therefore
it may be impossible to create a cell line stably over-expressing hSFMBT.
40

.

Figure 8. hSFMBT functions in a multi-protein complex.

(A) K562 nuclear extract was fractionated by Superdex 200 column chromatography and
analyzed by Western blotting using hSFMBT specific antibody. Protein bands
corresponding to 440KDa and 700KDa were determined by running protein standards.
hSFMBT from K562 cells is part of a 440~700KDa multi-protein complex. (B) Western
analysis of AH109 yeast cells transfected with Myc-tagged pGBKT7-null, SFMBT or
PR-Set7. pGBKT7-SFMBT was properly expressed and therefore could be used in Y2H
system. pGBKT7-PR-Set7 serves as positive control.

41

As an alternative, Y2H was performed. Y2H system is a powerful tool to identify
novel protein-protein interactions. This system requires a bait protein which is fused to
GAL4 DNA binding domain (GAL4-BD) and a prey protein fused to GAL4 DNA
activation domain (GAL4-AD). When the bait and prey fusion proteins directly interact,
the DNA-BD and AD are brought into a close proximity leading to the activation of
specific reporter genes controlled by GAL4-responsive upstream activating sequences
(UASs). Y2H was performed using MATCHMAKER GAL4 Two-Hybrid System 3
(Clonetech). This system allows either medium or high stringency selection for positive
interactions. The bait protein was created by inserting full length hSFMBT into yeast
expression plasmid pGBKT7-GAL4-BD. Figure 8B indicated that pGBKT7-SFMBT was
properly expressed in AH109 yeast cells, therefore could be used in the Y2H system.
Next pGBKT7-SFMBT was co-transfected into AH109 strains with human mammary
epithelium cDNA library, and the most stringent growing condition/media was used to
screen for positive clones. The screening revealed over 100 colonies indicating strong
interaction with hSFMBT. Table 2 displays a selection of the putative binding proteins,
and amongst them there are two major groups. One group (#2-10) is composed of
proteins closely related to protein translation including translational initiation and
elongation factors; the other group (#11-18) contains proteins which have enzymatic
activities including ubiquitin enzyme UBC9 and several kinases. This study suggested
that hSFMBT may be involved in translational processes and/or participate in regulating
certain enzyme’s catalytic functions.

42

Table 2. List of putative hSFMBT binding proteins from Y2H screening.
Y2H was performed using pGBKT7-SFMBT as bait protein against a mammary
epithelial derived cDNA library. 100 Positive clones were selected from the most
stringent media and sequenced. This table contains a selection of the putative binding
proteins. Clone Number marked with * indicates ≥ 2 reads of the same protein suggesting
true positive. These proteins were categorized into four groups based on their functions
and separated by grey bars.

43

1.2.9. hSFMBT is associated with HDAC activity
Histone acetyltransferase enzymes (HATs) and histone deacetyltransferase enzymes
(HDACs) function cooperatively to regulate the reversible acetylation status on lysine
residues of histone proteins. HATs are responsible for acetylating histones resulting in
transcriptional activation. By contrast, HDACs deacetylate histones leading to
transcriptional repression. Therefore the association of a protein with either HATs or
HDACs can shed light on its potential function in either transcriptional activation or
repression. My previous studies indicate that hSFMBT is a potent transcription repressor.
However the expression microarray data showing that 62% genes were down-regulated in
the absence of hSFMBT suggesting its possible role in gene activation. To solve this
discrepancy, I wanted to investigate the potential interaction of hSFMBT with HATs or
HDACs.
In vitro HAT assay was performed using HAT kit (Active Motif). FLAG-HA-
SFMBT was transiently expressed in HeLa cells and immunoprecipitated using anti-
FLAG agarose beads. The bound material was eluted off beads by FLAG peptides and
quantified by Bradford protein quantification assay. 0.1μg or 1μg immunoprecipitated
material was incubated with Acetyl-CoA and histone H3 peptide. The reaction was
stopped by adding stop solution, and after adding the developing solution, the developer
reacted with free sulfhydryl groups on the CoA-SH to give a fluorescent reading of
acetyltransferase activity. Therefore, elevated fluorescence reading indicates HAT
activity. As shown in Figure 9A, p300 histone acetyltransferase, served as a positive
control, had a high fluorescence reading at 0.1μg indicating a strong HAT activity, while
44

neither SFMBT nor GFP (as negative control) induced any significant changes in
fluorescence reading even with 1μg immunoprecipitates. Thus, SFMBT complex does
not appear to contain any HAT activity. Next, in vitro HDAC assays were carried out
using HDAC kit (Active Motif) to determine the possible association of hSFMBT with
HDACs. A short peptide substrate containing an acetylated lysine residue that can be
deacetylated by all classic HDACs was used as substrate. Once the substrate is
deacetylated, the lysine residue reacts with the developing solution and releases the
fluorophore resulting in a fluorescent product that can be easily measured by fluorescent
plate reader. As shown in Figure 9B, HeLa Nuclear Extract (HNE), the positive control
for this assay, displayed a dose-dependent increase in fluorescence reading, while GFP,
the negative control, only showed a trace amount of fluorescence reading even at higher
dose. Interestingly SFMBT also displayed a dose-dependent fluorescence reading, similar
to the trend of HNE indicating a strong HDAC activity. This result suggested that
hSFMBT may recruit HDAC activity via interaction with members from HDAC family.
To define which HDAC interacts with hSFMBT, co-immunoprecipitation assays using
anti-HA agarose beads were performed in HEK-293 cells expressing HA-SFMBT and
FLAG- HDAC1, -2, -3, -10 and -11, which come from three “classic” HDAC classes (see
Introducation). As shown in Figure 9C, Western analysis of the HA-SFMBT
immunoprecipitates revealed that SFMBT binds preferentially to HDAC1 and HDAC3,
but not HDAC2, -10 or -11. SFMBT did not bind non-specifically to FLAG beads either.
This study indiates that hSFMBT recruits HDAC activity by interacting with HDAC1 and
HDAC3.
45

Figure 9. hSFMBT recruits HDAC activity by interacting with HDAC1 and
HDAC3.

(A) FLAG-HA-SFMBT was transfected into HeLa cells and anti-FLAG argarose beads
were used to immunoprecipitate FLAG-HA-SFMBT. 0.1μg or 1μg of the
immunoprecipitated materials were applied to in vitro HAT assay using H3 peptide as
substrate. p300 histone acetyltransferase serves as positive control, and FLAG-HA-GFP
serves as negative control. (B) 1μg, 3μg or 10μg of the immunoprecipitated materials
prepared from (A) were applied to in vitro HDAC assays using a short peptide containing
acetylated lysine as substrate. HeLa Nuclear Extract (HNE) serves as positive control,
and FLAG-HA-GFP serves as negative control. (C) Immunoprecipitation assays were
performed using anti-HA agarose beads in HEK293 cells co-transfected with HA-
SFMBT and FLAG-HDAC1, -2, -3, -10, -11 or empty null. The input and bound
fractions were separated by SDS-PAGE followed by Western blotting using indicated
antibodies.

46

1.3. Discussion and future directions
Although Drosophila SFMBT was recently found to be a PcG protein required for
Hox gene repression, the biological function of human SFMBT remained unknown
(Klymenko et al., 2006). In this chapter, I have demonstrated for the first time that
hSFMBT is a nuclear matrix-associated protein that acts as a potent transcription
repressor. The finding that hSFMBT binds the N-terminal tail of histone H3 suggests
that this interaction is required for targeting hSFMBT to specific chromatin regions
destined for repression. Due to its functional similarity to dSfmbt, it is likely that
hSFMBT also plays a role in PcG-mediated Hox gene repression. However, the gene
expression microarray data from K562 cells failed to reveal any Hox genes as hSFMBT
targets. Instead, several putative hSFMBT target genes are involved in cell proliferation
and apoptosis, such as GADD45B and FOSB (Table1). This is consistent with the
observation that cells lacking hSFMBT displayed a slow growth phenotype (Fig. 7B, C).
For future studies, I propose:
1) Verify the bona fide hSFMBT targets by conventional Real-Time-PCR using
specific primers to selected genes from Table1.
2) Once the target genes are determined, Chromatin Immunoprecipitation assays
(ChIP) can be performed to validate that hSFMBT protein is physically targeted
to this genomic regions.
The amino acid sequence comparison between human and drosophila SFMBT
revealed a high degree of conservation in the four tandem MBT repeats, suggesting that
this region plays a critical role in protein function. Consistent with this hypothesis, we
47

found that the four tandem MBT repeats of hSFMBT were necessary and sufficient for
nuclear matrix-association, histone binding and transcriptional repression. Importantly,
the lack of any one of the four MBT domains resulted in the abolishment of repressor
activity indicating that all four repeats are required to form a functional structural unit
that is necessary for biological activity. These findings are consistent with the crystal
structure of hL(3)MBT where each of its three MBT repeats formed tight globular
modules that interdigitate to create a novel three-leaved propeller-like structure (Wang et
al., 2003). Based on these findings, it is likely that the four tandem MBT repeats of
hSFMBT also create a novel propeller-like structure that is required for functional
activity. One possible role of this structure is to interact with other members of a putative
multi-protein complex. This is likely since most PcG family members are part of larger
multimeric chromatin-associated complexes (Satijn and Otte, 1999; Simon and Tamkun,
2002). Consistent with this, we found endogenous hSFMBT in a multi-protein complex
with molecular weight of 440KDa ~700KDa (Fig. 8A). To determine hSFMBT binding
proteins, I performed Y2H assay and found about 50 putative binding proteins (Table2).
The first four listed proteins showed up in multiple interactions suggesting true bindings
to hSFMBT, therefore I would start with these proteins. For future studies, I propose to:
1) Verify the binding between the putative binding proteins and hSFMBT in AH109
yeast strain. The plasmid DNA containing putative binding protein, referred to as
protein X, will be co-transformed into AH109 with bait protein pGBKT7-
SFMBT. The interaction between them can be verified by colonies formed on the
most stringent selection media.
48

