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A regulatory transcription module of ZBRK1/KAP1 complex and its signaling network in regulating DNA damage-responsive genes expression
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A regulatory transcription module of ZBRK1/KAP1 complex and its signaling network in regulating DNA damage-responsive genes expression
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Content
A REGULATORY TRANSCRIPTION MODULE OF ZBRK1/KAP1 COMPLEX
AND ITS SIGNALING NETWORK IN REGULATING DNA DAMAGE-
RESPONSIVE GENES EXPRESSION
by
Yung-Kang Lee
A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(MOLECULAR PHARMACOLOGY AND TOXICOLOGY)
December 2006
Copyright 2006 Yung-Kang Lee
DEDICATION
To my beloved parents and wife
who give me enormous support and strength.
ii
ACKNOWLEDGEMENTS
I would like to express my sincere gratitude to all of the people who have
supported me, inspired me and helped me along the road to a science Ph.D.
First and foremost I would like to thank Dr. David Ann for his great support in
my protein SUMOylation study, which was a wild-open field just a couple of years
back. Completely new to this field, we have been through some serious struggles and
yet have made our way through. At the time of writing this dissertation, I hope I have
helped to create a little niche for our future research. I also heartily appreciate the
invaluable guidance and help I have received from my committee—Drs. Roger
Duncan, Sarah Hamm-Alvarez, Wei Li, Jean Shih, and Austin Yang. I would like
also thank Drs. Wei-Chiang Shen and Michael Stallcup for their help and suggestion
along the course of my study.
I am honored to have been working with a group of great people in Dr. Ann’s
lab, and especially I would like giving my special thanks to Dr. Helen Lin for helping
me in clearing all the technical bumps all these years.
iii
TABLE OF CONTENTS
DEDICATION
ACKNOWLEDGEMENTS
LIST OF TABLES
LIST OF FIGURES
ABSTRACT
CHAPTER ONE: INTRODUCTION
CHAPTER TWO: IDENTIFICATION OF GLOBAL
SUMOYLATION TARGETS BY AFFINITY
PURIFICATION AND TANDEM MASS
SPECTROMETRY
Summary
Background
Materials and methods
Results
Discussion
CHAPTER THREE: DOXORUBICIN DOWNREGULATES KAP1
SUMOYLATION THAT RELIEVES ITS
TRANSCRIPTIONAL REPRESSION ON P21
IN BREAST CANCER MCF-7 CELLS
Summary
Background
Materials and methods
Results
Discussion
CHAPTER FOUR: THE TRANSCRIPTIONAL CONTROL OF
THE KAP1 SUMOYLATION BY
DOXORUBICIN ON OTHER
ZBRK1-REGULATED GENES
Summary
Background
Materials and methods
ii
iii
vi
vii
ix
1
8
8
15
17
25
28
29
33
38
60
66
66
71
iv
Results
Discussion
CHAPTER FIVE: THE INVOLVEMENT OF THE SUMO-
SPECIFIC PROTEASE SENP1 AND THE DNA
DAMAGES-RESPONSIVE PROTEIN KINASE
ATM IN THE DEREPRESSION OF P21 AND
GADD45 α TRANSCRIPTION VIA KAP1
Summary
Background
Materials and methods
Results
Discussion
CHAPTER SIX: CONCLUSIONS AND FUTURE DIRECTIONS
BIBLIOGRAPHY
73
75
78
78
83
85
91
95
101
v
LIST OF TABLES
TABLES
1 Global SUMOylation targets identified from proteomic
study
2 Fragmented ions identified from tandem mass
spectrometry
3 Putative SUMOylation sites in PP2C, STK38, KAP1, and
the results of the in vivo and in vitro SUMOylation assays
4 Primers used in the real-time PCR and the chromatin
immunoprecipitation experiments
5 Primers used in the real-time PCR experiments
PAGE
20
21
24
36
72
vi
LIST OF FIGURES
FIGURES
1 Flowchart of affinity purification and tandem mass
spectrometry used in the identification of global
SUMOylation substrates
2 Transcriptional co-repressor KAP1 is subject to
multi-SUMOylation
3 Mapping SUMO-1 acceptor sites in KAP1
4 KAP1 SUMOylation-mimetic SUMO-1-KAP1 and
SUMOylation-defective KAP1 (3K/R) exhibit comparable
binding to ZBRK1
5 Suppression of Dox-induced p21 transcription by
SUMO-1-KAP1 and alleviation of transcriptional
repression by KAP1 (3K/R)
6 SUMOylation-mimetic and SUMOylation-defective KAP1
mutant exhibit similar affinity to ZBRK1 response
elements at the p21 promoter
7 Modulation of H3-K9 and H3-K14 acetylation and
methylation at the p21 promoter by KAP1 SUMOylation
8 KAP1 SUMOylation-mimetic, SUMO-1-KAP1,
desensitizes Dox-induced breast cancer cell death
9 The p53-responsive element and putative ZBRK1-binding
elements at the promoters of bax, puma, and noxa genes
10 The assessment of mRNA levels of p21, gadd45 α, bax,
puma, noxa in response to Dox treatment in MCF-7 cells
PAGE
19
40
43
45
49
52
54
58
74
76
vii
11 SENP1 but not SENP2 regulates the activation of p21
transcription that mimics the down-regulation of KAP1
SUMOylation by Dox treatment
12 ATR is not required for the Dox-induced gadd45 α
transcription
13 DNA damage-responsive protein kinase ATM is required
for the Dox-induced gadd45 α transcription
14 Proposed model of ZBRK1/KAP1 complex regulating the
transcription of their targets gene via KAP1 SUMOylation
in response to DNA double strand break (DSB) damages
86
88
89
97
viii
ABSTRACT
In pursuit of new SUMOylation targets that regulate the activities of cell
cycle progression and mediate cellular apoptosis, a proteomic screening that
combined affinity chromatography and tandem mass spectrometry was launched to
identify novel targets from SUMO-1 stably-expressed HEK293 cells. With a series of
discreet screens and analyses, this effort yields twenty-three SUMOylation
candidates, which are found to carry out distinct cellular functions. KAP1 was
verified as a bona fide SUMO-1 substrate and a following literature research pointed
KAP1 to a novel role in regulating the transcription of a cluster of cell cycle
regulator genes and pro-apoptotic genes via its interaction with a transcriptional
factor ZBRK1 that bound a 15-bp DNA sequence motif.
Subsequently, the lysines 554, 779 and 804 in KAP1 were identified as the
major SUMOylation sites. Using two KAP1 mutants--one deficient in SUMOylation
and the other mimics constitutive SUMOylation, the Dox-mediated induction of cell
cycle regulator p21
WAF1/CIP1
transcription is differentially regulated by KAP1
SUMOylation status. The SUMOylation-dependent modulation in the p21
transcription was achieved through changes in the lysine acetylation and methylation
of histone 3 at the p21 promoter. Furthermore, the KAP1 SUMOylation level was
transiently decreased upon Dox-exposure, and the introduction of constitutively
SUMOylated KAP1 desensitized breast cancer MCF-7 cells to Dox-elicited cell
death. Taken together, I provide a novel mechanistic basis underlying the
ix
Dox-induced de-repression of p21 transcription, and my results suggest that
Dox-induced decrease in KAP1 SUMOylation is essential for Dox-induced p21
expression and subsequent cell growth inhibition in MCF-7 cells.
Further tracking the upstream signaling cascade that leads to the presumed
Dox-induced KAP1 de-SUMOylation, my preliminary results indicated that
SUMO-specific protease SENP1 and DNA damage-responsive protein kinase ATM
might have served for this purpose. Lastly, a proposed future studies and the
rationale underlying it was described herein.
x
Chapter One
INTRODUCTION
Both ubiquitylation and SUMOylation are protein post-translational
modification processes through which small polypeptides ubiquitin and SUMO are
covalently linked to their target substrates. While ubiquitylation and SUMOylation
virtually employ the same logic in conjugation—a polypeptide chain transferring
system comprised of E1 activation enzyme, E2 conjugation enzyme, and E3 ligase,
the most prominent distinction between the two falls on the different biological
consequences they impose on their targets. Speaking about ubiquitylation, it is well-
established that ubiquitin-modified substrates can be recognized by 26S proteasome,
and subsequently degraded. The well-characterized lysine 48 (K48) poly-
ubiquitylation is specific for this particular process, although another well-studied
K63 poly-ubiquitylation has been shown to mediate DNA repair activity and other
signal transduction events (Weissman, 2001). Although the three-dimensional
structures of crystallized ubiquitin and SUMO-1 are highly homologous, their
primary amino acid sequences shares only about 30% identity, making distinct
surface charge distributions of the core global domain between the two. Due to this
reason it is not surprising to find specific sets of conjugation enzymes are needed for
each of modification processes(Ulrich, 2005). The SUMOylation E1 enzyme is
comprised of a heterodimer SAE1/SAE2 (Aos1/Uba2), while Ubc9 is the only E2
1
enzyme identified by far for SUMOylation. A number of E3 ligase specific for
protein SUMOylation have been reported. Among them a large number of proteins
from PIAS family that regulates Stat activity are found to enhance SUMOylation
efficiency. It is worth nothing where the ubiquitin E3 ligase complexes are the key
components that virtually endow the conjugation activity and specificity over their
substrates, it does not necessarily apply to SUMOylation E3s (Kerscher et al., 2006).
Many SUMOylation substrates are found to be SUMOylated relatively well even in
the absence of their corresponding E3 enzymes in vivo and in vitro.
Among other post-translational modifications, SUMOylation is a relatively new
discovery. A glut of proteomic studies aimed to unveil its substrates sprang up just
recently (Li et al., 2004; Vassileva and Matunis, 2004; Vertegaal et al., 2004;
Wohlschlegel et al., 2004). Despite of different strategies in the identification process,
these reports seems to reach two consensuses. First of all, a large number of novel
substrates for SUMOylation are involved in transcription, DNA metabolism, nuclear
organization and biogenesis, RNA metabolism. Secondly, the majority of the
substrates identified are resided in the nuclei, nucleoli, or nuclear periphery. Thus
these findings are in agreement with the fact that in large part, SUMOylation weighs
itself more in regulating nuclear events. Indeed, of the four major functions of
protein SUMOylation, two of them are to activate transcription and repress
transcription. The other two major functions can be either nuclear or cytoplasmic
events. For instance, protein inhibitor I κB α, which retains NF κB in the cytoplasm by
masking its nuclear translocalization signal, is found to be SUMOylated (Desterro et
2
al., 1998). Of the two lysines—lysines 21 and 22--in I κB α to be poly-ubiquitylated,
lysine 21 is also proved capable of being SUMOylated. Therefore SUMOylation
competes with ubiquitylation for the same lysine, thus directly inhibiting the rate of
poly-ubiquitylation-mediated I κB α degradation. The same principle applies to
nuclear proteins proliferating-cell nuclear antigen (PCNA) and the pathogenic
protein of Huntington disease Huntingtin, therefore altering their responses to DNA
damages and transrepressive ability respectively (Hoege et al., 2002; Steffan et al.,
2004). SUMOylation can also regulate protein translocalization between different
cellular compartments or subdomains. For example, in the absence of SUMOylation
the GTPase-activating protein RANGAP1 resides in the cytoplasm, and the
SUMOylated RANGAP1 is translocalized from cytoplasm to the cytosolic face of
the nuclear pore complex, where it activates RAN GTPase (Joseph et al., 2002). The
other fine example is the formation of promyelocytic leukemia (PML) protein. Once
it is SUMOylated, it recruits a wide selection of proteins to form specific nuclear
speckles—PML nuclear bodies (Ishov et al., 1999; Zhong et al., 2000). Yet there is
no unified theory that can summarize how SUMOylation regulates protein transport
and localization. For example, MAPK kinase MEK1 was shown to be SUMOylated
upon nutrient deprivation, consequently exporting it from nucleus (Sobko et al.,
2002).
As mentioned before, SUMOylation is also found to regulate gene transcription
and a large number of reports support this notion. Contradictory as it may seem,
SUMOylation of certain transcriptional factors and co-factors can lead to either
3
transcriptional activation or repression. Although SUMO itself, when fused to Gal4
DNA binding domain, can suppress Gal4-mediated promoter activity, it is obvious
that the outcomes of SUMOylation is determined by both the conjugation of SUMO
and the change in transcriptional activity of transcriptional factors or co-factors.
Since most proteins are found to be SUMOylated in between their functional
domains, it is considered that SUMOylation may mediate the interactions between
these domains intramolecularly, therefore modulating the activities of these proteins.
Also SUMOylation can influence protein-protein interactions, by which it regulates
specific protein activities recruited by the substrate proteins it modifies. In only a
limited number of cases, SUMOylation of transcriptional factors were found to
increase their transcriptional activities. Among those cases, the DNA binding activity
of heat shock transcriptional factors 1/2 (HSF1/2) increases after they are
SUMOylated (Goodson et al., 2001; Hong et al., 2001).
In most cases that involve transcriptional regulation, except those described
above, SUMOylation helps to repress the gene transcriptions mediated by the
transcriptional factors or co-factors that are subjected them to SUMOylation. There
are two models that explain the SUMOylation-mediated transcriptional repression. In
the first model, the SUMOylation of a transcriptional factor or co-factor can directly
recruit co-repressor or silencer activity to the target gene it is regulating. For example,
the direct recruitment of enzymes that modify post-translational modifications of
histones can change the “histone codes”, which are then translated by transcription
machinery and other factors to modulate local nucleosome structure, thus switching
4
gene transcription between different states. For example, the acetylation at lysines 9
and 14 of histone 3, a combination of methylation at lysine 4 and acetylation at
lysine 14 of histone 3, or a combination of methylation at arginine 3 and acetylation
at lysine 5 of histone 4, relaxes the compacted nucleosome structure and exposes
specific DNA response elements at local promoters, thereby making promoters more
accessible to transcriptional activators and co-activators. On the other hands, the
methylation at lysine 9 of histone 3 or the acetylation of lysine 12 of histone 4 results
in the condensation of its associated nucleosomes. While the chromatin condensation
itself blocks the access of transcriptional activators and co-activators to their target
promoters, these modifications of histones can further bring about DNA-modifying
activities such as DNA methylases (DNMTs) to exert another level of epigenetic
control on the transcription of those target genes (Prives and Manley, 2001). The
transcriptional co-regulator p300 is SUMOylated in its cell-cycle regulated domain
(CRD1), and this SUMOylation is proved necessary for its efficient transcriptional
repression by CRD1 (Girdwood et al., 2003). This observation was further supported
by the findings that the HDAC inhibitor trichostatin A (TSA) relieved CRD1-
meditaed repression and CRD1 bound to HDAC6. In another example, the repression
(R) domain of transcriptional factor Elk-1 efficiently represses transcription in an
artificial Gal4 system when it is SUMOylated, and the SUMOylated Elk-1 was
demonstrated to recruit HDAC2 (Yang et al., 2002; Yang and Sharrocks, 2004).
Furthermore, phosphorylation of Elk-1 by MAPK sequentially reduces the Elk-1
5
SUMOylation, importantly demonstrating its relevance in signaling cascade by the
sense of signal transduction.
The other model for the transcriptional regulation by SUMOylation depicts that,
instead of actively recruiting co-repressor activities to their target promoters,
SUMOylated transcriptional factors can call upon themselves into subnuclear
domains, where these SUMOylated factors utilized repressive environment, which is
pre-assembled by a number of factors, to execute their repressive functions. The
most well-known study is the SUMOylation of promyelocytic leukemia protein
(PML). PML itself is required for the formation of the nuclear subdomain called
PML nuclear body (PML NB), as well as the recruitment of many components into
PML NB. SUMOylation of PML matures the NBs by recruiting other components
into NBs. Given many PML NB constituents such as homeodomain-interacting
protein kinase (HIPK2), Daxx, Sp100 are found to be SUMOylated, PML NB has
been proposed as a hub that dynamically recruits these SUMOylated factors, where
they modulate different nuclear events (Duprez et al., 1999; Kamitani et al., 1998).
For example, SUMOylation of Sp100 was found to enhance its interaction with
heterochromatin protein 1 (HP1), through which a number of chromatin remodeling
activities such as H3-K9 methylase Suv39h1 and methyl-CpG binding domain
protein 1 (MBD1) are recruited to silence its target gene expression (Seeler et al.,
2001).
It is particularly worth nothing that PIASy, one of the five PIAS proteins that
function as SUMO E3 ligases, was shown to induce cellular senescence and
6
apoptosis (Bischof et al., 2006). PIASy stimulates p53 SUMOylation and its
transcriptional activity, while it represses E2F-mediated transcription by recruiting
tumor suppressor Rb to E2F response elements. Also PIASy was found to localize in
the subcellular domain senescence associated heterochromatin foci (SAHF), which
are transcriptionally silent and enriched with the target genes of E2F, HP1, and
methylated histone 3 (Narita et al., 2003).
7
Chapter Two
IDENTIFICATION OF GLOBAL SUMOYLATION TARGETS BY AFFINITY
PURIFICATION AND TANDEM MASS SPECTROMETRY
Summary
Since its discovery in 1996, a modest number of proteins have been
uncovered for their SUMOylation and for the role of SUMOylation in modulating
their cellular functions. Yet due to its natural low abundance, it has been suspected
that a good portion of SUMOylation substrates are yet to be identified. Along with
the increasing popularity of proteomic technology, a proteomic screening system
consisting of affinity purification and liquid chromatography-coupled tandem mass
spectrometry (LC-MS/MS) was set up to achieve this goal. With the stable
expression of FLAG-tagged SUMO-1 in HEK293 cells, more than forty proteins
were seized after the application of very strict filtering environment. One of the three
candidates was proved a good SUMOylation substrate both in vitro and in vivo.
Background
John B. Fenn, the inventor of electrospray ionization for biomolecules and
the 2002 Nobel Laureate in Chemistry, probably gave the best notion to what the
mass spectrometry is all about:
8
Mass spectrometry is the art of measuring atoms and molecules to determine their
molecular weight. Such mass or weight information is sometimes sufficient,
frequently necessary, and always useful in determining the identity of a species. To
practice this art one puts charge on the molecules of interest, i.e., the analyte, then
measures how the trajectories of the resulting ions respond in vacuum to various
combinations of electric and magnetic fields.
Over the past decade, mass spectrometry has undergone tremendous
technological improvements allowing for its application to proteins, peptides,
carbohydrates, DNA, drugs, and many other biologically relevant molecules. With
the advance in ionization sources such as electrospray ionization (ESI) and matrix-
assisted laser desorption/ionization (MALDI), mass spectrometry has become a
reliable and invaluable tool in the biological sciences.
A mass spectrometer determines the mass of a molecule by measuring the
mass-to-charge ratio (m/z) of its ion. Ions are generated by inducing either the loss or
gain of a charge from a neutral species. Once formed, ions are electrostatically
directed into a mass analyzer where they are separated according to m/z and directed
to a mass detector in an orderly manner. The actions of molecular ionization, ion
separation, and ion detection result a spectrum that can provide molecular mass and
even structural information. In a mass spectrometer the generated ions are separated
in the mass analyzer, digitized and detected by an ion detector (McCloskey J.A.,
1997).
9
Four basic components are standard in all mass spectrometers: a sample inlet,
an ionization source, a mass analyzer and an ion detector. Some instruments combine
the sample inlet and the ionization source, while others combine the mass analyzer
and the detector. Nevertheless, all sample molecules undergo the same processes
regardless of instrument configuration. Sample molecules are introduced into the
instrument through a sample inlet. Once inside the instrument, the sample molecules
are converted to ions in the ionization source, before being electrostatically propelled
into the mass analyzer. Ions are then separated according to their m/z in the mass
analyzer. The detector converts the ion energy into electrical signals, which are then
transmitted to a computer.
Ionization method refers to the mechanism of ionization while the ionization
source is the mechanical device that allows ionization to occur. The different
ionization methods work by either ionizing a neutral molecule through electron
ejection, electron capture, protonation, cationization, or deprotonation, or by
transferring a charged molecule from the condensed phase to the gas phase (Busch
K.L., 1989).
Prior to the 1980s, electron ionization (EI) was the primary ionization source
for mass analysis. However, EI limited chemists and biochemists to small molecules
well below the mass range of common bio-organic compounds. This limitation
motivated a number of great scientists to develop the new generation of ionization
techniques, including fast atom/ion bombardment (FAB), matrix-assisted laser
desorption/ionization (MALDI), and electrospray ionization (ESI). These techniques
10
have revolutionized biomolecular analyses, especially for large molecules. Among
them, ESI and MALDI have clearly evolved to be the methods of choice when it
comes to biomolecular analysis.
Electrospray ionization is a method routinely used with peptides, proteins,
carbohydrates, small oligonucleotides, synthetic polymers, and lipids. ESI produces
gaseous ionized molecules directly from a liquid solution. It operates by creating a
fine spray of highly charged droplets in the presence of an electric field. The sample
solution is sprayed from a region of the strong electric field at the tip of a metal
nozzle maintained at a potential of anywhere from 700 V to 5000 V. The nozzle to
which the potential is applied serves to disperse the solution into a fine spray of
charged droplets. Either dry gas, heat, or both are applied to the droplets at
atmospheric pressure thus causing the solvent to evaporate from each droplet. As the
size of the charged droplet decreases, the charge density on its surface increases. The
mutual Coulombic repulsion between like charges on this surface becomes so great
that it exceeds the forces of surface tension, and ions are ejected from the droplet
through a Taylor cone. Another possibility is that the droplet explodes releasing the
ions. In either case, the resulted ions are directed into an orifice through electrostatic
lenses leading to the vacuum of the mass analyzer. Because ESI involves the
continuous introduction of solution, it is suitable for using as an interface with HPLC
or capillary electrophoresis.
Electrospray ionization is efficient in the formation of singly charged small
molecules, but is also well-known for producing multiply charged species of larger
11
molecules. This is an important phenomenon because the mass spectrometer
measures the mass-to-charge ratio (m/z) and therefore multiple charging makes it
possible to observe very large molecules by an instrument with a relatively small
mass detection range. Fortunately, the software available with all electrospray mass
spectrometers facilitates the molecular weight calculations necessary to determine
the actual mass of the multiply-charged species. Multiple charging has other
important advantages in tandem mass spectrometry. One advantage is that upon
fragmentation you observe more fragment ions with multiply charged precursor ions
than with singly charged precursor ions.
Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS)
was first introduced in the late 1980s. It has since become a widely-used analytical
tool for peptides, proteins, and most other biomolecules (oligonucleotides,
carbohydrates, natural products, and lipids). The efficient and directed energy
transfer during a matrix-assisted laser-induced desorption event provides high ion
yields of the intact analyte, and allows for the measurement of compounds with sub-
picomole sensitivity. In addition, the utility of MALDI for the analysis of
heterogeneous samples makes it very attractive for the mass analysis of complex
biological samples such as proteolytic digests.
While the exact desorption/ionization mechanism for MALDI is not known,
it is generally believed that MALDI causes the ionization and transfer of a sample
from the condensed phase to the gas phase via laser excitation and ablation of the
sample matrix. In MALDI analysis, the analyte is first co-crystallized with a large
12
molar excess of a matrix compound, usually a UV-absorbing weak organic acid such
as α-cyano-4-hydroxycinnamic acid, 3,5-dimethoxy-4-hydroxycinnamic acid, and
2,5-dihydroxy-bezonic acid. Irradiation of this analyte-matrix mixture by a laser
results in the vaporization of the matrix and the co-crystallized analyte molecules
also vaporize, but without having to directly absorb energy from the laser. Molecules
sensitive to the laser light are therefore protected from direct UV laser excitation.
Once in the gas phase, the desorbed charged molecules are then directed
electrostatically from the MALDI ionization source into the mass analyzer. Time-of-
flight (TOF) mass analyzers are often used to separate the ions according to their
mass-to-charge ratio (m/z). The pulsed nature of MALDI is directly applicable to
TOF analyzers since the ion’s initial time-of-flight can be started with each pulse of
the laser and completed when the ion reaches the detector (Cotter, 1997).
The utility of MALDI for biomolecular analyses lies in its ability to provide
molecular weight information on intact molecules. The ability to generate accurate
information can be extremely useful for protein identification and characterization.
For example, a protein can often be unambiguously identified by the accurate mass
analysis of its constituent peptides, which are produced by either chemical or
enzymatic treatment of the sample.
When two or more sets of mass separators and analyzers are configured in a
series, they are able to separate different molecular ions, generate fragment ions from
a selected ion, and then mass measure the fragmented ions. The fragmented ions are
used for structural determination of parental molecular ions. Typically, tandem MS
13
experiments are performed by colliding a selected ion with inert gas molecules such
as argon or helium, and the resulting fragments are mass analyzed. Tandem mass
analysis is used to sequence peptides, and structurally characterize carbohydrates,
small oligo-nucleotides, and lipids (Kinter, 2000).
SUMOylation was first discovered in 1996 when a RAN GTPase named
RNAGAP1 was found to be modified by SUMO-1 protein. Following this discovery,
a limited number of other proteins that involves in a variety of cellular functions and
resides in distinct subcellular domains were reportedly subjected to this modification.
It was then understood only after these studies that, unlike other proteins post-
translational modifications, SUMOylation exists in a very low abundance in nature,
with only a few exceptions. In overall, in vivo SUMOylation of substrates beyond
those exceptions could only be detected in the condition of overexpressing both
those substrates and SUMO-1 protein. Therefore this low abundance in
SUMOylation does pose a huge barrier for revealing the identities of SUMOylation
targets. Nonetheless with the advance of various proteomic technologies—
improvement in the sensitivity and the automation of mass spectrometry, scientists
were now able to identify SUMOylated substrates in less quantity than previously
needed by, for example, Edman degradation, thus overcoming this natural problem.
As mentioned previously, a number of proteins that are subjected to
SUMOylation had been reported before the start of this project, but a large-scale
screening for SUMOylation targets was yet to be done. The goal of this project is to
provide reliable information about the SUMOylation targets on a global basis.
14
Materials and methods
Cell Culture. HEK293 cells were maintained in Dulbecco’s modified Eagle’s
medium with 10% fetal bovine serum and antibiotics in a humidified atmosphere of
37°C and 5% CO
2
. HEK293 cells stably transfected with FLAG-SUMO-1 were
maintained in the same condition with the additive of 300 μg/ml Gentamycin.
Antibodies. The anti-FLAG agarose, FLAG peptide, and anti-FLAG mouse
monoclonal antibody were purchase from Sigma-Aldrich Inc.
Construction of FLAG-SUMO-1 stably transfected HEK293 cells. Full-length
SUMO-1 in pCMV Tag 2A vector (Stratagene) was transfected into HEK293 cells
with lipofectamine 2000 according to the manufacturer’s manual, followed by the
addition of 300 μg/ml Gentamycin sulfate starting at the 30th hour post-transfection
for two weeks to generate the pool of Flag-SUMO-1 stably integrated cells. The
expression of FLAG-SUMO-1 was verified by immunostaining with the antibody
(Sigma) against FLAG epitope and a green-fluorescence (GFP)-conjugated
secondary antibody against mouse IgG. The GFP signal was detected in nearly 100%
of cells examined.
Affinity purification of cellular SUMOylated proteins. Whole cell extract was
prepared with RIPA buffer (50 mM Tris-HCl, 50 mM NaCl, 0.5% NP-40, 0.5%
sodium deoxycholate, 0.1% SDS, pH 7.4) supplemented with 10 mM NEM (N-
15
ethylmaleimide, Sigma). Flag affinity purification was performed with anti-Flag M2
agarose (Sigma) and non-specific binding was washed off with twenty bed volumes
of RIPA buffer for three times. Immunoprecipitates were then eluted with 150 μg/ml
Flag peptide (Sigma) in PBS, and concentrated with Microcon YM-50 concentrator
where Flag peptide and Flag-SUMO-1 were removed. Prior to subjecting to mass
spectrometric analysis, eluted proteins were digested with sequencing-grade
modified trypsin (Promega) at 100:1 ratio twice, and the digestion mixture was
desalted by passing through ZipTip C18 resins.
Liquid chromatography-ion trap mass spectrometry. Mass spectrometric analysis
of the trypsin-digested, SUMOylated proteins was performed using a Thermo
Finnigan LCQ Deca XP Plus mass spectrometer with RP-LC implemented with an
Ultra Plus II LC system (Micro-Tech Scientific) using a 150 mm x 75 μm C-18
reverse-phase (RP) column (5 μm 300 Å particles) from Micro-Tech Scientific.
Peptides were loaded onto a Michrom Bioresources peptide cap trap at 95% solvent
A (2% acetonitrile, 0.1% formic acid) and 5% solvent B (95% acetonitrile, 0.1%
formic acid) and then eluted with a linear gradient of 5-60% solvent B for 65 min
and 60-90% solvent B for 10 min. Tandem MS/MS spectra were acquired with
Xcalibur 1.2 software. A full MS scan was followed by three consecutive MS/MS
scans of the top three ion peaks from the preceding full scan. Dynamic exclusion was
enabled such that after three occurrences of an ion within 1 min, the ion was placed
on the exclusion list for 3 min. Other mass spectrometric data generation parameters
16
were as follows: collision energy 35%, full scan MS mass range 400-1800 m/z,
minimum MS signal 5x10
4
counts, minimum MS/MS signal 5x10
3
counts. The mass
spectrometer was equipped with a nanospray ion source (Thermo Electron) using an
uncoated 10 μm-ID SilicaTipTM PicoTipTM nanospray emitter (New Objective,
Woburn, MA). The spray voltage of the mass spectrometer was 1.9 kV and the
heated capillary temperature was 180°C.
Analysis of MS spectra. A Beta test version of Bioworks (Bioworks 3.1) on a nine
node (2-cpu/node) cluster computer from Thermo Electron utilizing the SEQUEST
algorithm was used to determine cross correlation scores between acquired spectra
and human KAP1 protein sequence (NCBI Accession #NP_005753). SEQUEST
parameters included threshold: 1000; monoisotopic; enzyme: trypsin; charge state:
auto. For peptide identification, spectra passing a threshold of cross-correlation vs.
charge state (1.5 for +1 ions, 2.0 for +2 ions, 2.5 for +3 ions) were then inspected to
verify that all major ions were identified. MS/MS spectra were also manually
validated.
Results
The basic strategy for identifying SUMO-1-conjugated substrates is to pull
these covalently modified proteins from cell extract by immunoprecipitation. Due to
the lack of an antibody that can suffice this purpose, I introduced ectopic FLAG-
tagged SUMO-1 into HEK293 cells by stable transfection. In contrast to transient
17
transfection by which SUMO-1 is robustly expressed so that for the SUMOylation
reaction the production of SUMOylated substrates overrides its reversible reaction
(de-SUMOylation), therefore risking the forced SUMOylation on false substrates,
stable transfection is gradually adapted by HEK293 cells along the selection process
and ideally will reach homeostasis, thus minimizing the risk of the forced
SUMOylation. Reckoning the pull-down by antibody against FLAG tag brought
about proteins both covalently modified by SUMO-1 and also through the non-
covalent interaction, a control stable transfection with inactive SUMO-1 in which its
essential di-glycine at the C-terminus required for the conjugation is replaced by di-
alanine, therefore making it a mock SUMO-1, was created. An immunoprecipitation
was run alongside with the same amount of the cell extract from this control stable
transfection. The immunoprecipitates were then digested with trypsin and subjected
to quadruple ion-trap tandem mass spectrometry. The digested peptides were
resolved by a reverse-phase C18 column against the gradient of an organic phase
(acetone) before entering the mass spectrometer. A flowchart of the identification
strategy is shown in figure 1.
The twenty-three proteins identified by the tandem mass spectrometry are
summarized in table 1, where fragmented peptides recovered by MS/MS were shown
in table 2. Theses candidates shown here were seized in a relatively harsh screening
environment with the SEQUEST algorithm, by setting stringent thresholds of cross-
correlation for the fragmented ions of different charges (1.5 for +1 ions; 2 for +2 ions,
2.5 for +3 ions) recovered during the sequencing process. Only the candidates with at
18
SUMO substrates
Flag-SUMO1-GG
Affinity Purification
Tryptic Digestion
LC-MS/MS Identification
proteins identified with satisfactory recovery of
primary amino acid sequence but not found from
control purification
Flag-SUMO1-AA
Collaboration
with Dr. Austin Yang
(Inconjugatable)
Figure 1. Flowchart of affinity purification and tandem mass spectrometry used
in the identification of global SUMOylation substrates. Two expression constructs,
FLAG-SUMO-1 and FLAG-SUMO-1-AA that the C-terminal di-glycine of SUMO-1
required for its covalent conjugation to its targets was replaced by di-alanine, were
stably-transfected into HEK293 cells. The selection of cells that expressed FLAG-
SUMO-1 or FLAG-SUMO1-AA was done in the presence of 300 μg/ml gentamycin
for three weeks post-transfection. These two lines of cells were then harvested and
underwent anti-FLAG purification, and the eluted proteins were subjected to ESI-
quadrupole ion trap mass spectrometry.
19
Classification Name Function ΨKXE MW Disease Status
SUMOylation Components
UBL1 Yes
RANGAP1 Yes
SUMO2 Yes
RANBP2 Yes
Cell Surface Porteins
Neurexin 4 Yes 1381
Clathrin HC-like 2 Yes 1675
Claudin 6 Tight Junction Yes 220
PTK7 Multi 1070 colon carcinoma
Lipid Metabolism
Fatty acid synthase No 2509
Proteins with unknown function
KIAA0596 Multi 1910
Transcription Cofactors
Neurogenin 1 No
OneCut2 No 485
Translational Control
DDX9 RNA helicase
eIF4A1 Yes 406
Energy Metabolism
PKM2 Pyruvate kinase
Mitochondrial Protein
SLC25A6 ATP/ADP antiporter No
Ion Channel
CACNA1S Calcium Channel, Voltage dep Multi 1873
Junctophilin 3 Intracellular Ion Channel Multi 748 HD
Cytokines
GDF9 Oocyte growth & function No 454
Nucleic Acid Metabolism
RRM2 Catalyze DNA from RNA Yes 389
Signal Transduction
PP2C Ser/Thr phosphatase 479
PRKG2 Ser/Thr kinase Multi 762
STK38 Ser/Thr kinase Yes 465
Table 1. Global SUMOylation targets identified from proteomics study.
Abbreviation for the target protein names: PTK7, protein tyrosine kinase 7; DDX9,
DEAD/H (Asp-Glu-Ala-Asp/His) box polypeptide 9; eIF4A1, eukaryotic initiation
factor 4 isoform A1; PKM2, pyruvate kinase M2; SLC25A6, solute carrier family 25,
member A6; CACNA1S, calcium channel, L type, α1 polypeptide, isoform 3; GDF9,
growth differentiation factor 9; RRM2, ribonucleotide reductase M2 polypeptide;
PP2C, protein phosphatase 2C; PRGK2, cGMP-dependent protein kinase type II;
STK38, Ndr Ser/Thr kinase.