2) Verify the binding in mammalian cells. K562 cells or HeLa cells will be co-
transfected with epitope tagged SFMBT plasmid and plamid containing putative
binding protein X. Immunoprecipitation assays will be performed to test their
interaction.
3) Further define the minimum domains in hSFMBT and protein X required for
efficient binding between them. First, truncation mutants will be created in
hSFMBT and tested in in vitro binding assays with full length protein X. Second,
truncation mutants will be generated in protein X and tested in in vitro binding
assays with full length hSFMBT.
4) Investigate the biological significance of the binding between hSFMBT and
protein X in terms of hSFMBT-mediated gene silencing pathway. This can be
achieved by depleting cells of protein X followed by examining the expression
levels of hSFMBT target genes. In addition, protein X mutant which lacks the
minimal required binding region (as determined by 3)) can be created and over-
expressed in cells to disrupt the interaction of hSFMBT and protein X.
Expression levels of hSFMBT target genes will be investigated.
Another possible role of the MBT domains of hSFMBT is to bind to specific
modified residues on histone proteins. It has been reported that the MBT domain in
hL(3)MBT protein binds dimethylated H4 lysine 20; and the MBT repeats in dSfmbt
protein preferentially bind the mono- and di-methylated forms of H3 lysine 9 as well as
H4 lysine 20 in vitro (Kim et al., 2006; Klymenko et al., 2006). However, our initial
studies from FA and peptide microarray showed that hSFMBT does not have any
49

preference for modified histone peptides tested. Studies have demonstrated that the ligand
binding pockets of the Chromo and Tudor domains bind the methylated lysine via a
hydrophobic cage created by conserved aromatic residues within the motif (Botuyan et
al., 2006b; Jacobs and Khorasanizadeh, 2002). Indeed, the aromatic residues were also
found in the MBT repeats of hL(3)MBT and dSfmbt. By contrast, these aromatic residues
are not identified in any MBT repeat of hSFMBT protein, which possibly explained the
finding that hSFMBT binds histone tail in a modification-independent manner.
I discovered that hSFMBT recruits HDAC activity by association specifically with
HDAC1 and HDAC3 supporting its role as a transcriptional repressor. This can lead to
further interesting studies including:
1) Determine the nature of the interaction between hSFMBT and HDACs. In vitro
binding assays will be conducted to determine whether the interaction is direct or
indirect by using purified hSFMBT with HDACs. If hSFMBT directly binds
HDACs, truncation mutants in hSFMBT and HDACs will be created and used in
binding assays to map the minimal required binding regions.
2) Once the binding regions are defined, I would verify it by Co-IP in cells
expressing the truncation mutants.
3) Characterize the function of HDACs in hSFMBT-mediated gene silencing
pathway. This can be achieved by down-regulating HDACs in cells and
monitoring hSFMBT target gene expression levels. I would expect to be able to
detect the up-regulation of some of the target genes in the absence of HDACs.
50

4) Further determine the histone lysine residues which are deacetylated by HDACs
at hSFMBT target genes. This can be achieved by performing Chromatin
Immunoprecipitation (ChIP) assays in HDAC down-regulated cells using a panel
of histone acetylation specific antibodies, such as H3Ac14, H4Ac16, H4Ac12,
H4Ac8 and H4Ac5.

51

Chapter 2: Characterization and functional analysis of the
phosphorylation of PR-Set7 histone methyltransferase

2.1.Introduction
Eukaryotic cell division is tightly regulated by a sequential of events. During mitosis,
there are three essential mechanisms that govern these events: i) the phosphorylation of
key substrates mediated by the cyclin-dependent kinases (CDKs); ii) their subsequent
dephosphorylation by certain phosphatases; and iii) the degradation of target proteins
mediated by the anaphase promoting complex (APC), an E3 ubiquitin ligase (Sullivan
and Morgan, 2007). The mitotic CDKs drive early mitosis by phosphorylating critical
downstream cell cycle regulators required for chromosome condensation, nuclear
envelope breakdown and mitotic spindle assembly. During late mitosis, the
dephosphorylation of CDK substrates by certain phosphatases is critical for chromosome
and spindle movements, spindle disassembly and nuclei reformation. The APC complex
governs cell progression into anaphase and beyond by ubiquitinating key substrates,
including mitotic cyclins and securin, resulting in their proteasome-mediated degradation
which triggers chromosome segregation (Peters, 2006; Thornton and Toczyski, 2006).
Therefore, proper mitotic progression depends on the sequential steps of phosphorylation,
dephosphorylation and degradation of the key cell cycle regulatory proteins. Although
several of these proteins have been identified, the discovery of novel proteins regulated
by this pathway will gain new insights into the fundamental mechanisms of the
eukaryotic cell cycle.
52

We have previously co-discovered a histone-modifying enzyme, PR-
Set7/Set8/KMT5a, that specifically monomethylates histone H4 lysine 20 (H4K20me1)
(Fang et al., 2002; Nishioka et al., 2002). Subsequent studies demonstrated that PR-Set7-
mediated H4K20me1 is required for chromatin compaction and transcriptional repression
of specific genes including some genes involved in mammalian differentiation (Biron et
al., 2004; Kalakonda et al., 2008; Sims and Rice, 2008; Trojer et al., 2007). Recent
studies indicate that PR-Set7 also plays an essential role in mammalian cell cycle
progression (Houston et al., 2008; Huen et al., 2008; Jorgensen et al., 2007; Karachentsev
et al., 2005; Tardat et al., 2007; Yin et al., 2008). For instance, loss of PR-Set7 results in
a G2 arrest in human cells, and the PR-Set7-/- mice are embryonic lethal arresting at the
8-cell stage (Houston et al., 2008; Oda et al., 2009). During the cell cycle, PR-Set7
protein levels are regulated as well, with the lowest levels observed during S-phase and
the highest at G2/M. This observation suggests that the regulation of PR-Set7 is
important for proper cell division, although the underlying mechanisms responsible for its
regulation remained unknown.
Since the X. laevis orthologue of PR-Set7 was previously described to be a mitotic-
specific phosphoprotein, we hypothesized that PR-Set7 phosphorylation is a critical event
for its regulation during mitosis (Stukenberg et al., 1997). In this study, I found that
serine 29 (S29) of PR-Set7 is a major target of phosphorylation and it is mediated
specifically by the cdk1/cyclinB complex during prophase to early anaphase. While S29
phosphorylation does no alter the methyltransferase activity of PR-Set7, it causes the
removal of PR-Set7 from mitotic chromosomes to the extrachromosomal space. In
53

addition, S29 phosphorylation stabilizes PR-Set7 during mitosis by directly inhibiting
APC-mediated ubiquitination and the subsequent proteosome-mediated degradation of
PR-Set7. Furthermore, the rapid dephosphorylation of PR-Set7 in anaphase by the Cdc14
phosphatases is required for PR-Set7 degradation and proper mitotic progression.
Collectively, our findings elucidate a novel pathway that governs PR-Set7 regulation and
demonstrates that these events are required for normal cell division.

2.2. Results
2.2.1. Serine 29 is a major phosphorylated residue of human PR-Set7
To determine if PR-Set7 is phosphorylated in human cells, total cell lysates from the
kidney cell line HEK-293 were processed using a Qiagen Phosphoprotein Column which
specifically retains phosphorylated proteins. Western analysis was performed on both the
column-bound protein fraction and the unbound flow-through using a PR-Set7 specific
antibody. PR-Set7 was strongly detected in as little as 1% of the total bound material,
whereas the ubiquitously expressed UBC9 protein was detected only in the flow-through
(Fig. 10A). Similar experiments conducted in the K562 human myeloid cell line
confirmed that PR-Set7 is phosphorylated in human cells. To validate these findings, a
FLAG-tagged PR-Set7 plasmid was ectopically expressed in HEK-293 cells and the cell
lysates were processed using a Qiagen Phosphoprotein Column as described above.
Western analysis using a FLAG antibody confirmed that the FLAG-PR-Set7 protein is
detected in the column-bound fraction and, therefore, is a target for phosphorylation (Fig.
10B)
54

The X. laevis orthologue of PR-Set7 was previously shown to be phosphorylated
specifically during mitosis suggesting that the PR-Set7 phosphorylation is related to
mitotic kinase(s) (Stukenberg et al., 1997). Sequence analysis of PR-Set7 revealed that
the sequence surrounding serine 29 perfectly matches the consensus sequence for the
cyclin dependent kinase 1 (cdk1)/cyclinB complex (S/T-P-X-K/R) (Fig. 10C). To
confirm that S29 is a target for phosphorylation in vivo, a FLAG-tagged PR-Set7 S29A
mutant plasmid was transfected into HEK-293 cells and the cell lysates were processed
using the Qiagen Phosphoprotein Column as described above. In contrast to the wild
type FLAG-PR-Set7, Western analysis revealed that the majority of the FLAG-PR-Set7
S29A mutant protein was found in unbound flow-through (Fig. 10B). These findings
indicate that S29 is the major phosphorylated site in PR-Set7 and suggests that this
phosphorylation event is mediated by cdk1/cyclinB. In addition, phylogenetic analysis of
PR-Set7 demonstrated that this consensus sequence is highly conserved among different
species suggesting a functional significance for this phosphorylation event (Fig. 10C).

55

Figure 10. Serine 29 is the major phosphorylated residue of PR-Set7.

(A) Phosphorylated proteins were isolated from HEK-293 cells or K562 cells using a
phosphoprotein purification column. The indicated amounts of the input, column bound
and unbound material were fractionated by SDS-PAGE prior to Western analysis. (B)
HEK-293 cells expressing FLAG-PR-Set7 or S29A mutant proteins were processed using
phosphoprotein purification columns. Western analysis was performed using the
indicated amounts of the input, column bound and unbound material. (C) Peptide
sequences of PR-Set7 were aligned from the indicated animals using ClustalX.
Conserved cdk1/cyclinB consensus sequence (S-P-X-K/R) and APC recognition motif D-
box are illustrated.

56

2.2.2. Cdk1/cyclinB complex specifically and directly phosphorylates serine 29 of
PR-Set7
To determine if the cdk1/cyclinB complex is responsible for PR-Set7 S29
phosphorylation, three independent experimental approaches were taken. First, to
determine if cdk1 or casein kinase I (CKI) (which shares the same consensus site)
phosphorylates S29 in vivo, HeLa cells were treated for 2 hours with a cdk1 specific
inhibitor (CGP74514A), a CKI inhibitor (D4476) or DMSO vehicle control. Western
analysis of the cell lysates using a newly created rabbit polyclonal antibody that detects
PR-Set7 only when S29 is phorphorylated (pS29-PR-Set7) (Fig. 11A) revealed that pS29-
PR-Set7 was drastically reduced in the CGP74514A-treated cells compared to vehicle
control (Fig. 11B). In contrast, no visible reduction in phosphorylated S29 was observed
in the presence of D4476 even with extended treatment times (data not shown). These
findings indicate that cdk1, but not CKI, is responsible for PR-Set7 S29 phosphorylation
in vivo.
To investigate if cyclinB is the bona fide regulatory factor for cdk1-mediated
phosphorylation of PR-Set7 S29, HeLa cells were transfected with a control shRNA or
two shRNA constructs that target different regions of cyclinB (cyclinB_1 and cyclinB_2).
Western analysis of the cell lysates revealed that only the cyclinB_2 shRNA could
deplete cells of cyclinB whereas the cyclinB_1 shRNA had little effect (Fig. 11C).
Importantly, a marked decrease of pS29-PR-Set7 in the cyclinB_2 shRNA cells was
observed but not in the shRNA control or cyclinB_1 shRNA cells. These data indicate
57

that the cdk1/cyclinB complex is specifically required for PR-Set7 S29 phosphorylation
in vivo.
Lastly, to determine if cdk1/cyclinB can directly and specifically phosphorylate S29,
in vitro kinase assays were performed using recombinant full length, N- or C-terminal
truncations of PR-Set7 (aa1-191 and 191-352, respectively) as substrates in the presence
or absence of purified cdk1/cyclinB. The full length and N-terminal portion of PR-Set7,
but not C-terminal, were phosphorylated in the presence of cdk1/cyclinB (Fig. 11D). To
determine if S29 was the major phosphorylation site of cdk1/cyclinB, a PR-Set7 N-
terminal S29A mutant was created and used as the substrate in the kinase assay.
Consistent with our results above, the S29A mutant protein failed to be phosphorylated
by cdk1/cyclinB in vitro (Fig. 11D). Importantly, CKI failed to phosphorylate PR-Set7
S29 despite sharing the consensus sequence of S29 (Fig. 11E). Collectively, these
findings demonstrate that cdk1/cyclinB directly and specifically phosphorylates S29 of
PR-Set7.