20
Name Peptides identified Name Peptides identified
PP2C K.HIYFINC*GDSR.A KAP1 K.IVAERPGTNSTGPAPMAPPR.A
NM_152542 R.SGSTAVGVMISPK.H NM_005762 K.VTEGQQER.L
K.SGSALELSVENVK.N R.APGPLSK.Q
R.QLLEEMLTSYR.L Claudin 6 R.DFYNPLVAEAQK.R
K.GPTEQLVSPEPEVYEILR.A NM_021195 R.YSTSAPAISR.G
K.SRLEVSDDLENVC*NWVVDTC*LHK.G PTK7 R.VTC*LPPK.G
Fatty acid synthase K.LPEDPLLSGLLDSPALK.A NM_002821 R.AHVQLTVAVFITFK.V
NM_004104 R.DNLEFFLAGIGR.L K.VSALGLSK.D
R.DGVVRPLK.C KIAA0596 K.FWYLDDSK.T
PRKG2 K.PENLILDAEGYLK.L NM_014994 R.MISC*GADK.S
NM_006259 K.QQEHVYSEK.R R.FLLQVQTRPLR.E
K.LVDFGFAK.K Neurogenin 1 R.MHNLNAALDALR.S
eIF4A1 R.KGVAINMVTEEDKR.T NM_006161 R.GAPNISR.A
NM_001416 K.MFVLDEADEMLSR.G OneCut2 R.YSIPQAIFAQR.V
SLC25A6 K.LLLQVQHASK.Q NM_004852 R.LVFTDLQR.R
NM_001636 R.GNLANVIR.Y CACNA1S R.VLLSLFTTEMLMK.M
PKM2 R.LAPITSDPTEATAVGAVEASFK.C NM_000069 K.IDEFESNVNEVK.D
NM_182470 R.GDLGIEIPAEK.V K.IALVDGTQINR.N
STK38 R.IGAPGVEEIK.S R.VLGPHSKPC*VEMLK.G
NM_007271 K.EQVGHIR.D Junctophilin 3 K.AHGHGVC*TGPK.G
Clathrin HC-like 2 R.KFDVNTSAVQVLIEHIGNLDR.A NM_020655 K.AEAALTAAQK.A
NM_004859 K.EAIDSYIK.A GDF9 R.GQETVSSELK.K
R.AMLSANIR.Q NM_005260 K.WDNWIVAPHR.Y
DDX9 K.TTQVPQFILDDFIQNDR.A RRM2 K.AAAPGVEDEPLLR.R
NM_001357 K.LAQFEPSQR.Q NM_001034 R.EIIINAVR.I
R.LSMSQLNEK.E R.LMLELGFSK.V
Table 2. Fragmented ions identified from tandem mass spectrometry. Sites of the
tryptic cleavage were indicated by dots, and asterisks denoted the cysteines of a
given proteins that were alkylated during the tryptic digestion process. NM numbers
right below each given protein names are the gene accession numbers for the NCBI
Entrez database.
21
least three fragmented ions that passed the SEQUEST screen were shown, thus
greatly bolstering the confidence level on the authenticity of their SUMOylation and
at the same time minimizing the risk of reporting false positives. Among those with
the most ions identified by MS/MS, each of SUMO-1 (a.k.a, Ubl1), SUMO-2,
RANGAP1, and RANBP2 were recovered with more than 40% of their primary
amino acid sequences. Since RANGAP1 and RANBP2 have been well-known for
their SUMOylation, my result evidences the success in the specificity of this
purification and identification strategy. The recovery rate of the fragmented ions
from the other candidates listed in table 1 ranges from 20% (95 out of 479 amino
acids) for protein phosphatase PP2C to 1.5% (28 out of 1910 amino acids) for
hypothetical protein KIAA0596. On the other hand, most of the fragmented ions
retained from the control immunoprecipitation fell through the SEQUEST screen
with the only exception of one peptide from GDF9 (
330
KPLGPASFNLSEYFR
344
),
indicating that GDF9 may have interacted with SUMO-1 through non-covalent
interaction. This proved the immunoprecipitation with high ionic-strength RIPA
buffer did yield a high-quality purification and identification of bona fide
SUMOylation substrates at the same time eliminating the co-purification of those
interactions due to the non-covalent binding. The fragmented ions from one of the
candidates, PKM2, matched different isoforms, so it needs further investigation to
clarify whether all of these PKM2 isoforms are capable of being SUMOylated.
Looking into the functions of those SUMOylation candidates, they are sorted
into twelve categories. Despite of those candidates with nuclear functions—for
22
example, Neurogenin 1, OneCut 2, and RRM2, I do find that those candidates
participate in a variety of cellular functions, and have different appearances in term
of their cellular localizations. Interestingly enough, the identification even yielded
some cell surface proteins, translational control components, mitochondrial proteins,
and ion channel constituents that are never reported to be subjected to protein
SUMOylation.
Despite of its superior specificity of this affinity purification-tandem mass
spectrometry system, a big shortcoming does exist: This system can only yield
“candidates” of protein SUMOylation rather than the immediate and direct
identification of SUMOylation sites in these candidates. Hence these candidates are
still needed to be manually verified for their SUMOylation by either in vivo or in
vitro SUMOylation assay. Of these candidates, our laboratory is particularly
interested in cell signaling molecules like PP2C and STK38, since SUMOylation has
been proposed to modulate protein-protein interaction, therefore regulating the
biological outcomes through the change of their interactions. The putative
SUMOylation sites in these two proteins and the results of the in vivo and the in
vitro SUMOylation assays are summarized in table 3. It surprises me that, even with
high confidence scores yielded by MS/MS screening procedure, these two candidates
did not seem to be SUMOylated, if not at all. Especially in the case of PP2C, the
sequence coverage after MS/MS identification reached a high 20%, making it
extremely unlikely to be false positive, and yet the detection of its SUMOylation
failed. This result probably reflects two facts: (1) the extremely low abundance of
23
Name Putative
SUMOylation sites
SUMOylated
in vivo
SUMOylated
in vitro
PP2C
302
VKKE
No No
STK38
57
LKDE,
213
IKPD
No No
KAP1
554
VKEE,
575
ETKP,
676
LKEE,
779
EDKA,
804
DTKE
Yes Yes
Table 3. The putative SUMOylation sites and the results of in vivo and in vitro
SUMOylation assay for the signal transduction molecules PP2C, STK38, and
KAP1. The positions of those putative SUMOylation sites in their target proteins are
as indicated.
24
most protein SUMOylation makes the detection by conventional immunoblotting
very difficult, even with the high-quality antibody (e.g., anti-FLAG antibody); (2)
mass spectrometry is very sensitive in picking up trace amount of bio-molecules or
oligopeptides. The failure in detecting PP2C SUMOylation may also indicate that the
high coverage rate of the fragmented ions from tandem mass source need not
positively correlate with its capability of being SUMOylated.
Next a transcriptional co-repressor named KAP1 (KRAB domain-associated
protein 1) was snapped off the list for testing. From numerous previous studies in
protein SUMOylation, it was concluded that most SUMOylation occurs to
transcriptional factors, co-factors, or nuclear proteins, consequently modulating their
functions or nuclear localization. Indeed, KAP1 was proved a SUMOylation target in
my in vivo and in vitro SUMOylation assays (table 3).
Discussion
From my study on the search for the targets of SUMO-1-mediated global
SUMOylation, twenty-three proteins were seized through the stringent SEQUEST
filtering criteria following FLAG affinity purification and tandem mass spectrometry.
In agreement with several recent reports employing the same proteomic strategy that
in general discloses that a substantially higher fraction of the SUMOylation targets
are nuclear or nucleolar proteins, my result concurs this conclusion. About half of the
candidates (12 of 23) identified herein are nuclear or nucleolar proteins. Nonetheless
the overall number of candidates pulled by this strategy seems much less than those
25
from other reports. This could be largely due to the harsh affinity purification
procedure I used, in which high-strength RIPA buffer was used throughout the
purification process. Those putative SUMOylation substrates with fewer SUMO-1
modifications—say, single or double SUMO-1 conjugations—may have been lost in
the RIPA buffer during the washes. Or those SUMOylated proteins just simply did
not make the cut of the stringent filtering criteria. Indeed, if lowering the screening
threshold, the SEQUEST program does offer substantially more hits that includes
nuclear and nucleolar proteins, cytoplasmic, and cell-surface proteins. Nonetheless
my goal for this multi-stage screening is to acquire high-quality SUMOylation
candidates, and lowering the screening threshold does undermine the statistical
confidence on the identification itself. Also the control purification and identification
using the inactive SUMO-1 mutant led to only one identification hit, demonstrating
the non-covalent interactions between SUMO-1 and its interacting partners have
been effectively eliminated under the experimental conditions, further bolstering the
specificity and significance of the discovery in those twenty-three SUMOylation
targets.
Given the high statistical confidence and the need to validate the
SUMOylation of the individual candidates revealed by tandem mass spectrometry, I
went ahead testing the SUMOylation of three of them. Surprisingly, two known
signaling molecules—a protein phosphatase PP2C and a protein kinase STK38—
failed in the in vivo SUMOylation assay, while the SUMOylation of the
transcriptional co-repressor KAP1 was successfully validated by both in vivo and in
26
vitro assays. PP2C and STK38 are known nuclear proteins, where PP2C regulates
cell-cycle activity and STK38 controls the completion of mitosis. Given that they are
all nuclear proteins and yields comparable statistical scores from SEQUEST data
processing, it is very interesting there is a huge contrast in the level of their
SUMOylation, where KAP1 is SUMOylated fairly nice and the other two could
barely be detected for their SUMOylations. In the biological sense this great
discrepancy could be the result of the distinct temporal or spatial control, or both, of
their respective SUMOylation machineries, and that put the SUMOylation of PP2C
and STK38 in very tight regulation. It is known many ubiquitylation and
SUMOylation E3 ligases possess RING domain that serves as a platform for the
assembly of macromolecular complexes, and under certain circumstances these
macromolecular assemblies can be induced by a wild array of stimuli and stresses--
some of these nuclear domains have even been found existing in specific types of
sub-nuclear structures (Kentsis et al., 2002). Therefore it is plausible that the simple
overexpression of SUMO-1 does not suffice the requisite of activating functional
SUMOylation machineries and of forming their corresponding sub-nuclear structures
to SUMOylating PP2C and STK38. With this in mind, giving cells the right cue may
be nothing but essential for the SUMOylation of these two proteins.
In a nutshell I believe all these twenty-three proteins are bona fide
SUMOylation targets, and this study exemplifies how diverse in cellular functions
the SUMOylation can participate in.
27
Chapter Three
DOXORUBICIN DOWNREGULATES KAP1 SUMOYLATION THAT
RELIEVES ITS TRANSCRIPTIONAL REPRESSION ON P21 IN BREAST
CANCER MCF-7 CELLS
Summary
The role of post-translational modification, such as SUMOylation, in
modulating the efficacy of doxorubicin (Dox)-treatment remains unclear.
Transcriptional co-factor KAP1 has been shown to complex with the KRAB zinc
finger protein, ZBRK1, to repress the transcription of target genes. Through a
combination of proteomic screening and site-directed mutagenesis approaches, we
have identified lysine 554, 779 and 804 as the major SUMOylation sites in KAP1.
We then present evidence that Dox-mediated induction of cell cycle regulator p21
expression is differentially regulated by KAP1 SUMOylation status. Moreover, the
KAP1 SUMOylation level was transiently decreased upon Dox-exposure, and
transfection with KAP1 SUMOylation mimetic, SUMO-1-KAP1, desensitizes breast
cancer MCF-7 cells to Dox-elicited cell death. The SUMOylation-dependent
stimulation of KAP1 function is achieved by enhancing the methylation of H3-K9
and attenuating the acetylation of H3-K9 and H3-K14 at the p21 core promoter. We
also show that occupancy of ZBRK1 response elements located at the p21 promoter
by ZBRK1/KAP1 is independent of KAP1 SUMOylation. Hence, SUMOylation of
28
KAP1 represses p21 transcription via a chromatin silencing process without affecting
interaction between KAP1/ZBRK1 and DNA, thus providing a novel mechanistic
basis for the understanding of Dox-induced de-repression of p21 transcription. Taken
together, our results suggest that Dox-induced decrease in KAP1 SUMOylation is
essential for Dox to induce p21 expression and subsequent cell growth inhibition in
MCF-7 cells.
Background
In the previous chapter I reported the successful identification of twenty-three
SUMOylation targets, and the authentic SUMOylation of the transcriptional co-
repressor KAP1 as well. In this chapter a follow-up study is reported here on how its
SUMOylation influence the KAP1-mediated transcriptional activity.
KAP1 (KRAB domain-associated protein 1) was first identified in a yeast two-
hybrid screening using the KRAB (Krüppel boxes A and B) domain of KOX1 as the
bait (Friedman et al., 1996; Kim et al., 1996; Le Douarin et al., 1996; Moosmann et
al., 1996). About a third of all 750 zinc finger proteins belong to KRAB zinc finger
(KZF) protein family. Although some of KZF proteins are found to repress the
transcription mediated by RNA polymerases I and II, most member of this family are
not yet known for their target genes and their regulatory mechanisms have not been
well-studied (Urrutia, 2003). The transcriptional repressor activity of KZF proteins is
dictated by KRAB domain, and interestingly, shuffling of the KRAB domain of
Kid1with that of KOX1—Kid1 represses RNA polymerase I-mediated transcription
29
while KOX1 represses RNA polymerase II-mediated transcription--failed to uphold
that repression (Moosmann et al., 1997). In addition to their KRAB domain, which is
unequivocally located in the N-terminus, the C-terminus of KZF proteins contain 10-
34 C2H2 zinc finger repeats, which bind to the specific DNA sequences of their
target genes and possibly their interacting proteins as well (Urrutia, 2003). Two of
the KZF proteins have been demonstrated to specifically interact with KAP1. KS1
(KRAB suppressor of transformation 1), which possesses ten zinc fingers at its C-
terminus, specifically binds to a 27-bp KS1 binding element (KBE) (Gebelein and
Urrutia, 2001). Its expression was found to suppress Ha-ras- and G protein-induced
transformation, probably though its suppression on the transcription of some cell
cycle regulators (e.g., CDKs and Cyclins). The other KZF protein, ZBRK1, was
identified as a BRCA1-interacting partner from a yeast two-hybrid screening, and
possesses eight zinc fingers (Chen et al., 1996). A follow-up study revealed that
ZBRK1 was able to bind a 15-bp ZBRK1 binding element, and its physical
association with BRCA1 enhanced its repression in Gal4-based transcription assays
(Zheng et al., 2000). This 15-bp ZBRK1 binding element has been found in the
regulatory regions of genes such as Gadd45 α, Gadd153, Ki-67, bax, p21, and some
tissue inhibitors of metalloproteases (TIMPs), therefore implying its function in
regulating the transcription of these genes. A recent report documented the triangular
interactions between ZBRK1, KAP1, and Epstein-Barr viral protein BBLF2/3 (Liao
et al., 2005).
30
KAP1, also known as TIF-1 β (transcriptional intermediary protein 1 β), was
originally identified from a yeast two-hybrid screening by using the KRAB domain
of KOX1 as the bait. The hallmark characteristics of the TIF family member proteins
includes their N-terminal RBCC (RING, B-Boxes 1 and 2, coiled-coil) domain,
which is also named as tripartite motif, and several well-defined domains that have
been known to function in the formation of heterochromatins. Studies on its
biochemical property revealed that the RBCC domain is capable of forming
macromolecules through homo-oligomerization or hetero-oligomerization, making it
an ideal scaffold module to assemble multifunctional subcellular domains (Peng et
al., 2002). Other RING-containing proteins like PML (promyelocytic leukemia
protein) and BRCA1 have been shown their importance in formation of particular
nuclear structures, and mutations associated with these proteins that cause the
disruption of these structures have helped explain how leukemia and breast cancers
arise (Borden, 2000). In a report that exhaustively studied the RBCC domains of
TIF-1α, β, and γ, TIF-1 α and TIF-1 γ were found to form hetero-oligomers where
TIF-1β was unable to complex with either TIF-1 α or TIF-1 γ, indicating that despite
of their homology, their RBCC domains are very selective and specific to their
interacting partners (Peng et al., 2002). Indeed, in another report swapping of the
RBCC domain of KAP1 with that from MID1 abrogated the binding of KAP1 to
KOX1, reassuring the individual specificity that lies in these RBCC-containing
proteins (Peng et al., 2000).