58

Figure 11. Cdk1/cyclinB phosphorylates serine 29 of PR-Set7.
(A) Characterization of the pS29-PR-Set7 specific antibody. Western analysis was
performed using the PR-Set7 antibody or pS29-PR-Set7 antibody on cell lysates from
HEK-293 cells transfected with either FLAG-PR-Set7 (lane 1) or FLAG-PR-Set7 S29A
mutant (lane 2), HEK-293 nuclear extracts (NE; lane 3) were also analyzed following
treatment with calf intestine alkaline phosphatase (CIP) (lane 4). Increasing amounts of
unmodified recombinant PR-Set7 (lanes 5 and 6) were used as negative controls. (B)
Western analysis for PR-Set7 and pS29-PR-Set7 on lysates from cells treated with
vehicle DMSO, the cdk1 inhibitor CGP74514A or the casein kinase I (CKI) inhibitor
D4476. A general H4 antibody was used to control for loading. (C) Western analysis
using the indicated antibodies were performed on cells transfected with a control shRNA
or two different cyclinB shRNA plasmids. (D) Recombinant wild type PR-Set7 (WT),
N-terminal (aa1-191) or C-terminal (aa192-352) truncations were used as substrates in in
vitro kinase assays with purified cdk1/cyclinB. The reactions were fractionated by SDS-
PAGE followed by autoradiography. (E) Western analysis of in vitro kinase assays using
wild type PR-Set7 as the substrate and either cdk1/cyclinB or CKI.

59

Figure 11: Continued

60

2.2.3. Phosphorylation of serine 29 does not affect PR-Set7 methyltransferase
activity
Since PR-Set7 functions to monomethylate histone H4K20, we hypothesized that S29
phosphorylation may affect PR-Set7 methyltransferase activity. To test this possibility,
in vitro histone methyltransferase (HMT) assays were performed using
3
H-SAM as the
radiolabeled methyl donor, HeLa histones or nucleosomes as substrates and either full
length recombinant PR-Set7 or in vitro phosphorylated PR-Set7 as methyltransferase
(Fig. 12A). Methyl incorporation was measured by autoradiography and scintillation
counting. As shown in Fig. 12A, core histones were not methylated by PR-Set7 as it is a
nucleosome-specific methyltransferase (Nishioka et al., 2002). Nucleosomal substrates
were methylated by S29-phosphorylated PR-Set7 but displayed no visible or significant
differences compared to non-phosphorylated PR-Set7. To confirm this result, HeLa cells
were transfected with either a FLAG-tagged wild-type PR-Set7, the S29A mutant or an
R265G catalytically dead mutant (CD). Purified FLAG immunoprecipitates were
examined for HMT activity using nucleosome as substrate. Similar to the above findings,
the immunoprecipitates from PR-Set7 wild-type and S29A mutant displayed no
significant differences of H4 methylation whereas the control and PR-Set7 CD
immunoprecipitates failed to methylate H4 (Fig. 12B). These findings indicate that S29
phosphorylation has no direct effect on PR-Set7 enzymatic function. To ensure that S29
phosphorylation would not indirectly affect PR-Set7 enzymatic activity in cells, HeLa
cells were immunostained with an H4K20me1-specific antibody following incubation
with the cdk1 inhibitor (CGP74514A) or DMSO vehicle control. Similar staining levels
61

of H4K20me1 were detected between the two samples at all phases in the cell cycle (Fig.
12C). Collectively, these findings demonstrate that cdk1/cyclinB-mediated
phosphorylation of S29 does not significantly alter PR-Set7 enzymatic function.

62

Figure 12. PR-Set7 methyltransferase activity is not altered by serine 29
phosphorylation.
(A) Histone methyltransferase (HMT) assays were performed using recombinant PR-
Set7 or in vitro phosphorylated PR-Set7 on core histone or nucleosomal substrates and
analyzed by autoradiography or scintillation counting. Error bars represent standard
deviation generated from three independent biological replicates. (B) The indicated
FLAG-PR-Set7 fusion proteins were immunoprecipitated prior to an HMT assay using
nucleosomal substrates and analyzed by autoradiography or scintillation counting. Error
bars represent standard deviation generated from three independent biological replicates.
(C) Cells treated with vehicle DMSO or the cdk1 inhibitor CGP74514A were
immunostained with an H4K20me1-specific antibody (red) and counterstained with
DAPI (blue).

63

Figure 12: Continued

64

2.2.4. Cdk1/cyclinB-mediated phosphorylation of PR-Set7 S29 occurs from
prophase to anaphase
Since the active cdk1/cyclinB complex is formed in late G2 and degraded in anaphase
during mitosis, we speculated that cdk1/cyclinB-mediated PR-Set7 S29 phosphorylation
occurs during these times. To test this, HeLa cells were synchronized at G1/S by a
thymidine-mimosine double block, released and collected for Western analysis at specific
time points in the cell cycle (Fig. 13A). Consistent with our previous reports (Rice et al.,
2002), PR-Set7 and H4K20me1 were lowest at S-phase but gradually increased during
cell cycle progression, reaching maximal levels at late G2 (Fig. 13B top panel). In
contrast to total PR-Set7, pS29-PR-Set7 was undetectable through S-phase, although
small amounts could be observed at longer exposures (Fig. 13B bottom panel), visible at
G2 followed by a dramatic increase during M that was rapidly reduced as the cells exited
mitosis. Therefore, the majority of cdk1/cyclinB -mediated S29 phosphorylation occurs
specifically during mitosis subsequent to the accumulation of PR-Set7 and global H4K20
monomethylation in G2.

65

Figure 13. Cdk1/cyclinB mediated PR-Set7 S29 phosphorylation occurs at mitosis.

(A) Fluorescence activated cell sorting (FACS) of HeLa cells chemically arrested at
G1/S, released and collected every 2 hours. The x-axis represents DNA content and the
y-axis represents the number of cells counted. Extrapolation of the peaks in
asynchronously dividing cells (not shown) indicates that an x-axis value of 60 represents
2N and 120 represents 4N. Quantitative real time PCR was performed on RNA extracted
from cells at each time point for PR-Set7 and Lamin A/C (control) gene expression.
Values were generated by normalizing PR-Set7 expression to Lamin A/C expression and
plotted as the fold change relative to the 0 hour time point. The error bars represent
standard deviation from three technical replicates. Student T test was performed by
comparing time point 8hr, 10hr, 12hr or 14hr to time point 6hr. P= 0.06 (8hr), 0.1 (10hr),
0.8 (12hr) and 0.6 (14hr). (B) Western analysis using a panel of antibodies on cells from
(A). Longer exposure of the same films was shown below.

66

To detail the precise phases of mitosis when PR-Set7 S29 phosphorylation occurs,
HeLa cells were immunostained with PR-Set7 or pS29-PR-Set7 antibodies and
counterstained with DAPI to visually determine cell cycle phase. Consistent with the
other findings, pS29-PR-Set7 staining was restricted to mitotic cells (Fig. 14A) compared
to PR-Set7 (Fig. 14B). These images also revealed that pS29-PR-Set7 S29 was detected
from prophase to early anaphase, but was greatly reduced by late anaphase and telophase
(Fig. 14A a-e). Importantly, in the presence of the cdk1 inhibitor (CGP74514A), pS29-
PR-Set7 was not detected in mitotic cells (Fig. 14A). Therefore, cdk1/cyclinB -mediated
phosphorylation of PR-Set7 S29 occurs predominantly from prophase to early anaphase.

67

Figure 14. PR-Set7 S29 phosphorylation occurs from prophase to anaphase.

(A) Immunostaining using the pS29-PR-Set7 antibody (red) or PR-Set7 antibody (B; red)
in the presence of vehicle DMSO or cdk1 inhibitor CGP74514A. Counterstaining with
DAPI (green as pseudo color) was used to identify cells in interphase (a), prophase (b),
metaphase (c), early anaphase (d) and telophase (e). Scale bar is 20 μm. (C) Zoomed in
images from (B;e) anaphase cells.

68

2.2.5. PR-Set7 is removed from mitotic chromosomes following serine 29
phosphorylation
Since PR-Set7 is targeted to specific chromosomal regions to monomethylate histone
H4K20, it remained unclear why pS29-PR-Set7 was paradoxically localized to the
extrachromosomal space during metaphase and early anaphase (Fig. 14A, c and d). Since
the bulk of H4K20 monomethylation had already occurred at G2 (Fig. 13B), we
hypothesized that the cdk1/cyclinB -mediated phosphorylation of PR-Set7 S29 may
function to remove PR-Set7 from chromosomes during mitotic progression. If this were
the case, then unphosphorylated PR-Set7 should be localized specifically to mitotic
chromosomes. To test this, HeLa cells were either treated with the cdk1 inhibitor
(CGP74514A) to block phosphorylation of S29 or DMSO vehicle control before staining
for PR-Set7. While the majority of PR-Set7 was localized to the nuclear periphery
during mitosis in the control cells, similar to pS29-PR-Set7 (Fig. 14A), PR-Set7 was
detected mainly on the chromosomes of mitotic cells in the absence of S29
phosphorylation (Fig. 14B; magnified by increasing contrast shown on the right). The
reduction in PR-Set7 staining intensity in mitotic cells was most likely due to the
abnormal enhanced degradation of PR-Set7 (see below).
To confirm these findings biochemically, HeLa nuclei were partially digested with
micrococcal nuclease (MNase) for 2, 4 or 8 minutes before isolating the various
chromatin fractions (Fig. 15A) (Wu et al., 2007). The MNase-sensitive soluble S1
fraction is composed of mono- and dinucleosomes typically associated with euchromatin
while the insoluble S2 fraction is composed of MNase-resistant oligonuclesomes
69

typically associated with heterochromatin (Fig. 15B). The pellet (P) contains the material
that remains bound to the nuclear matrix. With increased MNase digestion time, more of
the S2 and P fractions become soluble and shift towards the S1 and S2 fractions.
Western analysis of these fractions revealed that PR-Set7 was enriched more within the
heterochromatic S2 fraction than the P fraction but was not detected in the euchromatic
S1 fraction, even at the longer MNase digestion times (Fig. 15C). In contrast, pS29-PR-
Set7 was present and remained highly associated with the P fraction at extended MNase
digestion times indicating that pS29-PR-Set7 is preferentially enriched within the nuclear
matrix. Based on these findings, we speculated that PR-Set7 detected within the P
fraction was mainly composed of pS29-PR-Set7. To test this, the cells were treated with
the cdk1 inhibitor (CGP74514A) prior to a 4 minute MNase digestion and biochemical
fractionation. Western analysis of these fractions demonstrated that the absence of S29
phosphorylation resulted in a near ablation of PR-Set7 in the P fraction (Fig. 15C).
Collectively, these data indicate that S29 phosphorylation results in the removal of PR-
Set7 from mitotic chromosomes to the nuclear periphery.

70

Figure 15. Phosphorylation of serine 29 results in removal of PR-Set7 from mitotic
chromosomes to nuclear periphery.

(A) Flowchart of nuclear fractionation used to isolate euchromatin (S1), heterochromatin
(S2) or insoluble chromatin (P). (B) Nuclei were digested with micrococcal nuclease
(MNase) for the indicated times prior to fractionation and DNA electrophoresis. The
bands correspond to the expected sizes of mono-, di- tri- and oligonucleosomes. (C)
Western analysis of the different MNase digested fractions from cells treated with or
without the cdk1 inhibitor CGP74514A.

71

2.2.6. Phosphorylation of serine 29 prevents PR-Set7 degradation
It was consistently observed that upon inhibition of S29 phosphorylation, there was a
decreased total PR-Set7 level, suggesting that phosphorylation of S29 mastabilizes PR-
Set7 by preventing its degradation (Fig. 11B, 11C and 14B). To test this, the turnover of
wild type PR-Set7 was first examined in HeLa cells by monitoring its protein levels after
inhibiting translation of new proteins using cyclohexamide (CHX). Western analysis
demonstrated that PR-Set7 protein levels diminished rapidly within one hour of CHX
treatment, however, PR-Set7 degradation was ablated in the presence of the MG132
proteosome inhibitor (Fig. 16A). Interestingly, longer exposure of the Westerns revealed
that degradation of PR-Set7 reached a plateau around 5 hour CHX treatment (Fig. 16B).
Based on our hypothesis above, we speculated that the remaining PR-Set7 would be
phosphorylated at S29. Western analysis performed on the same lysates demonstrated
that pS29-PR-Set7 levels remained unchanged even after seven hours of CHX treatment
(Fig. 16B). These results indicate that the unphosphorylated PR-Set7 is rapidly degraded
whereas pS29-PR-Set7 is highly resistant to degradation. It also implies that the
remaining PR-Set7 at the 5-7 hour CHX plateau is pS29-PR-Set7. To determine if
phosphorylation of S29 is required for preventing PR-Set7 degradation, HeLa cells were
treated for 2 hours with the DMSO vehicle control or the cdk1 inhibitor (CGP74515A)
that depletes phosphorylated S29 prior to the addition of CHX (Fig 16C). While the rate
of PR-Set7 degradation in the DMSO control cells was similar to the wild type cells, the
absence of phosphorylated S29 resulted in a visible decrease in PR-Set7 within 45
minutes of CHX treatment; PR-Set7 was undetectable by 90 minutes.
72

Figure 16. Phosphorylation of serine 29 prevents PR-Set7 degradation.

(A) Western analysis of cells treated with cyclohexamide (CHX) for the indicated time
points in the presence or absence of MG132. A general H4 antibody was used as a
loading control. (B) Western analysis of cells treated with CHX for the indicated time
points. (C) Cells were treated with vehicle DMSO or cdk1 inhibitor CGP74514A prior to
CHX treatment. Cells were collected at the indicated time points for Western analysis.

73

To confirm these findings, HeLa cells were transfected with a GFP-tagged wild PR-
Set7, an S29A mutant or an S29D, which mimics phosphorylated serine, mutant plasmid
and visualized by fluorescence microscopy. As expected, both the wild type GFP-PR-
Set7 and S29D mutant displayed intense nuclear accumulation. By contrast, the S29A
signal was significantly reduced and was excluded from the nucleus (Fig. 17A and 17B).
Based on previous results, we speculated that the lack of S29A mutant nuclear
accumulation was caused by its proteasome-mediated degradation. Indeed, treatment of
the cells with the MG132 proteosome inhibitor rescued nuclear accumulation of GFP-PR-
Set7 S29A while MG132 had little to no effect on WT or S29D nuclear localization (Fig.
17C). Collectively, these findings indicate that phosphorylated S29 prevents the nuclear
degradation of PR-Set7.

74

Figure 17. S29A mutant undergoes rapid nuclear degradation.

(A) Cells transfected with GFP-PR-Set7 wild type, an S29A mutant or an S29D phospho-
mimic mutant were counterstained with DAPI and visualized by fluorescence
microscopy. (B) Quantitative analysis of GFP proteins localization from (A). 200 cells
per slide were blindly counted and plotted. (C) GFP-PR-Set7 S29A, WT or S29D
transfected cells were treated with vehicle DMSO or MG132 proteosome inhibitor and
visualized by fluorescence microscopy.

75

2.2.7. Serine 29 phosphorylation directly inhibits APC
cdh1
-mediated ubiquitination
and degradation of PR-Set7
Western analysis of the FLAG-tagged PR-Set7 fusion proteins revealed a high
molecular weight smear detected only in the S29A mutant compared to the wild type
suggesting that it was preferentially ubiquitinated; this was confirmed using an ubiquitin
antibody (Fig. 18A). This finding strongly suggested that the rapid degradation of PR-
Set7 following anaphase was most likely due to ubiquitin-mediated proteolysis. Since
the anaphase promoting complex (APC) is an E3 ubiquitin ligase required for proteolysis
of key mitotic regulators and completion of mitosis, we hypothesized that APC was
responsible for PR-Set7 ubiquitination (Sullivan and Morgan, 2007). Sequence analysis
revealed that PR-Set7 contains a conserved D-box flanking S29, a stretch of amino acids
recognized by APC, suggesting that APC ubiquitinates PR-Set7 (Fig. 10C). To test this
in vitro, recombinant N-terminal PR-Set7 which contains the D-box was incubated with
the purified APC
cdh1
complex, the predominant E3 ligase during anaphase (Kramer et al.,
1998). Western analysis revealed a ladder of higher molecular weight PR-Set7 with
increasing incubation times consistent with APC
cdh1
-mediated ubiquitination (Fig. 18B).
To determine the effect of ubiquitination on PR-Set7 degradation,
35
S-labeled PR-Set7
was incubated for different times with early G1 HeLa S3 cell extracts which contains
highly active APC
cdh1
complex. Autoradiography demonstrated that
35
S-PR-Set7 was
rapidly degraded whereas the addition of the APC inhibitor Emi1 greatly reduced PR-
Set7 degradation (Fig. 18C). Collectively, these findings indicate that proteolysis of PR-
Set7 is mediated by the APC
cdh1
E3 ubiquitin ligase.
76

Figure 18. PR-Set7 is ubiquitinated and degraded by APC
cdh1
E3 ligase.

(A) The indicated FLAG-PR-Set7 fusion proteins were immunoprecipitated from cells
followed by Western analysis. The asterisk indicates poly-ubiquitinated PR-Set7. (B)
Recombinant N-terminal PR-Set7 (aa1-191) was incubated with the purified APC
cdh1

complex at the indicated time points prior to Western analysis. Increasing degrees of PR-
Set7 ubiquitination are shown. (C) Autoradiography of
35
S-labeled wild type PR-Set7
incubated with HeLa G1 cell extracts at the indicated time points in the presence or
absence of the APC inhibitor Emi1.

77

Based on our observations above, we hypothesized that phosphorylation of S29 could
directly inhibit APC
cdh1
-mediated degradation of PR-Set7. To test this,
35
S-labeled wild
type PR-Set7, the S29D phospho-mimic mutant or the S29A mutant were incubated with
the G1 HeLa cell extracts for increasing periods of time. Autoradiography revealed that
the PR-Set7 S29A mutant was rapidly degraded, similar to wild type PR-Set7 (Fig. 19A).
However, proteolysis of PR-Set7 S29D mutant was nearly abolished, similar to Emi1-
treated PR-Set7. Since Cdh1 activates APC E3 ubiquitin ligase, we speculated that over-
expression of this protein could constitutively activate APC resulting in decreased PR-
Set7 levels. To test this, PR-Set7 levels were measured in HeLa cells over-expressing
Cdh1 (Fig. 19B). As expected, bulk PR-Set7 levels significantly decreased indicating a
rapid Cdh1 mediated degradation. Importantly, the S29 phosphorylated form of PR-Set7
remained unaltered, consistent with the findings that phosphorlated PR-Set7 interferes
with the APC
cdh1
-mediated degradation. It also implied that the remaining PR-Set7 upon
Cdh1 over-expression is mainly composed of pS29-PR-Set7. Since Cdh1 directly targets
specific substrates for APC
cdh1
-mediated ubiquitination, we speculated that the non-
degradable S29D mutant was caused by its decreased binding affinity for Cdh1 (Fang et
al., 1998). To test this, FLAG-tagged PR-Set7 wild-type or S29D mutant was co-
transfected into HeLa cells with an HA-tagged Cdh1. Western analysis of the epitope
tagged Cdh1 immunoprecipitates revealed a robust interaction with wild-type PR-Set7
but not S29D mutant (Fig. 19C). To confirm this observation conversely, anti-FLAG
conjugated beads were used to immunoprecipitate FLAG-tagged PR-Set7. Western
analysis of the immunoprecipitates indicated that Cdh1 was preferentially associated with
78

wild-type PR-Set7, with barely detectable binding to S29D mutant (Fig. 19D).
Collectively, these findings demonstrate that phosphorylation of S29 directly inhibits
APC
cdh1
-mediated ubiquitination and degradation of PR-Set7.

79

Figure 19. APC
cdh1
-mediated ubiquitination and degradation of PR-Set7 is directly
inhibited by serine 29 phosphorylation.
(A) Autoradiography of
35
S-labeled wild type PR-Set7, the S29A or S29D phospho-
mimic mutants incubated with cell extracts from G1-arrested HeLa cells at the indicated
time points in the presence or absence of the APC inhibitor Emi1. (B) Western analysis
of HeLa cells expressing HA-Cdh1. (C) HeLa cells co-expressing FLAG-PR-Set7 wild-
type or S29D mutant with HA-Cdh1 were immunoprecipitated using anti-HA agarose
beads. Western analysis of the input and bound material was performed using the
indicated antibodies. (D) HeLa cells co-expressing FLAG-PR-Set7 wild-type or S29D
mutant with HA-Cdh1 were immunoprecipitated using anti-FLAG agarose beads.
Western analysis of the input and bound material was performed using the indicated
antibodies.

80

Figure 19: Continued

81

2.2.8. Cdc14 directly dephosphorylate PR-Set7 serine 29

The findings above indicate that an unknown phosphatase is required to
dephosphorylate S29 following anaphase for rapid APC
cdh1
-mediated ubiquitination and
degradation of PR-Set7. The Cdc14 phosphatase preferentially dephosphorylates
proteins that are modified by proline-directed kinases, such as cdk1/cyclinB, and plays a
key role in mitotic exit suggesting that it may dephosphorylate PR-Set7 (Kaiser et al.,
2002; Mailand et al., 2002; Gray et al., 2003; Cho et al., 2005). Mammalian cells express
two Cdc14 isoforms, Cdc14A and Cdc14B whose specific substrates are poorly defined
(Li et al., 1997). To investigate the roles of Cdc14A or Cdc14B in dephosphorylating
S29, an HA-tagged PR-Set7 was co-transfected into HeLa cells with either FLAG-tagged
Cdc14A, Cdc14B or their corresponding phosphatase dead point mutants (PD). Western
analysis of epitope tagged PR-Set7 immunoprecipitates demonstrated that both Cdc14A
and B robustly dephosphorylated S29 in contrast to the PD mutants or the negative
control PTEN phosphatase (Fig. 20A). Consistent with these findings, Western analysis
of lysates from HeLa cells ectopically expressing the FLAG-tagged Cdc14 proteins
confirmed significant reductions in endogenous PR-Set7 S29 phosphorylation compared
to the PD mutants and PTEN phosphatase (Fig. 20B). Interestingly, a slight reduction in
total PR-Set7 protein levels was reproducibly detected in the wild type FLAG-Cdc14
lysates consistent with the findings above demonstrating that loss of S29 phosphorylation
results in PR-Set7 degradation.