31
Several distinctive domains located in the C-terminal half of KAP1 that
interacts with a spectrum of chromatin remodeling activities are important for its
transcriptional co-repressor activity. The HP1 binding domain carried a minimal 5-
amino-acid PxVxL motif that was previously reported to bind HP1 (heterochromatin
protein 1) (Ryan et al., 1999). HP1 was found having high binding specificity to
methylated histone 3 lysine 9 (H3-K9), and also found interacting with a H3-K9
specific methyl-transferase Su(var)3-9 (Li et al., 2002). In a model that delineates the
HP1-mediated spreading of heterochromatin regions, HP1 was first bound to the
starters of methylated H3-K9, where overall H3-K9 methylation is low at the
beginning. In the next Su(var)3-9 is recruited to these regions and H3-K9 in the
vicinity are subsequently methylated, leading to a bi-direction rolling of H3-K9
methylation and finally the formation of heterochromatin foci at these regions
(Shilatifard, 2006). It is worth noting that the transcription at the euchromatic genes
can also be repressed by HP1(Hediger and Gasser, 2006), even though whether H3-
K9 methylation is necessary for this repression is in debate. Two other domains—
PHD finger and Bromo domains--at the C-terminus of KAP1 also mediated
transcriptional silencing by modulating histone codes. In specific, these two domains
were found cooperatively to recruit two HDACs (histone de-acetylases)—Mi-2 α of
the NuRD HDAC complex and N-CoR1 complex and another H3-K9 methyl-
transferase SETDB1 (SET domain, bifurcated 1) (Schultz et al., 2002; Schultz et al.,
2001; Underhill et al., 2000). The deacetylation and methylation at certain lysines of
histones 3 and 4 are found tightly correlated to the silencing status of local
32
chromatins, providing great support to the role of these KAP1-interacting proteins in
gene silencing.
In Gal4 reporter system, KAP1 exerts its transcriptional co-repressor activity
through Gal4-DBD-fused KRAB of KOX1 in a dose-dependent manner (Friedman et
al., 1996). Exogenous expression of KAP1 in this reporter system further reduced
KRAB-mediated transcriptional repression to half, to about 20% of that with the
reporter only. A model depicting how KAP1 mediates its target gene expression was
proposed in a follow-up report by the same group (Ryan et al., 1999). In this chapter
I present several lines of evidence that further extend the application of this model. I
found the KAP1 SUMOylation acting as a switch that determines the local chromatin
status of its target genes. In specific, the methylation of histone 3 lysine 9 and the
acetylation of histone 3 lysines 9 and 14 at the p21 proximal promoter are
reciprocally regulated by the KAP1 SUMOylation. A decrease in the KAP1
SUMOylation induced by the topoisomerase II inhibitor doxorubicin (Dox) was
linked to the de-repression at the p21 proximal promoter.
Materials and methods
Cell Culture. HEK293 cells were maintained in Dulbecco’s modified Eagle’s
medium with 10% fetal bovine serum and antibiotics and MCF-7 cells were
maintained with the same medium formulation with an additive of 0.01 mg/ml
recombinant human insulin in a humidified atmosphere of 37°C and 5% CO
2
, while
33
T47D cells were maintained in RPMI 1640 medium with 10% fetal bovine serum
and antibiotics otherwise as the same conditions above.
Antibodies. The anti-FLAG agarose, FLAG peptide, and anti-FLAG mouse
monoclonal antibody were purchase from Sigma-Aldrich Inc. Mouse monoclonal
antibody against c-Myc was purchased from Santa Cruz Biotechnology Inc. Rabbit
polyclonal antibodies against acetylated histone 3 and di-methylated histone 3 lysine
9 were obtained from Upstate USA Inc.
In vivo and in vitro SUMOylation assays. The in vivo SUMOylation assay was
carried out with co-transfection of Flag-KAP1 and GFP-SUMO-1 in one-to-four
ratio. SUMOylated KAP1 was detected either by immunoblotting of whole cell
lysates or immunoprecipitation of Flag-KAP1 followed by immunoblotting against
Flag- or GFP-tag. The in vitro SUMOylation assay was performed with the
SUMOylation kit purchased from LAE Biotech International.
Co-immunoprecipitation of KAP1 mutants and ZBRK1- Transfection of Flag-
ZBRK1 and Myc-KAP1 expression construct was done with Lipofectamine 2000
(Invitrogen) according to the manufacturer’s manual. Whole cell lysates were
prepared by lysing cells with lysis buffer (50 mM Tris-HCl, pH 7.8, 100 mM NaCl,
0.5 mM MgCl
2
, 1
mM EDTA, 0.2% Nonidet P-40, 1 mM dithiothreitol, 1X protease
inhibitor cocktail from Roche) supplemented with 100 μM MG-132 (Calbiochem).
For each sample, 30 μl of anti-Flag M2 agarose was mixed with 5 mg of whole cell
34
lysates, incubating on ice for 2 h, washed with 600 μl lysis buffer three times.
Immunoprecipitates were then eluted in 50 μl of SDS sample buffer, and half of the
elution was subject to immunoblotting analysis.
Reverse transcription and real-time PCR. Total RNA from MCF-7 and T47D cells
were extracted with TRIzol reagent (Invitrogen). The p21 mRNA transcript was
reverse transcribed with the p21-specific primer (Table 4) by ThermoScript RT kit
(Invitrogen) according to its manual. A fraction of RT reaction was then amplified
with IQ SYBR Green supermix (BioRad) and the p21 specific primer pair by My IQ
real-time PCR detection system (BioRad). The fold change in p21 mRNA level was
calculated by ΔΔCt method against 18S rRNA.
Luciferase assay- The p21 promoter was PCR-cloned from a total DNA pool of
HeLa cells by DNeasy Tissue Kit (Qiagen), and was subcloned into pGL3b reporter
(Promega) as described previously (25). MCF-7 cells were co-transfected with
pGL3b-p21 reporter, internal control reporter pRL-TK, ZBRK1, KAP1, or its
mutants. Firefly luciferase activity was assayed with Dual-Luciferase Reporter
Assay System (Promega) and normalized against Renilla luciferase activity.
Chromatin immunoprecipitation (ChIP) assay. MCF-7 cells were cross-linked
with 1% formaldehyde in growth medium for 15 min. Cross-linking was stopped by
addition of glycine to a final concentration of 125 mM. Cells were rinsed with cold
PBS buffer and harvested, and subsequently swelled in 1ml RSB buffer (10 mM
35
Primer Purpose Sequence (5’-to-3’)
18S rRNA FP Real-time PCR CGGCGACGACCCATTCGAAC
18S rRNA RP RT & real-time
PCR
GAATCGAACCCTGATTCCCCGTC
p21 FP Real-time PCR TTTCTCTCGGCTCCCCATGT
p21 RP RT & real-time
PCR
GCTGTATATTCAGCATTGTGGG
p21 -20
amplicon FP
ChIP (real-time
PCR)
TATATCAGGGCCGCGCTG
p21 -20
amplicon RP
ChIP(real-time
PCR)
CTTCGGCAGCTGCTCACACCT
p21 -713
amplicon FP
ChIP(real-time
PCR)
TTTCCCTGGAGATCAGGTTG
p21 -713
amplicon RP
ChIP(real-time
PCR)
GGAAGGAGGGAATTGGAGAG
p21 -3038
amplicon FP
ChIP(real-time
PCR)
CAGGCTGGTCTCAAAACTCC
p21 -3038
amplicon RP
ChIP(real-time
PCR)
GCCTGTAATCCCAGCACTTT
Table 4. Primers used in the real-time PCR and the chromatin immuno-
precipitation experiments. RT, reverse transcription. Refer to figure 5 for position
of the amplicons used in ChIP experiments.
36
Tris-HCl, 3 mM MgCl
2
, 10 mM NaCl, 0.1% IGEPAL CA-330, pH 7.4)
supplemented with protease inhibitor cocktail (Roche). Nuclei were then collected
and resuspended in nuclear sonication buffer (50 mM Tris-HCl, 10 mM EDTA, 1%
SDS, pH 8.1). Sonication was then performed to shear DNA to 500-bp or less.
Samples were then diluted with 4 volumes of dilution buffer (6.7 mM Tris-HCl, 167
mM NaCl, 1.2 mM EDTA, 1.1% Triton X-100, 0.01% SDS) before precleared with
20 μl 50% protein A/G agarose. Approximate 3-5 μg of antibodies against acetylated
histone H3 or di-methylated H3-K9 were incubated with post-clearance supernatant
overnight at 4°C, and protein A/G or Flag M2 agarose that has been pre-incubated
with 100 μg/ml tRNA and 0.1% BSA were added to the mixture the following day
and incubated for additional 2 h. Washes were performed with LiCl buffer twice
(100 mM Tris-HCl, 500 mM LiCl, 1% deoxycholate, 1% NP-40, pH 8.0), high salt
buffer twice (20 mM Tris-HCl, 500 mM NaCl, 2 mM EDTA, 0.5% Triton X-100,
0.2% SDS, pH 8.0), and low salt buffer twice (same as high salt buffer except NaCl
concentration at 150 mM), and 2X TE buffer twice (20 mM Tris-HCl, 2 mM EDTA,
pH 8.0). Immunoprecipitates were eluted in 2% SDS in 2X TE buffer, and
subsequently treated with RNase (1 h) and protease K (Sigma) (1 h), respectively.
Cross-linking was reversed by incubation at 65°C overnight, and recovered DNA
was extracted with phenol/chloroform and ethanol-precipitated. DNA of the
designated amplicons were amplified with specific primer pairs (Table 4) using My
IQ real-time PCR detection system and IQ SYBR Green supermix (BioRad).
37
Normal sera and input DNA values were used to subtract and normalize the values
from ChIP samples.
Cell viability assay. MCF-7 cells were seeded into 24-well plates to obtain a
confluency of 50% on the day of the experiment. The cells were treated with vehicle
or 2.5 μM Dox and medium was changed daily for 3 days. Seventy-two hours after
the start of treatment, 0.2 ml of 0.1 mg/ml 3-[4,5-dimethylthiazol-2-yl]-2,5-
diphenyltetrazolium bromide (MTT) (Sigma, St. Louis, MO) in OptiMEM I
(Invitrogen, CA) was added to each well and the plate was incubated at 37°C for an
additional 1.5 h. After the incubation, the MTT solution was aspirated and 0.2 ml
isopropanol was added to each well to dissolve the formazan crystals. Absorbance
was read immediately at 540 nm in a scanning multiwell spectrophotometer. The
results were depicted as percentage of cell viability, reported as the mean ± S.D. of
three independent experiments performed in triplicates.
Statistical analysis. The p values from two tailed t-test were calculated by using
Microsoft Excel spreadsheet program and assuming equal variances. The difference
between two data sets is significant when p value is smaller than 0.05.
Results
By liquid chromatography-linked tandem mass spectrometry, KAP1 was among
twenty-three proteins that were seized with strict filtering parameters by the
38
SEQUEST program (refer to experimental procedures for details). The mass
spectrum with y-ion and b-ion assignments of KAP1 amino acid 408-427 following
collision-induced dissociation is shown in figure 2A.
Next we verified this SUMOylation with both in vivo and in vitro SUMOylation
systems. Co-transfection of KAP1 with GFP-SUMO-1 results multiple molecular
mass shifts on top of the unmodified FLAG-KAP1, roughly matching the
incremental masses of 50-kDa of GFP-SUMO-1, as observed from immunoblotting
in figure 2B. Holding the expression of KAP1 constant, decreasing expression of
GFP-SUMO-1 attenuates the intensity of SUMOylation ladders, providing extra
evidence on the specificity of its SUMOylation (lanes 4-6). With purified KAP1
from HEK293 cells, the in vitro SUMOylation assay with SUMOylation
E1(SAE1/ASE2 heterodimers) and E2 (Ubc9) enzymes purified from bacteria, also
evidences this multiple mass shifts (lanes 5 and 6, figure 2C), thus reaffirming this
authentication.
Previously KAP1 has been demonstrated to exert its transcriptional co-repressor
activity through its interaction with KRAB-domain-containing zinc finger (KZF)
proteins. Since its N-terminus RBCC domain is primarily responsible for its
interaction with KZF proteins and its C-terminus harbors several domains recruiting
a number of chromatin remodeling activities, it presumes that SUMOylation can
regulate either the interaction between KAP1 and its cognate KZF partner, or
between KAP1 and its subordinate chromatin remodeling activities. Given the high
probability of SUMOylation occurring to consensus ΨKXE sequence, we scan
39
Figure 2. Transcriptional co-repressor KAP1 is subjected to multi-
SUMOylation. (A) Mass spectrum of fragmented KAP1 (amino acid residues 408 to
427) by de novo sequencing following anti-Flag affinity purification. (B) In vivo
SUMOylation of KAP1. Flag-KAP1 and GFP-SUMO-1 were co-transfected into
HEK293 cells, and immunoblotting was performed with an anti-Flag antibody.
UnSUMOylated KAP1 and SUMO-1-modified KAP1 are indicated by an arrow and
asterisks respectively. (C) KAP1 is SUMOylated in vitro. The reactions were
carried out with purified KAP1 from HEK293 cells, SUMOylation E1 (Aos1/Uba2
heterodimer), E2 (Ubc9), and E3 (RANBP2) enzymes purified from E. coli. in
ATP/Mg
2+
buffer.
40
Figure 2
A
B
M.W.
(kDa)
250
150
100
WB: anti-Flag
Flag-KAP1
Flag-KAP1
++
1 2 3 4 5 6 7
-
++ + + -
GFP-SUMO-1
-
+ + -
B
M.W.
(kDa)
250
150
100
M.W.
(kDa)
250
150
100
WB: anti-Flag
Flag-KAP1
WB: anti-Flag
Flag-KAP1
Flag-KAP1
++
1 2 3 4 5 6 7
-
++ + + -
GFP-SUMO-1
-
+ + -
human KAP1 human KAP1 human KAP1 human KAP1
Relative Absorbance
m/z
human KAP1 human KAP1 human KAP1 human KAP1
Relative Absorbance
m/z
C
WB: anti-Flag
Flag-KAP1
WB: anti-Flag
Flag-KAP1
M.W.
(kDa)
250
150
100
ATP
+ -
++
SAE1/SAE2
+ +
+ +
+
UBC9
+ ++
SUMO-1 +
+
++ -
1 2 3 4 1 2 3 4
41
KAP1 for any match to this motif. Out of six consensus motives found, five are
located on the C-terminus of KAP1. Shown in figure 3A, among these five
sequences two are read in N- to C-terminus orientation (K554: VKEE, K676: LKEE)
and the others are read in reverse (K575: ETKP, K779: EDKA, K804: DTKE).
Mutations of these lysines to arginines, therefore abrogating the SUMOylation at
these sites, were made to pin the exact SUMO-1 acceptor lysines with the in vivo
SUMOylation assay. As shown in figure 3B, KAP1 mutants carrying alteration at
either K554R, K779R, or K804R, but not K575R or K676R, showed great reduction
in SUMOylation. K554R mutant not only showed extinguishment of the higher
SUMOylation mass shifts but the doubly SUMOylated band at around 230-kDa was
quenched. The 3K/R mutant that combines K554R, K779R, and K804R mutations
was come to an abrupt deprivation of all forms of SUMOylation. On the contrary
2K/R mutant that combines K575R and K676R preserved nearly intact
SUMOylation to the wild-type KAP1. Therefore we concluded that K554, K779, and
K804 are the prime sites for KAP1 SUMOylation.
As stated in the previous section, our takes in the function of KAP1
SUMOylation are that either it can modulate its physical interaction with KZF
proteins, therefore regulating KAP1 co-repressor activity available to KZF proteins,
or it can directly modulate the interaction between KAP1 and its associated
chromatin remodeling activities. Since the prime SUMOylation sites located in the
KAP1 C-terminus half, it is more likely the latter is true. However we’d still like to
crack down its interaction with KZF protein within the context of its SUMOylation.
42
B
A
K804: DTKF
65 418 627 669 697 801 835 1 483 510
K554: VKEE
K575: ETKP
K676: LKEE
K779: EDKA
RBCC
HP1-
BD
PHD Bromo
IP: Anti-Flag
IB: Anti-GFP
IB: Anti-Flag
1 2 3 4 5 6 7 8 9
KAP1
WT
K554R
K575R
K676R
K779R
K804R
3K/R (554, 779, 804)
2K/R (575, 676)
+ GFP-SUMO-1 + GFP-SUMO-1
WT
SUMOylated KAP1
KAP1
Figure 3. Mapping SUMO-1 acceptor sites in KAP1. (A) Consensus
SUMOylation sequences located in KAP1 outside the RBCC domain. (B) Lysines in
the putative SUMOylation sites of KAP1 were mutated to arginines individually or
in combination. Wild-type KAP1 and these mutants, together with GFP-SUMO-1
were transiently transfected into HEK293 cells for an in vivo SUMOylation assay.
KAP1 and its mutants were then enriched by immunoprecipitation with anti-Flag
agarose. SUMO-1-modified KAP1 was detected by immunoblotting against GFP
and unmodified KAP1 was probed with an anti-Flag antibody.
43
One of the KZF proteins, ZBRK1, has recently been indicated to interact with
KAP1. Several DNA damage-responsive genes were also implicated under the direct
transcriptional regulation of ZBRK1 through a 15-bp responsive element originally
identified in the third intron of gadd45α gene, thus making ZBRK1-KAP1 an
attractive system to investigate the role of the KAP1 SUMOylation in the
transcriptional control to these DNA damage-responsive genes. As shown in figure
4A, co-immunoprecipitation of ectopically expressed ZBRK1 with either SUMO-1-
KAP1 fusion protein in which active SUMO less its very C-terminus diglycines was
fused to KAP1 N-terminus, thus making it both mimetic to KAP1 SUMOylation and
inhydrolysable by the SUMO protease, or KAP1 3K/R mutant that rendered it
deficiency in SUMOylation, did not appear to change their interactions significantly,
with or without topoisomerase II inhibitor Doxorubicin. Therefore this result rejected
the idea that KAP SUMOylation regulates its interaction with ZBRK1, and
implicates that ZBRK1 and KAP1 form a stable transcriptional complex regardless
of its SUMOylation status.
ZRBK1 was previously reported to undergo ubiquitin-mediated proteasomal
degradation upon methyl methane-sulfonate (MMS) treatment (Yun and Lee, 2003).