82

Figure 20. Cdc14 specifically dephosphorylate PR-Set7 serine 29 in vivo.

(A) HeLa cells co-expressing HA-PR-Set7 with FLAG-Cdc14A, Cdc14B or their
corresponding phosphotase-dead mutants (PD) were immunoprecipitated using anti-HA
agarose beads. The beads bound material and the input were analyzed by Western
blotting (left panel). HeLa cells co-expressing FLAG-PR-Set7 and HA-PTEN were
immunoprecipitated using anti-FLAG agarose beads. The beads bound material and the
input were analyzed by Western blotting (right panel). (B) Western analysis of HeLa
cells expressing various FLAG-Cdc14 constructs or HA-PTEN.

83

Two approaches were taken to determine if Cdc14 could directly dephosphorylate
S29 of PR-Set7. Firstly, epitope tagged Cdc14 proteins were immunoprecipitated and
incubated with in vitro phosphorylated N terminal recombinant PR-Set7. As shown in
Fig. 21A, both Cdc14A and Cdc14B robustly reduced the phosphorylation levels of PR-
Set7. By contrast, the phosphatase-dead mutants failed to diminish PR-Set7 S29
phosphorylation. Next, to confirm the above finding, in vitro-translated (IVT) Cdc14A
and Cdc14B plasmids and their corresponding phosphatase dead point mutants (PD) were
incubated with in vitro phosphorylated recombinant N-terminal PR-Set7 (Fig. 21B).
Western analysis of the reactions demonstrated that both Cdc14A and B ablated S29
phosphorylation of PR-Set7 in contrast to the PD mutants and the negative control PTEN
phosphatase. These findings indicate that the Cdc14 phosphatases directly
dephosphorylate PR-Set7 S29.

84

Figure 21. Cdc14 directly dephosphorylate PR-Set7 serine 29 in vitro.

(A) In vitro dephosphorylation assays were performed using immune-purified FLAG-
Cdc14 proteins from HeLa cells with in vitro phosphorylated N terminal PR-Set7. The
reactions were separated on SDS gel followed by autoradiography or Coomassie staining.
(B) In vitro dephosphorylation assays were performed using in vitro translated (IVT)
FLAG-Cdc14 proteins or HA-PTEN protein with in vitro phosphorylated N terminal PR-
Set7. The reactions were separated on SDS-PAGE followed by Western Blotting or
Coomassie staining.

85

2.2.9. Constitutive PR-Set7 serine 29 phosphorylation impedes early mitotic
progression

The findings above suggest that PR-Set7 S29 dephosphorylation by Cdc14 and its
subsequent degradation may be important for proper mitotic progression; similar to what
was observed for other key cell cycle regulatory proteins. Therefore we reasoned that the
constitutive phosphorylation of PR-Set7 would inhibit its degradation resulting in
defective mitosis. To test this hypothesis, HEK-293 cells were transfected with either
FLAG-tagged wild type PR-Set7, the non-degradable S29D phosphomimic or the null
control plasmid prior to nocodazole treatment to arrest cells at metaphase. Cells were
then released into fresh media for increasing time points and the cell cycle progression
was analyzed by flow cytometry (Fig. 22A) and plotted (Fig. 22B). Within 30 minutes
following release into fresh media, the null and PR-Set7 wild type cells could be detected
entering G1. By 60 minutes, 15% of the synchronized null and wild-type PR-Set7 cells
had progressed to G1. In stark contrast, progression of the PR-Set7 S29D phosphomimic
mutant cells was significantly delayed; 3-fold less of these cells entered G1 at this time
point compared to wild type PR-Set7. At later times following release, however, the PR-
Set7 S29D cells progressed to G1 with similar kinetics as the null and PR-Set7 wild type
cells. These findings indicate that expression of PR-Set7 S29D induces a significant
mitotic delay, most likely by inhibiting entry to anaphase.

86

Figure 22. Sustained PR-Set7 levels results in defective mitotic progression.
(A) Representative graph of HEK293 cell cycle analysis by flow cytometry. HEK-293
cells transfected with empty vector, FLAG-PR-Set7 or FLAG-PR-Set7 S29D mutant
were nocodazole arrested at metaphase, released for 30, 60, 90 or 120 minutes before
ethanol fixation and propidium iodide staining to measure DNA content. The x-axis
represents DNA content and the y-axis represents the number of cells counted. Cells at
G1 phase and G2/M phase are indicated in each graph. (B) Quantitative analysis of cells
from (A). x-axis indicates time points, and y-axis indicates cells entering G1 phase. The
error bars represent standard deviation from three independent replicates. The Student t-
test was used to determine statistical significance (*p<0.05).

87

Figure 22: Continued

88

To further investigate the mitotic delay, identical experiments were performed in
HeLa cells in conjunction with live cell imaging. Thirty cells entering prophase were
visually identified from each group and photographed at 2 minute intervals to record
mitotic progression. Within 10-12 minutes, all cells had successfully achieved
prometaphase (Fig. 23A). The null and PR-Set7 wild type cells began to transition to
anaphase within an average of ~40 minutes (Fig. 23B). Although many of the PR-Set7
S29D cells displayed a similar pattern, we observed a distinct sub-population of cells (8
of 30) that exhibited a prolonged prometaphase/metaphase (Fig. 23A). Nearly three-times
more of the PR-Set7 S29D cells failed to enter anaphase within 60 minutes compared to
the control cells. Once anaphase was achieved, however, the PR-Set7 S29D cells
progressed with similar kinetics as the control cells (data not shown). Collectively, these
findings indicate that constitutive phosphorylation of PR-Set7 results in a substantial
delay to anaphase entry strongly suggesting that the dynamic regulation of PR-Set7 is
important for proper mitotic progression.

89

Figure 23. Sustained PR-Set7 levels results in defective mitotic progression.
(A) Phase contrast images of released HeLa expressing the indicated proteins were
recorded at 2 minute intervals by live cell imaging. Prophase cells were identified (t=0).
All cells achieved prometaphase by 10 minutes. The null and PR-Set7 wild type cells
exited metaphase (t=48) and progressed through anaphase (t=52) to cytokinesis (t=58).
The PR-Set7 S29D cell was delayed in early mitosis exiting metaphase at 126 minutes
but progressed normally thereafter. (B) Time in prophase through metaphase (y-axis) was
determined for 30 cells from each group (x-axis; black circles). Median values (open
circles) and standard deviation are indicated. (C) Proposed model for PR-Set7 regulation
during cell cycle progression. Black line represents DNA, grey circles are nucleosomes
and the pink circles depicts monomethylated H4K20 (H4K20me1). Following DNA
replication, PR-Set7 accumulates at G2 to methylated H4K20 at specific heterochromatic
loci. During prophase through metaphase, cdk1/cyclinB phosphorylates S29.

90

Figure 23: Continued

91

2.3. Discussion and future directions
This study illuminates the molecular mechanisms that govern the dynamic regulation
of PR-Set7 during mitosis and demonstrates that the orchestrated regulation of PR-Set7 is
required for the correct timing of mammalian cell cycle progression. Previous reports and
our new findings have led us to propose a model of how PR-Set7 is regulated (Fig. 23C).
PR-Set7 protein levels dramatically fluctuate during cell cycle progression with the
lowest levels observed during S phase before peaking at G2/M (Rice et al., 2002). While
this could be partially explained by modest changes in PR-Set7 transcription (Fig. 13A),
our results indicate that PR-Set7 is regulated predominantly at the protein level based on
its short half-life. Consistent with this, we found that PR-Set7 is ubiquitinated by APC
cdh1

during mitosis resulting in proteolysis of PR-Set7. Interestingly, a recent study also
determined that PR-Set7 ubiquitination by SCF
skp2
and subsequent degradation of PR-
Set7 at G1 was associated with S-phase entry (Yin et al., 2008). While the sustained
decreased levels of PR-Set7 through S-phase could be due to SCF
skp2
, it remains a formal
possibility that other E3 ubiquitin ligases may also participate in the degradation of PR-
Set7 during S-phase progression. These findings strongly suggest that the down
regulation of these ubiquitin ligases is largely responsible for the gradual but dramatic
nuclear accumulation of PR-Set7 observed at G2.
During G2 we demonstrated that PR-Set7 is targeted primarily to specific
heterochromatic regions of the genome resulting in histone H4 lysine 20
monomethylation (H4K20me1) (Congdon et al., 2010). Importantly, PR-Set7-mediated
H4K20me1 is required for cell cycle progression as ablation of H4K20me1 results in a
92

G2 arrest. The few cells that escape this arrest are marked by global decondensed
chromatin associated with aberrant chromosomal segregation defects that could result in
aneuploidy and oncogenesis (Karachentsev et al., 2005; Houston et al., 2008). It was
previously shown that the MBT repeats of the L3MBTL1 protein bind H4K20me1
resulting in chromatin condensation in vitro (Trojer et al., 2007) and mutations in these
MBT repeats correlates with mitotic defects in Drosophila (Yohn et al., 2003). In
addition, a recent report showed that N-CAPD3 and N-CAPG2, subunits of the condensin
II complex, also selectively bind H4K20me1 via their HEAT repeats (Liu et al., 2010).
These findings strongly suggest that PR-Set7-mediated H4K20me1 during G2/M
functions primarily to recruit chromatin condensation-promoting complexes, such as
condensin II and L3MBTL1, to ensure the proper timing of mitotic progression. Although
these hypotheses have yet to be experimentally validated, our data clearly demonstrates
that the accumulation of PR-Set7 on mitotic chromosomes is required for H4K20me1 and
normal mitotic progression.
During prophase through early anaphase, we discovered that PR-Set7 is
phosphorylated specifically at S29 by the cdk1/cyclinB complex. We observed that one
striking consequence of S29 phosphorylation was the removal of PR-Set7 from mitotic
chromosomes, although the mechanisms responsible for this remain unknown. There are
several possibilities including that S29 phosphorylation could physically decrease the
affinity of PR-Set7 for chromatin and/or the phosphorylation of S29 could create a
binding site for an unidentified protein complex that actively transports PR-Set7 from
chromosomes. Alternatively, the phosphorylation of S29 could potentially inhibit PR-
93

Set7 interaction with an unknown protein complex required for its recruitment to
chromatin. To identify these putative proteins, I propose to:
1) Create a tandem tagged PR-Set7 wild-type or S29D mutant plasmid, for example
FLAG-HA-PR-Set7 WT or S29D. Two-step purifications using anti-FLAG
agarose beads followed by anti-HA agarose beads can be performed in cells
expressing FLAG-HA-PR-Set7 WT or S29D mutant. The purified complexes will
be analyzed by mass spectrometry to define proteins which binds only to S29D
mutant but not wild-type PR-Set7. One potential pitfall of this experiment is that
the S29D mutant may not function exactly the same as phosphorylated S29
despite sharing the negative charges.
2) Alternatively, a biotinylated peptide surrounding S29 residue with S29 being
phosphorylated or not phosphorylated (as control) would be generated. These
peptides can be immobilized to Streptavidin beads followed by incubation with
HeLa cell extracts. The bound proteins can be eluted and analyzed by mass
spectrometry. One challenge of this approach is to optimize the length of the
peptides required for binding to its partners.
3)
One question raised in this study is the biological significance of the dismissal of PR-
Set7 off mitotic chromosomes. One possibility is that the removal of PR-Set7 from
chromatin may be required to expose H4K20me1 for binding to chromatin modifiers.
Previous studies in Drosophila demonstrated that chromatin maturation requires
dl(3)MBT which recognizes and directly binds H4K20me1 followed by recruiting a
94

histone deacetylase dRPD3 to the binding site (Scharf et al., 2009). Recent findings also
suggested that during mitotic progression H4K20me1 needs to be exposed in order to
recruit cohesion complex (Liu et al., 2010). Another appealing possibility is that the
nuclear periphery PR-Set7 participates in other processes following prophase, such as
mitotic spindle assembly, chromosome and spindle movements. Further investigations
are needed to elucidate the potential function of phosphorylated PR-Set7 in these
processes.
During anaphase through to G1, we discovered that the observed decrease in PR-Set7
was directly due to APC
cdh1
-mediated ubiquitination and subsequent proteolysis of PR-
Set7. Interestingly, we found that S29 phosphorylation inhibits Cdh1 interaction with PR-
Set7 thereby PR-Set7 ubiquitination and degradation during late mitosis. Due to the close
proximity of the D-box, it is possible that PR-Set7 S29 phosphorylation could directly
inhibit Cdh1 interaction. Since phosphorylated PR-Set7 is present in early mitosis, it is
highly likely that similar mechanisms prevent PR-Set7 ubiquitination by APC
cdc20
.
Alternatively, S29 phosphorylation-dependent binding of the postulated protein complex
responsible for removing PR-Set7 from chromosomes (see above) may indirectly inhibit
Cdh1 interaction.
The activation of APC
cdh1
at anaphase requires the dephosphorylation of Cdh1 by the
Cdc14 phosphatase (Visintin et al., 1998). We found that S29 was also dephosphorylated
by either Cdc14A or Cdc14B ultimately resulting in APC
cdh1
-mediated ubiquitination and
proteolysis of PR-Set7. Since the dephosphorylation and degradation of PR-Set7 seemed
to occur later in mitosis, we hypothesized that the degradation-resistant PR-Set7 S29D
95

phospho-mimic would display defects in anaphase and/or cytokinesis. Consistent with
this, a substantial delay in progression to anaphase was observed in the PR-Set7 S29D
cells compared to wild type PR-Set7 cells. Since the wild type PR-Set7 cells behaved
similarly to control cells during mitosis, the observed delay to anaphase entry is most
likely due to elevated levels of “phosphorylated” PRSet7 rather than increased levels of
bulk PR-Set7. However, once the PR-Set7 S29D cells eventually achieved anaphase they
displayed similar progression kinetics as the control cells.
The findings that protein phosphorylation regulates its APC-mediated degradation has
been proved to be true in several APC substrates. For instance, the phosphorylation of the
yeast securin orthologue Pds1(Wang et al., 2001), the mitotic kinase Aurora A
(Littlepage and Ruderman, 2002), the licensing factor Cdc6 (Mailand and Diffley, 2005)
and SCF regulator Skp2 (Rodier et al., 2008) protects them from APC
cdh1
-mediated
degradation. Besides cell cycle regulators, phosphorylation of the transcription factor
RUNX1 has been shown to promote its APC-mediated degradation as well (Biggs et al.,
2006). In the case of PR-Set7, we demonstrate that phosphorylation at a single site of a
histone-modifying enzyme contributes to its accumulation in mid M phase and regulates
its degradation at anaphase. Our study highlights the importance of substrate modification
in APC-mediated ubiquitination and degradation.
Taken together, our findings first revealed a novel mechanism that governs PR-Set7
mitotic regulation. The highly ordered regulation of PR-Set7, both temporally and
spatially, may reflect a general rule that higher eukaryotes employ to fine tune its cell
cycle regulation.
96

Chapter 3: Elucidating the roles of PR-Set7 and H4K20me1 in DNA
damage responses

3.1. Introduction
Genomic instability, the hallmark of human aging and cancer, is largely caused by
DNA double strand breaks (DSBs). Both exogenous DNA damaging agents and
endogenous sources including metabolites and naturally occurring processes (DNA
recombination and DNA replication) can cause DSBs. Since DSBs are potentially lethal,
eukaryotic cells have developed two major repair pathways to counteract it, namely
homologous recombination (HR) and nonhomologous end joining (NHEJ). These two
pathways, although distinct, share some commonality whereby they respond to DSBs
through quickly detecting the damage, recruiting proteins to the site, activating
checkpoints to stall the cell cycle progression and ultimately fixing the damaged DNA.
Since the DSBs occurs in the context of chromatin, a dynamic structure composed of
DNA and its associated histone proteins, the execution of these repair steps is partially
orchestrated by the modifications of histone proteins. For example, during DNA damage,
the histone variant H2AX is rapidly phosphorylated (γH2AX) at DSBs that, in turn, binds
MDC1 allowing for stabilization of the MRN complex at DSBs for effective DNA repair
(Stucki et al., 2005b).
Previous studies from our lab and others have demonstrated that PR-Set7 and
H4K20me1 are required to suppress aberrant DNA damage and genomic instability
(Houston et al., 2008; Huen et al., 2008; Jorgensen et al., 2007; Tardat et al., 2007).
97

Recent studies from our lab indicated that PR-Set7 directly interacts with the key
components of the NHEJ DNA repair machinery including Ku70, Ku80 and DNA-PKcs
(unpublished). Based on these findings, I propose that PR-Set7 and H4K20me1
participate in DNA damage response by targeting to damaged site and stabilizing the
DNA repair machinery for effective DNA repair. Results from my preliminary study
indicate that global levels of PR-Set7 and H4K20me1are elevated upon ionizing radiation
(IR)-induced DNA damage. In addition, depletion of PR-Set7-mediated H4K20me1 leads
to less 53BP1 foci formation suggesting an important role in DNA damage response.

3.2. Results
3.2.1. Global levels of PR-Set7 and H4K20me1 are elevated upon IR induced DNA
damage
To gain insights into the role of PR-Set7 and H4K20me1 in DNA damage response, I
first investigated whether the global levels of PR-Set7 or H4K20me1 change upon DSBs.
HeLa cells were exposed to 1Gy or 5Gy IR followed by recovery for increasing time
points (Fig. 24). Western analysis revealed that γH2AX, the sensor of DNA damage,
started to accumulate 30 minutes after IR indicating DNA damage responses.
Interestingly PR-Set7 protein level also increased at 30 minutes time point and
maintained until 2 hours after IR. A concomitant elevated H4K20me1 level was detected.
These changes were more pronounced and reamained longer in 5Gy treated cells. By
comparison, global level of H4K20me2 did not alter significantly despite its role in IRIF
formation (Yang et al., 2008) while H4K20me3 level decreased, reciprocally to the trend
98

of H4K20me1. I have previously showed that S29 phosphorylation of PR-Set7 stabilizes
PR-Set7 (Chapter 2). Therefore I speculated that S29 phosphorylation may contribute to
the elevated PR-Set7 protein level upon IR treatment. Indeed, pS29-PR-Set7 significantly
increased at 30 minutes time point, in sync with the changes of total PR-Set7 suggesting a
role in stabilizing PR-Set7. Together, these data demonstrate that global levels of PR-
Set7 and H4K20me1 increase upon IR-induced DSBs, and the elevated level of PR-Set7
may be attributed to pS29-PR-Set7.

99

Figure 24. Alterations in protein levels upon IR induced DNA damage.

Western analysis of HeLa cells at increasing time points after 1Gy or 5Gy Ionizing
Radiation (IR) treatment. An γH2A.X antibody was used as indicator of IR induced DNA
damage; an H4 general antibody was used to control for loading.

100

3.2.2. PR-Set7 is targeted to DNA damage loci upon micro-irradiation
It is known that exposure of cells to IR causes DSBs and results in the formation of
IR induced repair foci (IRIF). Key components of DNA repair machinery is recruited to
these sites for effective DNA repair including 53BP1, γH2AX and BRCA1. To further
investigate whether the global increased levels of PR-Set7 and H4K20me1 also occur
locally to form IRIF, immunofluorescence imaging for PR-Set7 was conducted on IR
treated Hela cells. As shown in Figure 25A, there was no apparent PR-Set7 foci
formation upon 5Gy IR exposure as compared to untreated cells. To confirm this
observation, IR treated cells were co-stained with PR-Set7 histone marker H4K20me1
and IRIF indicator 53BP1 (Fig. 25B). Upon IR treatment, 53BP1 displayed a punctate
staining pattern indicating the formation of IRIF, whereas H4K20me1 remained evenly
distributed within nucleus with no apparent overlapping with 53BP1 staining. This
finding implies that PR-Set7 and H4K20me1 are not targeted to DNA damage foci.
However, it is possible that this experimental system is not sensitive enough to be able to
detect moderate accumulation of PR-Set7 or H4K20me1 at IRIF.

101

Figure 25. PR-Set7 and H4K20me1 is not detected at DNA damage foci upon IR
treatment.
(A) HeLa cells exposed to 5Gy IR followed by 1 hour recovery were immunostained for
PR-Set7 (red) and counterstained with DAPI (blue). Non-IR treated cells serve as control.
(B) HeLa cells exposed to 5Gy IR followed by 1 hour recovery were co-immunostained
with 53BP1 (green) and H4K20me1 (red), and counterstained with DAPI (blue).
102

Next we decided to test whether PR-Set7 can be targeted to DNA damage foci. We
used a more delicate experimental system in collaboration with Dr. Kyoko Yokomori at
Univerisity of California, Irvine. This latest technology allows a controlled linear
induction of DNA damage in live cells by passing a laser beam through live cells
(Stephens et al., 2009). If a protein is directly involved in DNA damage responses, it may
be detected at the laser-induced DNA damage site. GFP-PR-Set7 was transfected to HeLa
cells. After 18 hours GFP positive cells were selected for laser exposure. The treated cells
were tracked by live cell imaging for the accumulation of GFP signals at laser cutting
sites. As shown in Figure 26 (top). 10 minutes after laser cutting, an increased GFP signal
was detected at the laser cutting sites in both laser treated cells, although the signal was
not robustly enhanced. This weak yet detectable signal may explain our previous failure
of detecting PR-Set7 or H4K20me1 recruitment to IRIF by conventional IR treatment
combined with fluorescence microscopy (Fig. 25). This observation is specific as GFP-
null failed to accumulate at laser cutting sites (Fig. 26 bottom). This data indicate that
PR-Set7 is targeted to DNA damage loci upon micro-irradiation and suggests that PR-
Set7-mediated H4K20me1 may play a role in IRIF formation.