Co-immunoprecipitation experiment demonstrates the stable complex of ZBRK1 and
KAP1 was lost after MMS treatment due to ZBRK1 degradation.
In an effort to assess the relationship between KAP SUMOylation status and its
transcriptional co-repressor activity, we have concluded in the previous section that
KAP1 SUMOylation-mimetic mutant (SUMO-1-KAP1 fusion protein) and
44
Figure 4. KAP1 SUMOylation-mimetic SUMO-1-KAP1 and SUMOylation-
defective KAP1(3K/R) exhibit comparable binding to ZBRK1. (A) HEK293
cells were transiently co-transfected with a combination of KAP1 and ZBRK1
expression constructs, as indicated. After recovery, cells were then treated with 1
μM Dox or 6 μM MMS at 27 h post-transfection for 3 h. Immunoprecipitation and
immunoblotting were then performed with antibodies as indicated. HC:
immunoglobulin heavy chain. (B) MCF-7 and T47D cells were treated with 1 μM
Dox for 3 h and their p21 mRNA levels were assessed by quantitative real time RT-
PCR. Result represents mean + standard deviation from three independent
experiments.
45
Figure 4
12 345 6
Dox –+ – + ––
MMS ––– –+ +
Flag-ZBRK1 +++ +++
Myc-SUMO-1-KAP1++– – +–
Myc-KAP1(3K/R) ––+ + –+
IP: Anti-Flag
IB: Anti-Myc
IP: Anti-Flag
IB: Anti-Flag
ZBRK1
KAP1
SUMO1-KAP1
KAP1 1/5 input
IB: Anti-Myc
SUMO1-KAP1
KAP1
A
B
Fold Induction Fold Induction
0
1
2
3
4
5
6
Dox MMS
p21 mRNA
MCF-7
T47D
0
1
2
3
4
5
6
0
1
2
3
4
5
6
Dox MMS
p21 mRNA
MCF-7
T47D
MCF-7 MCF-7
T47D
46
SUMOylation-defective mutant (KAP1 3K/R) did not change their interactions with
ZBRK1 in the presence or absence of Doxorubicin treatment. Next we went off
testing whether these two mutants that represent the two extremities in KAP1
SUMOylation status pose profound impact on p21 transcription. The p21 promoter
encompasses several crucial regulatory DNA elements: (1) TATA box at -24 to -27
(relative to the transcription start site at +1 nucleotide), (2) three ZBRK1 binding
elements at -318 to -332, -797 to -811l, and -910 to -924, (3) four p53 binding
elements at -1360 to -1379, -2242 to -2261, -2290 to -2312, -2303 to -2327. The
effect of KAP1 mutants was evaluated by co-transfecting their expression plasmids
and a luciferase reporter pGL3b-p21 harboring 2.4-kb p21 promoter. Shown in figure
5A, SUMO-1-KAP1 potently suppressed Doxorubicin-induced p21 promoter activity
(2.55-fold of bar 2 over bar 5), where unstimulated p21 promoter activities were
merely changed between MCF-7 cells transfected with KAP1 and SUMO-1-KAP1
(bars 1 and 4). Furthermore, this strong suppression exerted by the expression of
SUMO-1-KAP1 was virtually lost with MMS treatment, conveying that this
suppression was mandated by ZBRK1-KAP1 stable complex.
On the other hand, the expression of KAP1 SUMOylation-defective mutants
K554R and 3K/R de-repressed unstimulated p21 promoter activity (bars 7 and 9,
compared to bar 1), while mutant 3K/R marginally enhanced Doxorubicin-induced
p21 promoter activity (1.5-fold, bars 10 and 2) and mutant K554R posed no further
increase in this respect. KAP1 mutant 2K/R, which behaved alike its wild-type
counterpart in the previous SUMOylation assay, bore no change to either
47
unstimulated or Doxorubicin-induced p21 promoter activity, further strengthening
that the observations from mutant K554R and 3K/R were SUMOylation-dependent.
The de-repression of unstimulated p21 promoter activity by mutant K554R or 3K/R
was recapitulated in figure 5B: 2-fold from K554R and 3.14-fold from 3K/R, over
MCF-7 cells transfected with wild-type KAP1.
Previously we postulated, with the fact that all SUMO-1 acceptor lysines in
KAP1 are located in its C-terminus half, and given the knowledge that its C-terminus
interacts with a number of chromatin remodeling activities, KAP1 SUMOylation
may well be destined to regulate local chromatin structure where ZBRK1-KAP1
stable complex is present. Now our transcriptional assay seems to bolster this notion
since MMS treatment bypasses this point of regulation where ZBRK1-KAP1
complex is non-existent.
The chromatin remodeling activities with which KAP1 partners includes
ESET/SETDB1, which is a H3-K9 methyltransferase, NuRD HDAC complex, and
heterochromatin protein HP1, which complexes with H3-K9 methyltransferase
SUV39H1 to silent transcription. It has been established that H3-K9 and H3-K14
acetylation facilitates the transcriptional activation by de-condensing
transcriptionally inactive chromatin, thus making it more accessible to transcriptional
factors and transcription initiation complex at the proximal promoter, while H3-K9
methylation silences transcription in the converse way. Therefore next we elected to
investigate the underlying mechanism that explains the suppression of Doxorubicin-
induced p21 transcription by SUMO-1-KAP1 and the de-repression by KAP1 3K/R.
48
Figure 5. Suppression of Dox-induced p21 transcription by SUMO-1-KAP1 and
alleviation of transcriptional repression by KAP1(3K/R). (A) MCF-7 cells were
co-transfected with pGL3b-p21 reporter, pRL-TK, ZBRK1 and variousKAP1
mutants, as indicated. Cells were 1 μM Dox or 6 μM MMS at 30 h post-transfection
for 3 h, followed by luciferase assays. Result represents mean + standard deviation
from three independent experiments, after normalization for transfection efficiency.
∗ denotes p=0.08; ∗∗ denotes p=0.78; † denotes p=0.01; ‡ denotes p=0.009 between
corresponding samples. (B) The expression levels of transiently transfected KAP1
and its mutants are comparable in MCF-7 cells. (C) Un-stimulated p21 reporter
activities in MCF-7 cells transfected with various engineered KAP1 mutants are
compared with that of MCF-7 cells transfected with wild-type KAP1.
49
Figure 5
A
12 3 4 5 67 8 9 10 11 12
∗
∗
∗∗
∗∗
Dox
MMS - -+ - - + - - - -- -
ZBRK1
- + --+ - - + -+ - +
-- + - - + - - - - - -
++ + + + + + ++ + + +
KAP1 + + + - - - - - - - - -
SUMO-1-KAP1 - - - + + + - - - - - -
KAP1(K554R) - - - - - - + + - - - -
KAP1(3K/R) - --- - - - - + + - -
KAP1(2K/R) - --- - - - - - -+ +
Dox
MMS - -+ - - + - - - -- -
ZBRK1
- + --+ - - + -+ - +
-- + - - + - - - - - -
++ + + + + + ++ + + +
KAP1 + + + - - - - - - - - -
SUMO-1-KAP1 - - - + + + - - - - - -
KAP1(K554R) - - - - - - + + - - - -
KAP1(3K/R) - --- - - - - + + - -
KAP1(2K/R) - --- - - - - - -+ +
0
2
4
6
8
Relative light units
10
12
14
16
18
20
‡
†
‡
†
†
‡
†
1 2 3 4 5
KAP1
SUMO-1-KAP1
KAP1(K554R)
KAP1(3K/R)
KAP1 (2K/R)
B C
12 3 4 5
0
Relative Basal p21-Luc activity
0.5
1
1.5
3
3.5
2
2.5
ZBRK1
KAP1
SUMO-1-KAP1
KAP1(K554R)
KAP1(3K/R)
+
-
-
-
-
12 3 4 5
0
Relative Basal p21-Luc activity
0.5
1
1.5
3
3.5
2
2.5
ZBRK1
KAP1
SUMO-1-KAP1
KAP1(K554R)
KAP1(3K/R)
+
-
-
-
-
+ KAP1(2K/R)
+
+
-
-
-
-
+
+
-
-
-
-
+
-
+
-
-
-
+
-
+
-
-
-
+
-
-
+
-
-
+
-
-
+
-
-
+
-
-
-
+
-
+
-
-
-
+
-
50
By using chromatin immunoprecipitation (ChIP) assay with antibodies against H3-
K9 and H3-K14 acetylation and methylation, H3-K9 and H3-K14 acetylation and
methylation status at p21 proximal promoter was to be determined. Shown in figure
7A, Doxorubicin treatment induced a marked increase of H3-K9 and H3-K14
acetylation at p21 proximal promoter/-20 amplicon (4.5-fold, closed bars 5 and 1) in
MCF-7 cells transfected with wild-type KAP1. Substituting wild-type KAP1 with
SUMO-1-KAP1 drastically suppressed this inducible acetylation (2.7-fold, closed
bars 6 and 5), while KAP1 3K/R elicited a 2-fold increase of H3-K9 and H3-K14
acetylation at p21 proximal promoter without Doxorubicin treatment. On the
contrary H3-K9 and H3-K14 acetylation was comparable at -20 amplicon between
MCF-7 cells transfected with KAP1 and 2K/R mutant, with and without Doxorubicin
treatment, stressing that the modulation in H3-K9 and H3-K14 acetylation observed
was indeed due to the change of KAP1 SUMOylation status. Acetylation at far
upstream of p21 proximal promoter was merely changed as assessed at -3038
amplicon.
Next we employed the same approach to evaluate H3-K9 methylation in
relation to KAP1 SUMOylation status in MCF-7 cells. Due to low level of H3-K9
methylation at p21 promoter, we co-expressed a H3-K9-specific methyltransferase
ESET/SETDB1 to enhance global H3-K9 methylation. As opposed to H3-K9 and
H3-K14 acetylation, Doxorubicin treatment diminished H3-K9 methylation at p21
proximal promoter/-20 amplicon (4.7-fold, closed bars 1 and 5, figure 7B), whereas
SUMO-1-KAP1 largely blocked this Doxorubicin-induced de-methylation (3.9-fold,
51
Figure 6. SUMOylation-mimetic and SUMOylation-deficient KAP1 mutants
exhibit similar affinity to ZBRK1 response elements at the p21 promoter. (A)
Diagram of p21 promoter indicates the relative position of transcription start site,
TATA box, and ZBRK1 binding elements, and amplicons used in ChIP assay.
ZBRK1 RE depicts ZBRK1 response element. (B) The binding of KAP1, SUMO-1-
KAP1, KAP1(3K/R), and KAP1(2K/R) to p21 promoter in MCF-7 cells was
assessed by chromatin immunoprecipitation (ChIP) assays in the absence or presence
of Dox-treatment. Cells were transiently transfected with individual KAP1 wild type
and its engineered expression constructs, as indicated. Dox-treatment (1 μM) was
applied to cells at 30 h post-transfection for 3 h. ChIP was carried out as described
in the Experimental Procedure section. After genomic DNA was recovered,
individual amplicons were quantified by real time PCR, and values are expressed as
a percentage of input DNA immunoprecipitated. Result represents means + standard
deviation from three independent immunoprecipitations. ∗ denotes p=0.52; ∗∗
denotes p=0.176; † denotes p=0.244 between corresponding samples.
52
Figure 6
TATA
ZBRK1
RE
ZBRK1
RE
ZBRK1
RE
-20 amplicon
-713 amplicon -3038 amplicon
≈
-27 -24 1 -332 -318
-811 -797 -924 -910
Transcription
Start
-27 44
-749 -695 -3083 -3013
p21
A
B
Dox ––– –+ + + +
ZBRK1 +++ +++ ++
Flag-KAP1 +– – – +– – –
Flag-SUMO-1-KAP1 –+ – ––+ ––
Flag-KAP1(3K/R) ––+ ––– + –
Flag-KAP1(2K/R) ––– + –– –+
IP: Anti-Flag
-20 -713 -3038
∗
∗
∗∗ ∗∗
∗∗ ∗∗
†
†
0
1
2
3
4
5
6
12 34 56 78
DNA immunoprecipitated
(% input)
53
Figure 7. Modulation of H3-K9 and H3-K14 acetylation and methylation at the
p21 promoter by KAP 1 SUMOylation or not. (A) Breast cancer MCF-7 cells
were transiently transfected with a combination of expression constructs as indicated.
Dox treatment and quantification of amplicons by real time PCR are same as
described in Fig. 5B. H3-K9 and H3-K14 acetylation at the -20 amplicon of p21
proximal promoter was assessed by ChIP with an anti-acetylated H3-K9 antibody,
while distal -3038 amplicon upstream of the p21 promoter was immunoprecipitated
as a control. Result represents means + standard deviation from three independent
ChIP assays. ∗ denotes p=0.0002; ∗∗ denotes p=2.84e-5; † denotes p=0.046; ‡
denotes p=0.9 between corresponding samples. (B) H3-K9 methylation at the -20
amplicon of p21 proximal promoter was assessed by ChIP with an anti-di-methylated
H3-K9 antibody, while distal -3038 amplicon upstream of p21 promoter was
immunoprecipitated as a control. Result represents means + standard deviation from
three independent immunoprecipitations. ∗ denotes p=0.28; ∗∗ denotes p=0.005; †
denotes p =0.025; ‡ denotes p=0.035 between corresponding samples.
54
Figure 7
A
B
Dox – – – – ++++
hESET ++ ++++++
ZBRK1 ++ ++++++
KAP1 + ––– + –––
SUMO-1-KAP– + ––– + ––
KAP1(3K/R) – – + ––– + –
KAP1(2K/R) – –– + ––– +
∗
∗∗ ∗∗
∗∗ ∗∗
†
†
‡
‡
∗
IP: Anti-di-methylated H3-K9
-20 -3038
IP: Anti-di-methylated H3-K9
-20 -3038
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
5
1 2 345 6 7 8
DNA Immunoprecipitation
(% input)
Dox – – – – ++++
ZBRK1 + +++ ++++
KAP1 + ––– + –––
SUMO-1-KAP– + –– – + ––
KAP1(3K/R) – – +– – – +–
KAP1(2K/R) – –– + ––– +
∗
∗
∗∗ ∗∗
∗∗ ∗∗
‡
‡
0
2
4
6
8
10
12
14
1 234 567 8
DNA Immunoprecipitation
(% input)
IP: Anti-acetylated histone H3
-20 -3038
IP: Anti-acetylated histone H3
-20 -3038
†
†
55
closed bars 6 and 5), without much affecting proximal promoter methylation in the
absence of Doxorubicin (compare closed bar 2 to closed bar 1, p=0.28). On the other
hand, the reduction in H3-K9 methylation at p21 -20 amplicon brought by KAP1
3K/R was much greater in normal MCF-7 cells (1.6-fold, closed bars 1 and 3) than
the Doxorubicin-treated (20% lower, closed bars 7 and 5). Shown in figure 6, KAP1
and its mutants exhibited similar affinity to p21 proximal promoter and ZBRK1-
binding element (closed bars and hatched bars respectively), despite that after
Doxorubicin treatment, a minor reduction in their bindings to these two regions did
occur (about 10% in average). Since this reduction was universal among wild-type
KAP1 and its mutants, it was unaccounted for the H3-K9 and H3-K14 acetylation
and H3-K9 methylation seen in figure 6.
Considering the observations made from the reporter assay and from the ChIP
assays on H3-K9 and H3-K14 acetylation and methylation, we came to draw the
following conclusions. (1) When KAP1 swings to decrease its SUMOylation, local
H3-K9 and H3-K14 acetylation where ZBRK1 binding element is present increases
and H3-K9 methylation decreases, presumably through changing its interaction with
those known interacting HDACs and histone methyltransferases, thus de-repressing
p21 transcription, and vice versa. (2) The deSUMOylation of KAP1 is of prerequisite
and necessity to optimal induction of p21 transcription by Doxorubicin, as evidenced
that the expression of SUMO-1-KAP1 fusion protein blocks transcription activation,
H3-K9 and H3-K14 acetylation, and H3-K9 de-methylation to great extents.
56
Previously we have concluded that KAP1 deSUMOylation is required for
optimal p21 transcriptional induction by Doxorubicin, now we would like to attest
the physiological relevance of this idea. Given a treatment course of Doxorubicin for
eight hours, the SUMOylation ladders of ectopically expressed KAP1 in MCF-7
cells tapered off as shown and quantified in figure 8A. This wane reached its fullest
effect at between two and four hours after the treatment, and the triply and doubly
SUMOylated KAP1 were reduced to one half and three fifths, respectively, of those
of non-treatment KAP1. Afterwards KAP1 SUMOylation gradually returned to
pretreatment level, marking it a transient process.