103

From Xiangduo Kong, UCI

Figure 26. PR-Set7 is targeted to DNA damage foci upon micro-irradiation
treatment.
GFP-PR-Set7 or GFP-null was transiently transfected into HeLa cells. 18 hours later,
DNA damages were induced by green laser (indicated by yellow arrows in the field
view). Pictures were taken at different time points after laser micro-irradiation.
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3.2.3. 53BP1 recruitment to DNA damage foci is impaired in the absence of PR-
Set7
An early study implied that the depletion of PR-Set7 could impair the recruitment of
53BP1 to DNA damage foci upon IR treatment (Botuyan et al., 2006a). My above
findings also suggest a role of PR-Set7 in DNA damage foci formation. To confirm this,
HeLa cells transfected with null or PR-Set7 shRNA were exposed to 10Gy of IR and co-
stained for H4K20me1 and 53BP1 (Fig. 27A and C). Cells with decreased H4K20me1
staining indicated efficient depletion of PR-Set7. While the null cells displayed normal
levels of H4K20me1 and 53BP1 foci formation (~10), the cells lacking H4K20me1
showed significantly less 53BP1 foci (~3). To further determine whether the decreased
53BP1 foci is caused by PR-Set7 or H4K20me1 level, a dominant negative catalytically
dead PR-Set7 (PR-Set7 CD) was used to deplete the cells of H4K20me1 while maintain
the PR-Set7 level. As expected, over-expression of the PR-Set7 CD resulted in
significantly decreased H4K20me1 staining. In the same cells, less 53BP1 foci (~5) was
detected indicating that the defective foci formation in PR-Set7 depleted cells was caused
by the lack of H4K20me1 histone mark, not PR-Set7 protein. Since SUV4-20 mediated
H4K20me2 has also been shown to positively participate in 53BP1 foci formation upon
DNA damage (Yang et al., 2008), it serves as a positive control in this experiment (Fig.
25B). An SUV4-20 shRNA was used to deplete the cells of H4K20me2 followed by co-
staining for H4K20me2 and 53BP1. Consistent with previous findings, the cells lacking
H4K20me2 displayed significantly less 53BP1 foci (~2). This data suggest that PR-Set7-
mediated H4K20me1 functions positively in DNA damage foci formation.
105

Figure 27. 53BP1 recruitment to DNA damage foci is impaired in the absence of PR-
Set7.

(A) HeLa cells transfected with PR-Set7 shRNA, catalytically dead PR-Set7 (PR-Set7
CD) or control vector were exposed to 10Gy IR to induce DNA damage prior to staining
for H4K20me1 (green) or 53BP1 (red). (B) HeLa cells transfected with SUV4-20 shRNA
or control vector were exposed to IR as (A) prior to staining for H4K20me2 (green) or
53BP1 (red). (C) The histogram displays the average number of 53BP1 foci observed per
cell in 50 randomly selected cells.

106

3.3. Discussion and future directions
My preliminary findings demonstrate that upon IR-induced DNA damage, PR-Set7 is
recruited to DSBs, and PR-Set7 mediated H4K20me1 facilitates the damage foci
formation for effective DNA repair. Previous studies from our lab indicate that PR-Set7
directly interacts with key components of the NHEJ repair machinery. Based on this we
propose that PR-Set7 is targeted to DSBs via interaction with NHEJ proteins and
monomentylate H4K20 which could stabilize the repair machinery such as 53BP1 at
damage foci. Therefore for future investigations, I propose:
1) Aim: to determine whether PR-Set7 is targeted to DSBs via interaction with
NHEJ components.
Experiment description: First we need to define the minimal required regions
within PR-Set7 for binding to key components of NHEJ machinery (Ku70, Ku80
or DNA-PKcs). This can be achieved by creating a panel of PR-Set7 truncation
mutants followed by in vitro binding assays with NHEJ proteins. Once the
minimal region is defined, we can create a GFP tagged PR-Set7 binding mutant
and determine whether this mutant PR-Set7 can accumulate at DSBs using laser
micro-irradation technology (see section 3.2.2.).
2) Aim: to determine whether the disruption of PR-Set7 binding to indicated
proteins would lead to aberrant DNA damage and genomic instability.
Experiment description: This can be achieved by replacing endogenous PR-Set7
with the binding defective PR-Set7 mutant. Since lack of PR-Set7 is lethal to
cells, we can introduce siRNA targeting 3’-UTR of PR-Set7 into cells
107

simultaneouly with GFP tagged PR-Set7 mutant plasmid which should not be
targeted by this siRNA. Western analysis can be performed to confirm the
depletion of endogenous PR-Set7 and the expression of the exogenous PR-Set7
mutant. Once this system is established, first we will verify that the interaction of
PR-Set7 mutant with NHEJ proteins is abolished in cells by immunoprecipitation
assays. Next, the cells will be exposed to IR to induce DSBs and the ability to
repair DSBs will be determined by methods such as comet assays.

108

Chapter 4: Methods
4.1. Immunofluorescence Studies on cells (See Table 3 for antibody dilutions)
1. Sterilize coverslips by rinsing in 100% ethanol and quickly passing through a
flame to dry. Place sterilized coverslips into 6 well plates.
2. Plate cells onto sterilized coverslips to a density of 10
5
cells/mL (2mL/well).
3. Perform experiments on cells, e.g. drug treatment, IR, transfection.
4. Fix cells for 10 minutes at RT in 4% paraformaldehyde/PBS (Make 4%
paraformaldehyde by mixing with PBS and heating to 60
0
C and adding 10M
NaOH dropwise until paraformaldehyde goes into solution).
5. Wash cells 3×10 minutes with PBS.
6. Permeabilize cells for 5 minutes at RT with 0.2% Triton X-100/PBS.
7. Remove permeabilization solution and wash cells for 3×5 minutes with PBS.
8. Block cells 1 hour at RT in 5% donkey serum/PBS.
9. Add primary antibody (125μL/coverslip, diluted in 5% donkey serum/PBS).
Incubate the primary antibodies at 37
0
C for 1 hour in a humidified chamber
(Tupperware + damp paper towel lining the edges).
10. Wash cells 3×2mL 5% donkey serum/PBS for 10 minutes.
11. Add secondary antibodies (125μL/coverslip, diluted in 5% donkey serum/PBS).
Make sure that the coverslips are in the center of the dish and incubate at 37
0
C for
1 hour in a humidified chamber (Tupperware + damp paper towel lining the
edges).
109

12. Wash cells 3×2mL 5% donkey serum/PBS for 10 minutes (Incubate in the dark to
prevent decreasing fluorescence signal).
13. Mount coverslips onto slides using mounting medium containing DAPI
(VectaShield) by adding 1 drop of mounting medium to the slide and rinsing
coverslips briefly in dH
2
O before placing cell side down onto mounting medium.
14. To store the slides, seal the edges with fingernail polish to prevent the slides from
drying out. Store slides at 4
0
C in the dark.

**For dual staining on cells: if the different primary antibodies are from different
species (ex. mouse and rabbit), they can be mixed together before adding to cells.
The same is true for different species of secondary antibodies.* *

110

Table 3. Dilutions of antibodies used for Western Blotting and Immunofluorescence
experiments
Antibody Species Western Blotting (dilutions;
conditions)
IF on cells
H4K20me1 (liner) Rabbit 1: 5,000 1: 1,000
H4K20me2 (branched) Rabbit 1: 10,000 1: 500
H4K20me3 (branched) Rabbit 1: 10,000 1: 1,000
H2A.X (Millipore) Mouse 1: 40,000 1: 1,000
H3S10phos (Abcam) Rabbit 1: 25,000
H3 general (Abcam) Rabbit 1: 100,000
H4 general (Millipore) Rabbit 1: 40,000
GST (Millipore) Mouse 1: 5,000
UBC9 (Santa Cruz) Goat 1: 5,000
β-Actin (Sigma) Mouse 1: 40,000
GFP (Abcam) Rabbit 1: 10,000
53BP1 (Millipore) Mouse 1: 250
HA (Santa Cruz) Rabbit 1: 1,000
HA (Sigma) Mouse 1: 4,000
RNAP II (Covence) Mouse 1: 100,000
DBD (Santa Cruz) Mouse 1: 5,000
FLAG (Sigma) Mouse 1: 10,000
FLAG (Sigma) Rabbit 1: 2,000
CyclinB (Santa Cruz) Rabbit 1: 1,000
SETD8 (Cell Signaling) Rabbit (mAb) 1: 1,000; O/N at 4C 1: 50
pS29-PR-Set7 Rabbit 1: 1,000; 3hr at RT 1: 200
FITC secondary Rabbit or Mouse 1:200 1: 200
Cy3 secondary Rabbit or Mouse 1:150 1: 150
111

4.2. GST pull-down assay
1. Transfer 25μl Glutathione-conjugated Sephase 4B beads (GE Healthcare) into
tubes. Wash beads 3×250μl PBS at 1000g for 1 minute at 4
0
C. Resuspend beads
in 50% slurry.
2. Immobilize GST fusion proteins on beads by incubating 3-6μg GST fustion
proteins with beads in PBS-T (1% Triton X-100) to a final volumn of 500μl.
Incubate at 4
0
C for 15-30 minutes. Wash beads 3 times with PBS-T to remove
unbound proteins.
3. Add acid-extracted histones to the protein-beads in PBS-T to a final volumn of
500μl. Incubate at 4
0
C for 30 minutes. Wash 3×5 minutes with PBS-T at 4
0
C.
4. Elute the bound proteins by adding 40μl elution buffer (10mM reduced
glutathione, 50mM Tris pH8.0), and rotating at 4
0
C for 10 minutes.
5. Analyze by SDS-PAGE or/and Western blotting.

4.3. Western Blot Analysis
Preparation of Lysates
1. Collecting cells by scraping cells off the plates or trypsinization.
2. Resuspend the cell pellets in 2×Laemmli (0.2% SDS, 3mM Tris, 0.4M glycine) to
lyse the cells (final concentration of 10
7
cells/mL). Boil cells at 100
0
C for 10
minutes and homogenize cells using a 21-gauge needle after lysates cool down.
3. We usually load the gels by cell number/lane using ~10
5
cells/lane or ~10μL/lane.

112

Gel Transfer
1. Make “sandwich” of blot paper, membrane, and gel in the following order
( bottom to top):
• 9 pieces of blot paper soaked in 1× Tobin buffer + MeOH
• Membrane soaked in MeOH
• Gel soaked in 1×Tobin buffer + MeOH
• 6 pieces of blot paper soaked in 1×Tobin buffer + MeOH
2. Remove all air bubbles using a pipet to ensure good transfer.
3. Run transfer for 120 minutes at 54mA/gel (ex: 2 gels = 106mA), place a 1L bottle
filled up at least 500 mL on top of the apparatus to ensure that the sandwich is
held together tightly.
4. Disassemble “sandwich” and place membrane into dH
2
O. procede with WB or
store the membrane at RT.
Blot (See Table 1 for antibody conditions and dilutions)
1. Block for 30 minutes in 5% milk/TBS pH 7.5.
2. Incubate with primary antibody (diluted in 1% milk/TBS, 4mL/whole membrane)
in sealed pouch on Nutator for 1 hour at room temperature.
**The incubation time and temperature may vary according to primary
antibodies**
3. Wash membrane 3×10 minutes TBS/0.1% Tween-20.
4. Incubate with secondary antibody (diluted in 1% milk/TBS, 4mL/whole
membrane) in sealed pouch on Nutator for 1 hr at room temperature.
113

5. Wash membrane 3×10 minutes TBS/0.1% Tween-20.
6. Develop using ECL plus kit.

4.4. Luciferase Studies
1. Transfect 6 well plates of cells using 5ng of Renilla vector as control for
transfection efficiency with experimental DNA.
2. 24-48 hours after transfection, lyse cells in 1×Passive Lysis Buffer (PLB, 5×
stock provided by Promega Luciferase kit at –40
0
C) by trypsinizing cells off plate
and resuspending in 1×PLB.
3. Freeze/thaw the lysates 2×alternating between –80
0
C and room temperature (it
usually takes about 15 minutes to freeze).
4. Spin down the lysates 2 minutes full speed at room temperature.
5. Aliquot 20μL cleared lysate per well (×4 wells for quadruplicate reading) into
black luminometer plates.
6. Add 100μL LAR II reagent/well to read luciferase values, this is a time sensitive
process so make sure to add just before putting the plate in the TopCount.
7. After reading is finished, add 100μL Stop&Glo reagent (to read renilla values),
this is a time sensitive process so make sure to add just before putting the plate in
the TopCount.

114

**LAR II reagent is prepared by mixing 10mL Luciferase Assay Buffer II with
Luciferase assay substrate. 10mL LAR II is sufficient for 1 full 96 well plate. This
buffer can be stored for 1 year at –80
0
C.**
**Stop&Glo reagent is prepared by mixing 10mL Stop&Glo buffer with 200μL 50×
Stop&Glo substrate. 10mL Stop&Glo reagent is sufficient for 1 full 96 well plate. This
reagent must be made fresh every time. Both LAR II and Stop&Glo are light sensitive
and must be kept on ice when not in use.**

4.5. Electroporation to K562 cells
1. Collect K562 cells and spin down for 6 minutes at 600g RT.
2. Wash cells in PBS and respin.
3. Resuspend cells to a final concentration of 10
7
cells/mL in electroporation buffer
(100mM HEPES, pH 7.4, 10μg/mL DEAE/Dextran, Opti-MEM medium).
4. Mix 10μg DNA with 200μL of cells and transfer to 0.2mm electroporation cuvette
and incubate at 37°C for 2 minutes.
5. Electroporate at 1000 μF, 150V.
6. Incubate electroporated cells at 37°C for 10 minutes.
7. Transfer cells to one well of a 6 well plate containing 1.8mL RPMI 1640/10%
FBS media, then rinse out cuvette with media to ensure a good transfer of cells to
the plate. For this step, use a pastuer pipet to transfer the cells and try to avoid
foam of dead cells.
8. Change media 24-48 hours after electroporation to remove cell debris.
115

**This will result in ~10-25% transfection efficiency. The transfection efficiency can
be measured by electroporating a GFP construct into the cells and measuring GFP
expression by FACS analysis.**
**For better electroporation efficiency, you should use Nucleofector apparatus and
purchase a buffer specifically formulated for K562. It is expensive, but you can get up
to 40-50% gene delivery.**

4.6. Immunoprecipitation assay
IP lysis buffer (store at 4
0
C)
50mM Tris pH 7.5
150mM NaCl
0.5mM DTT
1% NP-40
protease inhibitors (1μg/mL pepstatin A, 1μg/mL leupeptin/aprotinin, 1mM PMSF)
Preparation of agarose beads for immunoprecipitation
Protein A or G beads
1. Transfer beads needed for IPs and spin down 30 seconds at 14000 rpm.
2. Wash beads 3 × 0.5mL IP lysis buffer and resuspend beads in 50% slurry of
IP lysis buffer.
Antibody conjugated beads
1. Transfer beads needed for IPs and spin down 30 seconds at 14000 rpm.
116

2. Add 10 volumes 100mM glycine pH 2.5 to remove unconjugated antibody.
3. Wash with 10 volumes 200mM Tris pH 8.
4. Wash with 10 volumes IP lysis buffer + protease inhibitors, and resuspend in
50% slurry in IP lysis buffer.
Immunoprecipitations from tissue culture cells
1. Trypsinize cells from 6 well plate and pellet cells for 5 minutes at 600g RT.
2. Wash pellet 1×PBS, and resuspend cells in 300-500μL cold IP lysis buffer +
protease inhibitors.
3. Spin down 10 minutes at 14000 rpm 4
0
C to pellet insoluable fraction. Save
supernatant for IPs.
**If using protein A or G beads, preclear lysates 4h-overnight using 40μL of 50%
slurry of appropriate beads. Spin down lysates 5 minutes 4000 rpm 4
0
C. Save the
supernatant as precleared lysates.**
4. Incubate beads and antibody or antibody-conjugated beads with 200-400μl IP
lysates in a final volumn of 500μl and rotate overnight at 4
0
C.
5. Spin down 5 minutes 4000 rpm at 4
0
C and save supernatent as unbound fraction.
6. Wash beads 2×500μL cold IP lysis buffer.
7. Resuspend beads in 35μL 6×SDS dye and either boil 10 minutes or elute at 55
0
C
for 10 minutes. Elution at 55
0
C allows for separation of the bound fraction
without getting too much of the heavy and light chain of the antibody to reduce
background.
117

8. Collect bound fraction by poking a hole in the top of the epi tub and in the bottom
and placing in a clean epitube to spin down. This allows for collection of the
bound material without getting any of the beads. Proceed with Western analysis
or save bound and unbound fractions at -40
0
C.

4.7. Nuclear Fractionation assay
1. Collect cells from one well in 6-well plate (around 4×10
6
cells). Wash with PBS,
and resuspend in 400μl NIB buffer (check thorough lysis by trypan blue staining).
2. Centrifuge 500g for 5 minutes at 4
0
C to get nuclei pellet.
3. Resuspend pellet in 400μl nuclear buffer. Aliquot into 80μl×4 reactions (for
different time points).
4. Add 1μl MNase (6U/ml) to each reaction and incubate at 37
0
C waterbath for
designated times, e.g. 2, 4, 8 and 16 minutes.
5. Terminate reactions by adding 1μl EDTA + 1μl EGTA (0.5M stock). Spin down
at full speed for 30 seconds. Collect supernatant (S1 fraction, mainly composed to
mononucleosomes).
6. Resuspend pellet in 80μl 2mM EDTA and put on ice for 15 minutes to lyse the
nuclei membrane.
7. Quick spin at full speed for 30 seconds to separate S2 and P fractions.
8. Resuspend P fraction in 80μl lysis buffer.
9. For each collect fractions, split to 50μl + 20μl:
50μl for WB analysis
118

20μl for DNA analysis (add 5ul 0.4% SDS to denature protein, then incubate with
1μl proteinase K at 37
0
C for 15 minutes to digest proteins. Add 2μl 65% glycerol
before loading on 1.3% agarose gel)

**protease inhibitors freshly added to the following buffers:**
NIB buffer: Nuclear buffer: Lysis buffer:
10mM Hepes, pH7.5 20mM Tris, pH7.5 50mM Tris, pH7.5
150mM NaCl 70mM NaCl 100mM NaCl
1.5mM MgCl2 5mM MgCl2 5mM EDTA
10mM KCl 20mM KCl 0.5% SDS
0.1% NP-40 3mM CaCl2

4.8. HMTase assay
1. Set up the following 25μl reaction for each sample:
-12.5ul 2×HMT buffer (100mM Tris, pH8.0, 20% glycerol, 2mM DTT, 2mM
PMSF)
- 1μl
3
H-SAM (adenosy-L-methionine-S-[methyl-
3
H])
- 1μg target substrate (histones, nucleosomes, peptide…)
- 1μg enzyme (PR-Set7…..)
-dH2O
2. Mix well and incubate at 30
0
C for 1 hour.
** The reaction can be analyzd by
3
H autoradiography or scintillation counting.**
119

3. Spot a designated amount of the reaction onto P-81 filter paper. Wash filter paper
3×200ml of 50mM sodium bicarbonate pH9.0 for 10-15 minutes by shaking.
Place filter paper in scintillation tubes with 2ml scintillation fluid (include blank
filter paper as background reading). Counts using Scintillation Counter.
4. Analyze by autoradiography (develop at -80
0
C for 3-6 days).

4.9. In vitro kinase assay
1. 1μg of purified protein (PR-Set7, N-termianl or C-terminal portion) is incubated
with 20units of cdk1/cyclinB (New England Biolabs) and 0.4mM
32
P-ATP in a
final volume of 30 μl for 30 minutes at 30°C.
2. Terminate reactions by adding adding 6×SDS load dye prior to fractionation by
SDS-PAGE and autoradiography.

4.10. APC ubiquitination and degradation assay (by Weiping Wang)
1. APC is purified from early G1 HeLa cell extracts (according to Rape et al., 2006)
2. Incubate recombinant protein with purified APC for 30 minutes or more
depending on experiments.
3. Terminate the reactions by adding 6x loading dye and followed by Western
blotting analysis.
4. For degradation assay, early G1 Hela S3 extracts was added to
35
S-labeded full
length PR-Set7 wild-type, S29A mutant or S29D mutant in the absence or
120

presence of 0.25mg/ml APC inhibitor Emi1. Reactions were fractionation by
SDS-PAGE and visualized by autoradiography.

4.11. Cell Cycle Synchronization (HeLa cells)
G0/G1 block by serum starvation
1. Plate the cells so that the next day they get to 60-80% confluency.
2. The next day, Change the medium to 0% serum DMEM before leaving the lab.
3. Check the cell phenotype: cells become bigger and more spreading out.
**do seriel depletion of serum if the cells do not react well**
Early S phase block by thymine-mimosine double treatment (by Judd Rice)
1. HeLa cells were split into 100mm plates at 5×10
5
cells/plate.
2. 18 hours later, change to new media with 2mM thymidine; first block at G1/S.
3. 17 hours later, release cells into fresh media without drug.
4. 7.5 hours later, change to new media with 400μM mimosine; second block at
G1/S.
5. 16.5 hours later, wash cells thoroughly to remove any drug residues, and add fresh
media; second release.
**Time points taken every 2.5 hours for 20 hours total**.
For each time point:
1. Place supernatant into 50ml conical tube.
2. Wash plates 2×PBS.
3. Trypsinize off cells and place into the same 50ml conical tubes as 1.
121

4. Count cells.
5. Spin 500g at RT for 5 minutes, wash cell pellet in PBS.
6. Resuspend pellet in PBS TO 5×10
5
cells/ml.
** Cells can be used for FACS analysis, histone extraction……..**
Mitotic arrest by nocodazole treatment
1. Plate HeLa cells so that they can reach ~70% confluent before treatment.
2. Dilute nocodazole into fresh media at final concentration 0.4μg/ml, then replace
the old media with fresh media containing nocodazole.
3. Wait for at least 12 hours to get a satisfying mitosis arrest.

122

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Abstract (if available)

Abstract Eukaryotic chromatin, a dynamic structure composed of DNA and chromatin-associated proteins, plays an essential role in key biological processes including gene transcription, cell cycle progression and DNA repair. In this thesis, I wanted to gain insights into the molecular mechanisms of these processes by focusing on: 1) a novel human protein SFMBT-mediated gene silencing pathway

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Asset Metadata

Creator Wu, Shumin (author)

Core Title Characterization and functional study of a novel human protein SFMBT, and PR-Set7 histone methyltransferase

School Keck School of Medicine

Degree Doctor of Philosophy

Degree Program Biochemistry and Molecular Biology

Degree Conferral Date 2010-12

Publication Date 11/11/2010

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

Tag APC,Cdc14,Cdk1,cell cycle,chromatin,OAI-PMH Harvest,PR-Set7

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Rice, Judd C. (committee chair), Johnson, Deborah L. (committee member), Laird-Offringa, Ite A. (committee member), Li, Wei (committee member), Stallcup, Michael R. (committee member)

Creator Email luckywsm@gmail.com,shumin@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m3525

Unique identifier UC1193611

Identifier etd-wu-4158 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-410757 (legacy record id),usctheses-m3525 (legacy record id)

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Rights Wu, Shumin

Type texts

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

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Repository Location Los Angeles, California

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