As mentioned previously, DNA-responsive genes gadd45 α, p21, and bax all
possess consensus ZBRK1 binding element, therefore subjecting them to the
transcriptional regulation of ZBRK1-KAP1 complex. Since SUMO-1-KAP1 and
KAP1 3K/R mutants profoundly sway the state of p21 transcription, it is intriguing
to see whether these mutants exert a collective effect on apoptosis when cells are
challenged by Doxorubicin. In figure 8B we showed that continuous Doxorubicin
treatment to MCF-7 cells for three days drastically reduced cell viability in our MTT
assay (12.3% of cells survived the treatment, bar 2 compared to bar 1). Not
surprisingly, transfection of SUMO-1-KAP1 desensitized MCF-7 cells to cell death
(3.1-fold increase, bars 4 and 2), where non-treatment controls did not exhibit
significant difference in viability (p=0.13, bars 1 and 3). Also KAP1 3K/R partially
reduced non-treatment cell viability (67.6% of cells survived after three day culture),
57
Figure 8. KAP1 SUMOylation-mimetic, SUMO-1-KAP1, desensitizes Dox-
induced breast cancer MCF-7 cell death. (A) MCF-7 cells were transiently
transfected with Flag-KAP1 and GFP-SUMO1 and were treated with 1 μM Dox for
different time periods, as indicated. Immunoprecipitation and immunoblotting were
then performed with an antibody against Flag tag. Quantitation was done with
BioRad Quantity One volume report program. After normalization with transfection
efficiency, the relative KAP1 SUMOylation level is calculated by designating the
individual level of SUMOylated KAP1 prior to DOX treatment as 1. (B) MCF-7
cells were transiently transfected with various KAP1 expression constructs, as
indicated. These transfected cells were treated with 2.5 μM Dox in growth medium
for 3 days. Cell survival was then measured by MTT assays. Result represents
means + standard deviation from three independent experiments. ∗ denotes p=4.5e-5;
∗∗ denotes p=0.0065; † denotes p=0.13; ‡ denotes p=1.83e-7 between corresponding
samples.
58
Figure 8
B
0
20
40
60
80
100
120
140
12 3456 78
% C ell V iab ility
(R elative to M C F -7 tran sfected w ith K A P 1)
Dox − + − + − + − +
KAP1 + + − − − − − −
SUMO-1-KAP1−− ++ − − − −
KAP1(3K/R) − − − − ++ − −
KAP1(2K/R) − − − − − − ++
∗
∗
∗ ∗ ∗ ∗
∗ ∗ ∗ ∗
†
†
‡
‡
A
Vehicle-treatment 0 1 2 4 6 8 (h)
IP: Anti-Flag IB: Anti-Flag
Triple
Double
Single
Dox-treatment 0 1 2 4 6 8 (h)
Triple
Double
Single
Triple 100 70 54 48 77 90
Double 100 82 69 61 87 94
Single 100 82 76 82 90 84
Relative KAP1 SUMOylation Level
SUMOylated KAP1
SUMOylated KAP1
Vehicle-treatment 0 1 2 4 6 8 (h)
IP: Anti-Flag IB: Anti-Flag
Triple
Double
Single
Triple
Double
Single
Dox-treatment 0 1 2 4 6 8 (h)
Triple
Double
Single
Triple
Double
Single
Triple 100 70 54 48 77 90
Double 100 82 69 61 87 94
Single 100 82 76 82 90 84
Triple 100 70 54 48 77 90
Double 100 82 69 61 87 94
Single 100 82 76 82 90 84
Relative KAP1 SUMOylation Level
SUMOylated KAP1
SUMOylated KAP1
59
while with Doxorubicin treatment the viability of the cells transfected with KAP
3K/R was halved to 6.2% from 12.3% with wild-type KAP1 (p<0.05 for both cases).
On the other hand MCF-7 cells transfected with KAP1 2K/R just behaved same
as those with wild-type KAP1. Therefore we concluded, since a collective effect to
apoptosis by Doxorubicin from those DNA-responsive genes under ZBRK1-KAP1
regulation is obvious, SUMO-1-KAP1 fusion protein could have well inhibited the
transcriptional induction of these genes, thus desensitizing cells to apoptosis; on the
other hand, the de-repression of the transcription of these genes by KAP1 3K/R
might have somewhat activated their transcriptions, therefore curtailing cell viability.
Discussion
Herein, we report that KAP1 is a SUMOylation target and its SUMOylation level
is transiently decreased upon the exposure to chemotherapeutic agent Dox. Our
findings suggest that deSUMOylation or SUMOylation of KAP1 defines the extent
in which Dox induces the expression of cell cycle regulator p21 via ZBRK1/KAP1
response element and subsequent breast cancer MCF-7 cell death. We further
demonstrate that SUMOylated KAP1 decreases H3-K9 and H3-K14 acetylation and
augments H3-K9 methylation at the 21 promoter, and deSUMOylated KAP1 renders
an inverse methylation and acetylation profile. In addition, this represents the first
demonstration, to our knowledge, that de-SUMOylation governs KAP1 function and
modulates, at least in part, the effect Dox-treatment in MCF-7 cells.
60
We first identified and verified that KAP1 is subjected to SUMOylation
modification (Fig. 2). By using site-directed mutagenesis and in vivo SUMOylation
assays, we determined that K554, K779, and K804 are the prime SUMOylation sites
in KAP1 (Fig. 3). Among these lysines, while K779R or K804R mutations of KAP1
results in a decrease of KAP1 overall SUMOylation, K554R mutation not only
reduces high molecular mass SUMOylation species, but specifically abrogates the
presumable di-SUMOylated KAP1 (Fig. 3B). The exact mechanism underlying this
observation is still unclear. We speculate that the SUMOylation of lysines other than
K554 is dependent on that of K554. Alternatively, the conjugation of SUMO-1 to
K554 is capable of being poly-SUMOylated. Although no SUMOylation consensus
motif can be found in SUMO-1, it has been reported that SUMO-1 could form
polymeric SUMOylation chain (Cooper et al., 2005), supporting this possibility.
Another interesting observation is that SUMOylation can take place on consensus
sequences in both orientations; K554 is in N- to C-terminus direction of ΨKXE/D,
where K779 and K804 are positioned in a reverse orientation of ΨKXE/D.
Based on reports from several proteomic studies on the global SUMOylation, a
large portion of SUMOylation substrates are found to be located in the nucleus or
nucleolus. Further examination of these substrates reveals that many of them are
transcription factors or co-factors. In most cases, SUMOylation of these
transcription factors and co-factors enhances their transcriptional repression
capability on their target genes. Currently, the molecular mechanism(s) by which
conjugated SUMO elicits transcriptional repression remains largely unclear. Recent
61
studies of the transcription co-activator p300 and transcription factor Elk-1 revealed
that SUMOylation mediates the recruitment of HDAC6 and HDAC2 to the cell-
cycle-regulated domain 1 (CRD1) of p300 and the repression (R) domain of Elk-1,
respectively, leading to SUMO-dependent transcription repression. In this study, we
further demonstrate that SUMOylation of KAP1 leads to an enhanced H3-K9
methylation (Fig. 7B).
SETDB1, possessing H3-K9 methylase activity, has been reported to interact
with KAP1. Neither SETDB1 nor KAP1 binds to specific DNA sequences,
suggesting that observed increase in p21 de novo H3-K9 methylation is a result of
protein-protein interaction via ZBRK1. Although the interaction between ZBRK1
and KAP1 is independent of KAP1 SUMOylation (Fig. 3), the ZBRK1/KAP1-
mediated histone methylation is, at least in part, dependent upon KAP1
SUMOylation. In addition, SETDB1 is reportedly to interact with HDAC1/2,
consistent with that the level of H3-K9 and H3-K14 acetylation correlates inversely
with H3-K9 methylation level (Fig. 7).
Database and literature searches suggested the presence of conserved
ZBRK1/KAP1 binding motifs in a number of genes other than p21, including
gadd45α, bax, puma, and noxa (Lee, YK. and Ann, DK.; unpublished observation),
which are involved in cell cycle regulation or cell death pathways. Therefore, it is
not surprising to note that the transfection of a deSUMOylase-resistant SUMO-1-
KAP1 results in a marked reduction of Dox-induced cell growth inhibition (Fig. 8B).
Presumably, SUMO-1-KAP1 fusion protein, which mimics SUMOylated KAP1,
62
could have inhibited the transcriptional induction of all these genes, thus
desensitizing cell death, while KAP1(3K/R), analogous to deSUMOylated KAP1,
might have de-repressed the transcription of all these genes, therefore curtaining cell
viability, even in the absence of Dox-treatment. We have attempted to provide
evidence that endogenous KAP1 is SUMOylated in cells prior to Dox-treatment, thus
silencing the transcription of those DNA damage responsive genes. Unfortunately,
we realized that the detection of KAP1 SUMOylation by endogenous SUMO-1 could
be difficult without overexpression of SUMO-1. Obviously, elucidating the fraction
of endogenous KAP1 is SUMOylated in different cell contexts will be another
intriguing future work.
Taken together those results from p21-reporter assays and ChIP experiments, we
concluded that the optimal activation of p21 transcription by Dox, but not by MMS,
requires the downregulation of KAP1 SUMOylation level. Given that Dox is a
known Topoisomerase II inhibitor that induces DNA double strand breaks, whether
this modulation of KAP1 SUMOylation process is mediated by the effectors of DNA
damage signaling pathway responding specifically to DNA double strand breaks
warrants further investigation. Conceivably, the reduction of KAP1 SUMOylation
level by Dox-treatment (Fig. 8A) could be a result of activation of deSUMOylase
and is currently under investigation.
In addition, DNA CpG methylase DNMT3 also interacts with SETDB1(Li et al.,
2006), making it possible that KAP1 SUMOylation also promotes DNA methylation
in p21 promoter. Whether SUMOylated KAP1 could interact with additional gene
63
silencing machinery through SUMO-dependent protein-protein interaction remains
to be tested.
It is worth noting that Wafik El-Deiry and his colleagues have reported that the
activation of p21 transcription is largely dependent on p53 activity (el-Deiry et al.,
1993). The lack of p21 induction by Dox in p53-defective T47D cells (Fig. 4B)
supports this notion. However, it was intriguing to observe that the activation of p21
transcription by Dox, which activates p53 expression, is abrogated by SUMO-1-
KAP1 fusion protein in MCF-7 cells. It is tempting to speculate that SUMO-1-
KAP1 represses Dox-induced p21 expression by inhibiting p53 transcription factor
activity, but not p53 expression, through, for example, the de-acetylation of p53.
Alternatively, the deSUMOylation of KAP1 could be a downstream event of p53
activation, and SUMO-1-KAP1 functions in a dominant-negative manner on Dox-
mediated p21 induction.
In summary, we have demonstrated that the ZBRK1 response elements at the p21
promoter are occupied by KAP1/ZBRK1 complex, regardless of KAP1
SUMOylation status. Functionally, SUMOylation is required for KAP1 full
transcription repressor activity. Taken together, our results suggest a more detailed
scenario regarding Dox-mediated p21 transcriptional induction. In this process, the
ZBRK1/KAP1 complex is first recruited to p21 promoter through ZBRK1 response
elements. Histone tales are then subjected to repressive modifications and p21
transcription is repressed, presumably through SETDB1 recruitment to SUMOylated
KAP1. Upon Dox-exposure, deSUMOylated KAP1 accumulates (without affecting
64
the occupancy of ZBRK1 response elements) and histone acetylase is recruited to
p21 promoter, leading to the activation state of p21 transcription. It will be of
interests to examine whether KAP1 SUMOylation is enhanced in cancer or other
(patho)physiological conditions or regulated by other therapeutic agents.
65
Chapter Four
THE TRANSCRIPTIONAL CONTROL OF THE KAP1 SUMOYLATION BY
DOXORUBICIN ON OTHER ZBRK1-REGULATED GENES
Summary
In the previous chapter it was demonstrated that the down-regulation of
KAP1 SUMOylation in response to Doxorubicin treatment were directly linked to
the de-repression of the transcription of p21 through the changes in histone
acetylation and methylation regulated by ZBRK1/KAP1 complex. Here it is shown
that KAP1 SUMOylation-mimetic SUMO-1-KAP1 and SUMOylation-defective
KAP1 3K/R pose the same regulatory control over the transcription of four other
genes--namely gadd45, bax, puma, and noxa. Therefore the transcription of these
genes is likely under the same regulatory mechanism found in the case of p21.
Background
As discussed in the previous chapter, the KAP1 SUMOylation regulates the
transcription of the cell-cycle regulator p21 through its binding to the KRAB-zinc-
finger protein ZBRK1 upon DNA damage. The apoptosis of MCF-7 cells induced by
Doxorubicin were greatly suppressed by expressing SUMOylation-mimetic SUMO-
1-KAP1, giving an early indication that the swaying of the KAP1 SUMOylation
status may determine the fate of cells when encountering double-strand DNA
66
damage. Given that a number of other genes such as gadd45 α, bax, ki-67, and TIMPs,
also putatively possess this 15-bp nucleotide sequence that ZBRK1 specifically binds
to, I speculate this regulatory module with the core components of ZBRK1 and
KAP1 carrying some specific cellular function like apoptosis in response to DNA
damage.
The translational product of p21, also named CDKN1A, is the first identified
inhibitor to the cyclin-dependent kinase (CDK) complex. Many CDKs have been
discovered ever since, but CDKN1A remains the only inhibitor that virtually binds to
all of the CDK complexes (Perkins, 2002). The p21 expression is elevated in
response to a variety of stresses including ionizing radiation, DNA-damaging agents,
inhibitor to DNA replication and mitosis, and oncogenic Ras. The up-regulation of
p21 during these events is primarily p53-dependent, through the increase of its
expression or the upshot of its transcriptional activity (Harris and Levine, 2005). The
only exception is the inhibition of DNA replication by mimosine, where the stability
of p21 mRNA and its protein is regulated in p53-independent manner (Alpan and
Pardee, 1996).
Since p21 is the direct transcriptional target of tumor suppressor p53, it is
expected that the loss of p21 expression would contribute to tumorigenesis, and can
serve as prognostic markers for different cancers. From reports that examines a wide
selection of tissues and cell types, it is generally concluded that in small-cell lung
carcinoma, colorectal, cervical, head and neck cancers, lack of p21 did correlate with
the tumor progression and poor prognosis. However, in prostate, ovarian, breast, and
67
esophageal squamous cell carcinomas, increased p21 expression was associated with
tumor progression and poor prognosis (Rowland and Peeper, 2006). Confusing as
this may seem, a report done with extrahepatic bile duct carcinoma concluded both
high and low levels of p21 as the marker for poor prognosis, where moderate level in
p21 indicates more satisfactory, favorable prognosis. With these said, they just
highlight the dual role of p21 in tumor suppression and tumorigenesis. Despite of
those seeming contradictions in its role in tumorigenesis, p21 was also found to
inhibit apoptosis. At the onset of apoptosis, CDKN1A is cleaved by caspase 3,
consequently losing its ability to suppress apoptosis (Mahyar-Roemer and Roemer,
2001). This finding is further supported by that p21 knockout mice exhibit a decrease
in radiation-induced tumorigenesis, while loss of p21 in ATM-deficient mice
sensitizes them to radiation-induced apoptosis (Adnane et al., 2000; Bearss et al.,
2002; Martin-Caballero et al., 2001; Poole et al., 2004; Topley et al., 1999; Wang et
al., 1997; Yang et al., 2001).
The growth arrest and DNA damage-inducible gene gadd45 α is a member of
protein group whose expression is induced by growth arrest signal and DNA-
damaging agents. Reagents triggering gadd45 expression includes UV radiation,
methylmethane sulfonate (MMS), nitrogen mustard, melphalan, hydrogen peroxide,
hypoxia, many chemotherapeutic agents, ionizing radiation (IR), growth factor
withdrawal, and medium depletion (Zhan, 2005). Generally gadd45 induction
following DNA damages is rapid, transient, and dose-dependent. Rapid induction of
gadd45 has been observed in every cell types tested to date.
68
The signaling events that regulate gadd45 expression following DNA damages
are though to be complex and may involve different mechanisms in response to
different types of DNA damages—for example, single-strand or double-strand DNA
break damages. It was found that gadd45 was concomitantly expressed with wild-
type p53 upon ionizing radiation, and further examination of the gadd45 gene
structure revealed a p53-responsive element in its third intron (Kastan et al., 1992).
Both introduction of HPV E6 protein or ectopic expression of dominant p53 mutant
blocks the inducibility in gadd45 transcription (Butz et al., 1999). Moreover,
overexpression of Mdm2 that targets p53 for ubiquitin-dependent proteasomal
degradation also abolishes this inducibility, demonstrating that its transcription is
highly regulated by p53 (Chen et al., 1994). ATM kinase seems also involved in the
gadd45 induction by ionizing radiation, because gadd45 induction is greatly reduced
in lymphoblast from the patients of ataxia telangiectasia (AT), compared to
lymphoblasts from normal individual (Papathanasiou et al., 1991). In addition to its
role in growth arrest, gadd45 may also participate in the induction of apoptosis. The
evidence that the ectopic expression of gadd45 in tumor cells by transient
transfection autonomously induces apoptosis and UVB radiation-induced apoptosis
is defective in gadd45-deficient cells supports this notion (Hildesheim et al., 2002;
Takekawa and Saito, 1998). Nonetheless whether ionizing radiation-induced
apoptosis is mediated by gadd45 remains to be determined.
As demonstrated and discussed in previous chapter, it seems the KAP1
SUMOylation exerts a collective effect of apoptosis on MCF-7 cells, indicating that
69
the transcription and expression of the components of apoptotic pathways may also
be subjected to the control of KAP1 SUMOylation status. The apoptotic activity in
mammalian cells is largely controlled by the members of Bcl-2 family. Aligned by
the homology of their domain structures, members of Bcl-2 family are divided into
three categories: (1) the first group including Bcl-2, Bcl-xL, Bcl-w, Mcl-1, and A1,
are characterized with four repeats of Bcl-2 homology domains—BH1, BH2, BH3,
BH4, and is pro-survival, generally through antagonizing pro-apoptotic Bax, Bak,
and Bok; (2) this group of Bax, Bak, and Bok contains BH1, BH2, and BH3 domains
but not BH4 domain, and is capable of forming homo- or hetero-oligomers that insert
themselves into the outer membrane of mitochondria by which cytochrom C is
released followed by the activation of effector caspases, therefore tagged as pro-
apoptotic; (3) this group--including Bim, Bid, Bad, Bik, Bmf, Puma, Noxa--contains
only BH3 domain, and has been shown to bind to and sequester the pro-survival
members, thus leading to apoptosis (Cory et al., 2003). Of the potent activators to
apoptosis from the members of the second and third groups, studies have shown that
members from the third group are more prone to transcriptional and post-
transcriptional regulation, where the activation of their oligomerization of Bax, Bak,
and Bok is definitively more important for apoptotic activation rather than the up-
regulation on their transcriptions.
The apoptosis provoked by DNA damages requires p53 and the tumor
suppressor activity of p53 essentially prevents the dysregulation of cell growth and
replication, and therefore tumorigenesis. Pro-apoptotic Puma and Noxa are
70
reportedly regulated by p53 at the transcriptional level (Han et al., 2001; Nakano and
Vousden, 2001; Oda et al., 2000; Yu et al., 2001). Puma-deficient lymphocytes
exhibited resistance to DNA damage-induced apoptosis similar to that those deficient
in p53, where Noxa affected p53-dependent apoptosis to a lesser extent (Yu et al.,
2003). Here the transcriptional activation of gadd45, bax, puma, and noxa by
Doxorubicin was assessed by reverse transcription followed by real-time PCR, and
the SUMOylation-mimetic and SUMOylation-defective KAP1 were used to evaluate
the role of KAP1 SUMOylation in regulating their transcriptional activation.
Materials and methods
Total RNA extraction, reverse transcription, and real-time PCR. Total RNA was
extracted with TRIzol reagent (Invitrogen) according to its manufacturer’s protocol.
The total RNA from Dox-treated and control MCF-7 cells was then treated with
RNAase-free DNAase (Invitrogen) and extracted again with phenol-chloroform,
followed by ethanol precipitation. Reverse transcription and quantitative PCR in p21,
gadd45 α, bax, puma, and noxa mRNA was done with iTaq SYBR Green Supermix
(BioRad), a fraction of each total RNA samples, and specific pairs of gene-specific
primers listed in table 5. PCR amplification and fluorescence detection were done
with MyIQ real-time PCR detection system, and the threshold cycles were
determined by iCycler program with its default setting. Fold inductions were
determined by the ∆∆Ct method against 18S rRNA.
71
Primer Purpose Sequence (5’-to-3’)
18S rRNA FP Real-time PCR CGGCGACGACCCATTCGAAC
18S rRNA RP RT & real-time
PCR
GAATCGAACCCTGATTCCCCGTC
bax FP Real-time PCR CCGATTCATCTACCCTGCTG
bax RP RT & real-time
PCR
CAATTCCAGAGGCAGTGGAG
noxa FP Real-time PCR ATTACCGCTGGCCTACTGTG
noxa RP RT & real-time
PCR
GTGCTGAGTTGGCACTGAAA
puma FP Real-time PCR CTGTGAATCCTGTGCTCTGC
puma RP RT & real-time
PCR
AATGAATGCCAGTGGTCACA
Table 5. Primers used in the real-time PCR experiments. RT, reverse transcription.
72
Results
In the previous chapter I demonstrated the principle of KAP1 SUMOylation
in regulating the transcription of p21: SUMOylation-mimetic KAP1 enhanced
transcriptional repression by increasing the methylation of histone 3 lysine 9 and
decreasing the acetylation of histone 3 lysines 9 and 14, where SUMOylation-
defective KAP1 relieve transcriptional repression in the opposite way. It is also noted
that some other genes involving in cell-cycle control and apoptosis such as gadd45,
bax, puma, and noxa, might also be subjected to this regulatory mechanism due to
the presence of ZBRK1-biniding element at the promoters of these genes. To address
this question, I first examined whether these genes do possess both DNA-binding
elements for p53 and ZBRK1 through both literature search and motif scanning by
the ScanProsite program. All these five genes including p21 have been demonstrated
previously that their transcriptional induction in response to DNA damage is p53-
dependent. Previously gadd45 has been shown subjected to the transcriptional
control under ZBRK1 and BRCA1, where BRCA1 exerts its transcriptional co-
repressor activity via its binding to the C-terminus of ZBRK1 that does not directly
interact with KAP1. The putative ZBRK1-binding elements for bax were indicated
previously, and sequence scanning revealed several ZBRK1-binding elements in
both pro-apoptotic genes puma and noxa. Both the p53-binding elements and
ZBRK1-binding elements in bax, puma, and noxa are shown in figure 9, where the
relative positions of these elements to the transcriptional start site in these genes are
also indicated.
73
ZBRK1
BE
p53
BE
p53
BE
1 -161 -145 -249 -229 -795 -781
Transcription
Start
p53
BE
ZBRK1
BE
ZBRK1
BE
1 -114 -100
-126 -112
-211 -193
Transcription
Start
p53
BE
ZBRK1
BE
1 -233 -219 -335 -326
Transcription
Start
149
ATG
bax
ATG
282
ZBRK1
BE
-1244 -1230
puma
ATG
174
noxa
Figure 9. The p53-responsive element and putative ZBRK1-binding elements at
the promoters of bax, puma, and noxa genes. The positions of p53-responsive
elements were denoted according to previous reports, and the ZBRK1-binding
elements were searched against the 15-bp consensus motif G-G-G-X-X-X-C-A-G-X-
X-X-T-T-T with the permission of mismatches less than two nucleotides by
ScanProsite program. The transcription start site of each gene was defined as +1
position, and the corresponding ATG start codon was also shown. Note the two
consensus motifs found in noxa were partially overlapped.
74
To test whether the transcription of these give genes are regulated by the
SUMOylation status of KAP1 in response to Dox treatment, MCF-7 cells were
transfected with either wild-type, SUMOylation-mimetic, or SUMOylation-defective
KAP1. Reverse transcription followed by real-time PCR to detect their mRNA levels
four hours after 1 μM Doxorubicin treatment was carried out and fold changes of
their mRNA were presented in figure 10. At the fourth hour post-treatment, the
transcription of these give genes were all up-regulated, by 4.3-fold for p21, 3.0-fold
for gadd45, 1.6-fold for bax, 2.3-fold for puma, and 2.1-fold for noxa. In general, the
up-regulation was more prominent in p21 and gadd45, whose expressions are
important to pause cell-cycle progression and allow cells to repair DNA damages,
than in pro-apoptotic genes bax, puma, and noxa. In overall, the introduction of
SUMO-1-KAP1 suppressed the transcription of all these five genes four hours post-
treatment, while the expression of KAP1 3K/R enhanced the transcription of all these
five genes, albeit the enhancement by KAP1 3K/R was more modest than the
suppression by SUMO-1-KAP1.
Discussion
In the previous chapter we demonstrated that the transcription of p21 was
regulated by the SUMOylation status of KAP1 through ZBRK1/KAP1 stable
complex. Since a number of other genes involving in the control of cell-cycle
progression, DNA damage repair, and apoptosis also possess this ZBRK1-binding
element, it is speculated that the transcription of these genes could also be regulated
75
0
1
2
3
4
5
6
12 34 5
Fold induction
WT
SUMO-1-KAP1
KAP1 3K/R
ction du ld In Fo
mRNA detected: p21 gadd45 α bax puma noxa
Figure 10. The assessment of mRNA levels of p21, gadd45, bax, puma, and noxa
in response to Dox treatment in MCF-7 cells. MCF-7 cells transiently transfected
with wild-type KAP1, SUMO-1-KAP1, and KAP1 3K/R were treated with 1 μM
Doxorubicin for four hours, and total RNA were extracted by TRIzol reagent. Their
mRNA levels were then quantitated by one-step reverse transcription and real-time
PCR with gene-specific primers. The fold induction of each gene in response to Dox
treatment was calculated by ∆∆Ct method against 18S rRNA. The result represents
mean + standard deviation from three independent experiments.
76
by the same principle of KAP1 SUMOylation in the case of p21. Indeed, under the
same Doxorubicin treatment for four hours, here I showed that the transcription of
gadd45, bax, puma, and noxa were universally down-regulated at this specific time
point, indicating that the proposed mechanism by which KAP1 regulated the
transcription of p21 through modulating its SUMOylation states was also utilized in
the regulation of the transcription of the other four genes. That said, during the first
four hours of the Doxorubicin treatment it is likely to observe the same histone
modifications at the proximal promoters of these four genes—the de-methylation of
histone 3 lysine 9 and the acetylation of histone 3 lysine 9 and 14 as observed in the
p21 case, creating a local chromatin environment that is in favor of active
transcription.
77
Chapter Five
THE INVOLVEMENT OF THE SUMO-SPECIFIC PROTEASE SENP1 AND
THE DNA DAMAGES-RESPONSIVE PROTEIN KINASE ATM IN THE
DEREPRESSION OF P21 AND GADD45α TRANSCRIPTION VIA KAP1
Summary
To further understand the signaling events upstream of KAP1 SUMOylation,
I studied the effect of two SUMO-specific proteases—SENP1 and SENP2, and two
DNA damages-responsive PI3K-like protein kinases—ATM and ATR, in the de-
repression of p21 and gadd45 α transcription in response to Doxorubicin treatment.
The results showed that SENP1 is a legitimate de-SUMOylase for KAP1 and directly
contributes to the transcriptional de-repression upon Dox treatment; ATM, but not
ATR is required for the de-repression of gadd45 α transcription in response to Dox
treatment. These findings indicated that these two signal transducers may be key to
the de-repression regulated by KAP1 de-SUMOylation.
Background
In the chapter three it was demonstrated that the chemotherapy agent
Doxorubicin induced a transient decrease in KAP1 SUMOylation, which was linked
to the up-regulation of p21 transcription. The dynamics of the SUMOylation of any
given protein is immediately regulated by two forces: the activity of their
78
corresponding SUMOylation conjugation enzymes and de-conjugation enzymes.
There have been many examples in which the target gene disruption in SUMO E3
ligases was utilized to study the pathways and the biological consequences they were
regulating. Studies in PIAS1 -/- mice revealed the PIAS1 specifically regulated
interferon-inducible gene expression, which in turn activated innate immune
response (Liu et al., 2004). PIAS γ knock-out mice exhibited moderate defect in the
gene expression by interferon γ and Wnt agonists (Roth et al., 2004). Whether the
rest of the PIAS family members and the other unrelated SUMO E3 ligase—RANBP
and polycomb protein, subject their activities to the extracellular cues remain unclear.
There are six SUMO-specific proteases, known as SENPs (Sentrin/SUMO-
specific proteases), that have been discovered to date. SENP1 is a nuclear protease
and has been found to de-conjugate quite a number of proteins. A recent report
indicated that SENP1 participated in androgen receptor-mediated and c-Jun-mediated
transcriptions, through a probable mechanism of de-conjugating the SUMO residue
from the R domain of p300 that relieved the repressive ability of its R domain
(Cheng et al., 2004). In the same report the authors claimed the enhancement of AR-
mediated and c-Jun-mediated transcriptions resulted in cell proliferation and
observed prostate intraepithelial neoplasia when overexpressing SENP1. SENP2 is
found primarily nuclear envelop-associated, and its nucleo-cytoplasmic shuttling
modulates its activity and its ubiquitination-dependent turnover (Hang and Dasso,
2002; Itahana et al., 2006). An alternative-splicing variant of SENP2, named SuPr-1,
was found to alter the distribution of nuclear POD-associated proteins such as CBP
79
and Daxx (Best et al., 2002; Ross et al., 2002). Although SuPr-1 was reported to
regulate c-Jun-dependent transcription, this regulation is independent of its SUMO-
specific protease activity (Cheng et al., 2005). SENP3 and SENP5 are found
primarily associated with nucleoli and have preferred activity toward SUMO2 and
SUMO3 (Gong and Yeh, 2006). SENP6 and SENP7 are also nuclear but their
functions remain to be characterized.
In the previous chapter I discussed the role of KAP1 SUMOylation in the
activation of p21 transcription in response to Doxorubicin, which induces DNA
double strand break through inhibiting the activity of topoisomerase II. DSB, which
poses serious threat to normal cellular function if left untended, could result in
various genomic pathological states such as chromosomal deletion and translocation.
Accumulation of these pathological events of genome instability will finally lead to
the rise of cancers, and thus a comprehensive repair system is essential for cells to
survive different DSB insults. The primary activator and transducer of the DSB
repair response is the nuclear protein kinase ataxia telangiectasia mutated (ATM).
Patients with the genome instability syndrome ataxia telangiectasia (AT) are very
sensitive to ionization radiation (Shiloh, 1997). Upon the occurrence of DSB, ATM is
rapidly phosphorylated and its kinase activity subsequently phosphorylates a number
of substrates, resulting in the activation of cell cycle checkpoint control, DNA repair
machinery, and in the extreme case that cells are unable to restore the genetic
information, apoptosis (Sancar et al., 2004). ATM belongs to a conserved protein
family termed PI3K protein-like kinases (PIKKs), most of which possess
80
serine/threonine kinase activity and a domain that is characteristic of
phosphatidylinositol 3-kinase (PI3K) (Hartley et al., 1995; Manning et al., 2002;
Poltoratsky et al., 1995). In addition to ATM, ataxia telangiectasia and Rad3-related
(ATR), hSMG-1, mTOR, and the catalytic subunit of DNA-PK are the four other
protein kinases of this family identified by far. TRRAP, a component of a histone
acetyltransferase complex, is the only non-kinase member of this family. Whereas
ATM and DNA-PK primarily responds to DNA DSB, ATR mainly transduces DNA
damage signal resulted from UV irradiation (Abraham, 2001; Lees-Miller and Meek,
2003).
The hallmark of the ATM signaling network is that it contains protein kinases
Chk1 and Chk2 that are themselves capable of targeting several downstream
effectors simultaneously (Iliakis et al., 2003). A prominent example is ATM-
mediated activation and stabilization of the p53 protein, an important mediator for
both cell-cycle checkpoints and damage-induced apoptosis (Banin et al., 1998;
Canman et al., 1998). ATM mediates the transcriptional activity of p53 by regulating
various posttranslational modifications of p53. For example, ATM phosphorylating
Mdm2, an ubiquitin ligase of p53, thus targeting p53 for degradation (de Toledo et al.,
2000; Khosravi et al., 1999; Maya et al., 2001). Mdmx, an inhibitor to p53 activity,
undergoes several ATM- and Chk2-dependent phosphorylation events that enhance
its degradation (Chen et al., 2005; LeBron et al., 2006; Pereg et al., 2005).
A major response to DNA damage is represented by marked alterations in
gene expression program. A combination of microarray and computational analysis
81
supported evidence that p53 and the transcription factor NF- κB are two of the key
molecules involved in the ATM-dependent gene expression (Elkon et al., 2005;
Rashi-Elkeles et al., 2006; Stankovic et al., 2004). As counterintuitive as it may seem
at the first sight, p53 mediates the apoptotic response to DNA damage, whereas NF-
κB activates genes associated with cellular survival, thus offsetting the effects
brought about by each other. Yet the functional link between ATM and NF- κB lies in
the NF- κB essential modulator (NEMO) (Wu et al., 2006). NF- κB is held inactive in
the cytoplasm by its inhibitor I κB until an appropriate stimulus leads to activation of
the IkB kinase (IKK) complex, which phosphorylates I κB and thus targets it for
proteasome-mediated degradation. NF- κB is subsequently translocated into nucleus
where it activates its target genes. Since NEMO is the regulatory subunit of the IKK
complex, after DNA damage ATM-mediated phosphorylation of NEMO in the
nucleus leads to its ubiquitin-dependent nuclear export and subsequent activation of
cytoplasmic IKK, consequently leading to stimulation of NF- κB.
Another transcription factor that has been recently found to be a direct target
of ATM is the Ca2+/cAMP response element binding protein (CREB). It was
reported that genotoxic-stress-induced phosphorylation of CREB by ATM led to
inactivation of CREB, and a phosphorylation-ablation mutant of CREB showed
enhanced activity that was resistant to DNA damage (Shi et al., 2004). Despite of
that, ATM is involved in phosphorylation of CREB not only after DSB but in
response to UV damage (Dodson and Tibbetts, 2006).
82
One of the main lines of research attempts to understand how cells make the
crucial choice between activation of the survival response and apoptosis in the face
of heavy DNA damage. A recently identified ATM target – the BID protein, a
member of the ‘BH3-only’ pro-apoptotic member of the BCL-2 family – has thrown
light on this choice. ATM-mediated phosphorylation of BID has an anti-apoptotic
role in the damage response and is also involved in the S-phase checkpoint (Kamer
et al., 2005; Zinkel et al., 2005). This change in role after ATM-mediated
phosphorylation might be typical of other ATM effectors. Thus, activating
transcription factor 2 (ATF2), which is known to be involved in the JNK/p38 stress
response, has been found to be phosphorylated by ATM at DSB sites and is required
to mount an efficient DNA-damage response (Bhoumik et al., 2005). The role of
ATF2 in the DNA-damage response is completely uncoupled from its well-
documented function as a transcription factor.
Materials and methods
Cell culture. MCF-7 cells were maintained in Dulbecco’s modified Eagle’s medium
supplemented with 10% fetal bovine serum, antibiotics, and 0.01 mg/ml recombinant
human insulin in a humidified atmosphere of 37°C and 5% CO
2.
Both ATM-deficient
pEBS7 and ATM-proficient YZ5 cells were originally established at Yosef Shiloh’s
laboratory, Tel Aviv University, Israel (Ziv et al., 1997). In short, the primary A-T
fibroblast cells possesses a homozygous frameshift mutation at codon 762 of the
ATM gene that resulted a truncated ATM with reduced stability. YZ5 cells were
83
derived from the primary fibrobasts transfected with the full-length ATM open
reading frame, where pEBS7 cells were transfected with an empty control vector.
Both transfected cell lines were then selected and maintained in Eagle’s DMEM
medium supplemented with 15% fetal bovine serum, antibiotics, 2nM glutamine, 100
μg/ml hygromycin, and 1.25 U/ml nystatin in a humidified atmosphere of 37°C and
5% CO
2.
The doxycycline-inducible kinase-defective (kd) ATR U2OS (human
osterosarcoma) stable cell line was established previously (Nghiem et al., 2001). In
short, amino-terminal FLAG epitope-tagged full-length construct of ATR-kd was
inserted into the cytomegalovirus (CMV) promoter-based plasmid pcDNA4/TO
(Invitrogen), which contains two tetracycline operator binding sites. These constructs
were co-transfected with a 20-fold lower amount of pcDNA3.1, which contains the
neomycin resistance gene. Beginning 2 days later, G418-resistant clones (400 µg/ml)
were selected. Hygromycin was always present at 200 µg/ml to maintain expression
of the tetracycline repressor.
Luciferase assay. The p21 luciferase reporter construct was made by subcloning a
2.3-kb p21 promoter into pGL3-Basic as previously described. The gadd45 α reporter
was a gift from Dr. Wen-Hwa Lee at the University of California, Irvine. The
luciferase reporters were co-transfected with a firefly control reporter, pRL-TK, for
the normalization purpose. Transfection was done with Lipofectamine 2000
(Invitrogen) as the manufacturer’s manual indicated. Forty hours post-transfection
the assays were carried out with DualGlo Luciferase Assay Kit (Promega).
84
Result
As previously mentioned, the SUMOylation status of any given protein is the
delicate balance between its SUMOylation and de-SUMOylation. Therefore the
Doxorubicin-induced down-regulation of KAP1 SUMOylation could be the result of
the de-activation of its SUMOylation enzymes, the inactivation of its de-
SUMOylation enzymes, or both. To test whether the SUMO-specific protease play a
role in this process, a p21 reporter assay in MCF-7 cells in combination with the
exogenous expression of either SENP1 or SENP2 was employed for this purpose.
As shown in figure 11, p21 transcription in the breast cancer MCF-7 cells transfected
with wild-type ZBRK1 and KAP1 was induced by 4.6-fold (bars 1 and 2) upon
Doxorubicin treatment, whereas p21 transcription was held nearly unchanged upon
Dox treatment with the exogenous expression of SUMO-1-KAP1 fusion protein
(bars 3 and 4). Yet MCF-7 cells transfected with SENP1 exhibited 3.6-fold increase
in the basal p21 transcription (bars 5 and 1), whereas the co-transfection of
incleavable SUMO-1-KAP1 with SENP1 largely suppressed this phenomenon (bars
6 and 5), indicating that the de-SUMOylation of KAP1 by SENP1 could have
accounted for the Dox-induced p21 transcription and this induction is likely
dependent on the enzymatic activity of SENP1. In contrast to SENP1, SENP2 had no
effect on the p21 transcription in the absence of Dox, further supporting the
specificity of SENP1 in this observation.
In the previous chapter I demonstrated that Dox activated the transcription of
gadd45 α in a similar way as that of p21, where SUMO-1-KAP1 efficiently repressed
85
0
0.5
1
1.5
2
2.5
3
3.5
4
123 4567 8
Relative light uni
Dox - + - + ----
ZBRK1 ++++++++
KAP1 + +- - +- +-
SUMO-1-KAP1 - - + +- +- +
Figure 11. SENP1 but not SENP2 regulates the activation of p21 transcription
that mimics the down-regulation of KAP1 SUMOylation by Dox treatment.
MCF-7 cells were transfected with a 2.3-kb p21 luciferase reporter along with
ZBRK1, KAP1, SUMO-1-KAP1, SENP1, or SENP2 as indicated. Forty hours post-
transfection the luciferase activity was measured and normalized against a firefly
control reporter (pRL-TK). Result represents mean + standard deviation from three
independent experiments, after normalization for transfection efficiency.
S
S
ENP1 ---- + + --
ENP2 ------ + +
Relative light units
86
the Dox-induced gadd45 α transcription. Since ATM and ATR function as the sensors
for the DNA break repair, as well as the activators, I asked if ATM and ATR are
important for the Dox-induced gadd45 α transcriptional activation. Human
osteosarcoma U2OS cells implanted with a stably-transfected, doxycycline-inducible
expression system of an ATR kinase-defective mutant, termed GK41 cells, was used
to assess whether ATR is necessary for the KAP1-dependent gadd45 α transcription.
Shown in figure 12, in the absence of doxycycline such that the expression
endogenous wild-type ATR is intact in GK41 cells, the induction of gadd45 α
transcription by Doxorubicin (2.4-fold, bars 1 and 4) was similar to that we have
seen from MCF-7 cells, where the expression of SUMO-1-KAP1 mildly repressed
both basal and Dox-inducible gadd45 α transcription. With the inducible expression
of ATR-defective ATR by the addition of 1 μg/ml doxycycline, both basal and Dox-
inducible gadd45 α transcription with the exogenous expression of either the wild-
type KAP1 or SUMO-1-KAP1 behave nearly the same as their counterparts without
doxycycline, except Dox-induced gadd45 α transcription was a bit lower when ATR-
defective ATR was expressed, indicating that the ATR activation is not required for
the SUMOylation-dependent, KAP1-mediated gadd45 α transcription.
To address the question of whether ATM is required in the SUMOylation-
dependent, KAP1-mediated gadd45 α transcription, an ATM-deficient fibroblast cells
(pEBS7) and its counterpart (YZ5), in which the expression of wild-type ATM is
restored by stable transfection, is employed. Shown in figure 13, in ATM-proficient
YZ5 cells the Dox-induced gadd45 α transcription behaved alike to what we have
87
0
1
2
3
4
5
6
7
12 34
KAP1
lo SUMO-1-KAP1
hi SUMO-1-KAP1
1 2 3 4 5 6 7 8 9 10 11 12
Doxycycline
Doxorubicin - + - +
-+
Relative light units
Figure 12. ATR is not required for the Dox-induced gadd45 α transcription.
GK41 cells that were inducible in a kinase-defective form of ATR with the addition
of doxycycline were transfected with KAP1 or SUMO-1-KAP1. Thirty hours post-
transcription the cells received 1 μM Dox treatment for four hours, and the cells were
treated 1 μg/ml doxycycline continuously starting twelve hours before transfection
began. The gadd45 α luciferase assay was then carried out and the luciferase activity
was normalized against a firefly luciferase activity. Result represents mean +
standard deviation from three independent experiments, after normalization for
transfection efficiency.
88
Figure 13. DNA damage-responsive protein kinase ATM is required for the Dox-
induced gadd45 α transcription. ATM-proficient YZ5 cells and ATM-deficient
pEBS7 cells were co-transfected with a gadd45 α reporter, ZBRK1, KAP1, SUMO-1-
KAP1, or SUMO-1-KAP1 L306P, and treated with UV irradiation or Dox. The
luciferase activity was monitored as described previously. Result represents mean +
standard deviation from three independent experiments, after normalization for
transfection efficiency.
89
Figure 13
0
2
4
6
8
10
12
12 34
KAP1
lo SUMO-1-KAP1
hi SUMO-1-KAP1
SUMO-1-KAP1 L306P
0
0.5
1
1.5
2
2.5
1 234
KAP1
lo SUMO-1-KAP1
hi SUMO-1-KAP1
SUMO-1-KAP1 L306P
-UV +UV -Dox +Dox
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16
-UV +UV -Dox +Dox
YZ5 cells (ATM-proficient)
pEBS7 cells (ATM-deficient)
Relative light units
Relative light units
90
seen from MCF-7 cells, where the expression of SUMO-1-KAP1 exerted strong
repression on this inducibility and the L306P mutant of SUMO-1-KAP1, whose coil-
coiled domain is disrupted by a leucine-to-proline mutation, abolished this repression.
On the contrary, although UV radiation induced a comparable induction in the
gadd45 α transcription, SUMO-1-KAP1 has negligible effect on this UV-induced
transcription, indicating that the SUMOylation of KAP1 is only important in ATM-
mediated p21 and gadd45 α transcriptions.
Discussion
In this chapter I first demonstrated that SENP1, not SENP2, was able to
mimic Dox-induced down-regulation of KAP1 SUMOylation, thus activating p21
transcription in a magnitude comparable to that of Dox treatment. SENP1 is
exclusively a nuclear SUMO-specific protease, because the SUMOylated PML that
forms subnuclear structure called PML oncogenic domains (PODs) can be
selectively disrupted by the expression of SENP1, whereas the SUMOylated
RANGAP1, which resides in the cytoplasm, is not affected by SENP1. Although it
remains unclear whether SENP1 also involves in other subnuclear domains, it is of
no doubt that its functions are mainly nuclear. In a preliminary experiment by using
crude cell extract to test the activity of endogenous SENP1 and SENP2 upon Dox
treatment, it is found that only the SENP2 activity is significantly enhanced
(unpublished observation. Y.K. Lee, Y. Chen, D. Ann). Nevertheless this cannot rule
out the possibility of SENP1 in the Dox-induced activation of p21 and gadd45 α
91
transcriptions, especially if SENP1 is mainly functional in the specialized subnuclear
domain where it is highly concentrated.
More interestingly, a recent report showed that SENP1 participates in
androgen receptor (AR)-mediated where overexpressing SENP1 alone activates AR-
dependent transcription in LNCaP cells by humongous 25-fold. It is speculated that
this effect is done through the de-SUMOylation of AR-interacting partners such as
p300 and HDAC1 by SENP1, thus relieving their repressive ability endowed by their
SUMO conjugation. KAP1 has been shown to interact with Mdm2/Hdm2 and
overexpressing KAP1 reduced p53 acetylation, and decreased p53 ubiquitination and
its subsequent proteasomal-mediated degradation, thus in overall reducing p53
transcription activity. Nonetheless it remains unclear how this regulatory module will
act in the context of signaling. For example, the Dox-induced p21 or Dox-induced
gadd45 α transcription systems will be comprehensible systems to gain insights into
this issue. Furthermore, it remains highly interesting to see if p300 constantly
associates with KAP1-containing supramolecular complex that reportedly has a mass
of up to 10,000 kDa (Kentsis et al., 2002). If so, then the regulation between the
components within this supramolecular complex will be far more complex than it
appears now.
With the GK41 cells that provide the inducible expression of the ATR-
defective ATR, I provided evidence that Dox-induced gadd45 α transcription is
independent of ATR. It has been generally accepted that ATR exhibits some
redundancy with ATM in activating Chk1 and Chk2 upon DNA double-strand-break
92
damages, while UV damage signaling is primarily, if not exclusively, handled by
ATR. A minor reduction in the Dox-induced gadd45 α transcription was observed
when A TR-defective A TR was expressed, therefore agreeing with this point.
In ATM-proficient YZ5 and ATM-deficient pEBS7 cells, the activations of
gadd45 α transcription by UV irradiation and Dox were comparable. Nonetheless
SUMO-1-KAP1 was only able to repress Dox-induced but not UV-induced gadd45 α
transcription, indicating that the transcriptional inductions by Dox and UV were
achieved in distinct manners. It was previously shown that upon UV irradiation
ZBRK1 was subject to poly-ubiquitination and the proteasomal-dependent
degradation, and therefore the exogenous expression of SUMO-1-KAP1 would have
no effect on UV-induced gadd45 α transcription. On the other hand, in ATM-deficient
pEBS7 cells the gadd45 α transcription was induced by UV radiation just as the same
way as in YZ5 cells, concurring with the generally accepted concept that the DNA
damage response induced by UV irradiation is ATR-dependent. While UV-induced
gadd45 α transcription is ATM-independent, Dox-induced gadd45α transcription in
pEBS7 cells was largely blocked, implying that ATM is required for this
transcriptional activation. Since the activation of gadd45 α transcription has been
known to be p53-dependent, it is plausible to assume that ATM might target p53 or
its associated factors to increase its stability or transcriptional activity. Indeed, the
phosphorylation of p53 at serine 15 by ATM following DNA damages has been
shown to block its nuclear export and recruit CBP/p300 to its target genes, both
actions enhancing its transcriptional activity. Furthermore, p53 is reportedly
93
acetylated by p300 upon DNA damages and its acetylation enhances its sequence-
specific binding activity toward its genes. ATM also phosphorylates Mdm2 at serine
395, which blocks its ubiquitin E3 ligase activity toward p53. Adding more
complexity to this regulatory network, a possible scenario is that KAP1 itself may be
targeted by ATM and its phosphorylation upon DNA DSB directly affects its
SUMOylation status, removing its transcriptional co-repressor activity from its target
genes. In this sense the blockage of Dox-induced gadd45α transcription can then be
explained, at least in part, by the phosphorylation-dependent swing of KAP1
SUMOylation.
94
Chapter Six
CONCLUSIONS AND FUTURE DIRECTIONS
In this dissertation I first described a proteomic method to screening cellular
SUMOylation targets. Fortunately and unfortunately, one of the three subjects—
KAP1--identified from combined affinity purification and tandem mass spectrometry
seems as a bona fide SUMOylation target, while PP2C and STK38 failed in both in
vivo and in vitro SUMOylation assays. I have also developed a novel method aimed
to direct pinpointing the SUMOylation acceptor lysines in KAP1, however for some
reason, after several tries using both MALDI and ESI--the two most commonly used
ionization methods for mass spectrometric analysis, the recovery rates of KAP1 by
both methods were low (about 30% in amino acid sequence) and it was impossible to
recover nearly any putative SUMOylation site. Hence the KAP1 digest seems to
have certain characteristics preventing it from protonation or carrying hydrogen ion.
Since ionization with MALDI or ESI also produces other charged species through
de-protonation and cationization, a modified detection and search strategy at the
analyzer will enable the detection of these species so to solve this problem of low
recovery rate.
Failure in detecting the SUMOylation of PP2C and STK38 is most likely due
to reasons other than false positives given by the mass spectrometry, because these
candidates were seized by a set of stringent filtering parameters as mentioned in
95
chapter two. A hypothesis developed by Ronald Hay aimed to explain the low
cellular SUMOylation activity proposes that SUMOylation is a transient, transitional
state between two functional states. Unlike protein phosphorylation or acetylation
that directly regulate protein-protein interactions or protein activities and therefore
these modifications carry certain functionalities until a change back to the
unmodified state takes place, SUMOylation does not stay with a particular functional
state of that protein and therefore it is hard to catch on this short-lived modification.
Hence it is possible that PP2C and STK38 do get SUMOylated in a small timeframe
which I could not pin down.
The most exciting discovery of this dissertation must be that the down-
regulation of KAP1 SUMOylation by Dox directly regulates p21 transcription
through ZBRK1-KAP1 complex. In addition to that, I managed to prove ZBRK1 and
KAP1 are relevant regulators of the transcription of p21 by ChIP, p21 luciferase
assay, and RNA interference. The identification of sequence-specific binding sites of
ZBRK1 on the p21 promoter is currently underway and eight putative ZBRK1-
binding elements, each of which never mismatches more than two nucleotides from
the 15-bp consensus sequence G-G-G-X-X-X-C-A-G-X-X-X-T-T-T, has been
located through the motif screening. In addition, the cartoon in figure 14 summarizes
the mechanism which ZBRK1-KAP1 complex used to regulate its target genes by the
swing of the KAP1 SUMOylation status.
At the same time of writing this dissertation, Dr. Yosef Shiloh’s lab reported
that DNA damage-responsive protein kinase ATM phosphorylated KAP1 at serine
96
ZBRK1
KAP1
Chromatin
Remodeling
Activities
SUMO1
p53 BE
ZBRK1 BE
ZBRK1
KAP1
Chromatin
Remodeling
Activities
SUMO1
ZBRK1 BE
Nucleosome Nucleosome Nucleosome
Transcription
“ON”
p53
P Ac
histone histone histone
Me Me Me
histone histone histone
Ac Ac Ac
HMT
HDAC
Transcription
“OFF”
Figure 14. Proposed model of ZBRK1/KAP1 complex regulating the
transcription of their target gene via KAP1 SUMOylation in response to DNA
double strand break (DSB) damages. Upper panel: silenced/repressed transcription
under normal, resting state. Lower panel: de-repressed/activated transcription under
DSB. Me: lysine methylation. Ac: lysine acetylation. P: serine/threonine
phosphorylation. HMT: histone methyltransferase. HDAC: histone de-acetylase.
97
824 upon both double strand break inducer neocarzinostatin and ionization radiation,
and this phosphorylation was essential for chromatin de-condensation/relaxation
since the phosphorylation-defective KAP1 was incapable to carry out this function
(Ziv et al., 2006). Cells expressing this mutant were hypersensitive to DSB inducer
and more prone to cell death upon DSB challenges. Moreover, the serine-to-aspartate
mutant of KAP1 resulted in constitutive chromatin relaxation. The authors argues
that the temporary de-condensation of chromatin by KAP1may be attributed to its
novel function of “genome surveillance” that supports the notion made by Mats
Ljungman and David P. Lane in 2004 (Ljungman and Lane, 2004), in addition to its
role in the transcriptional regulation. This finding is reminiscent of the interplay of
phosphorylation and de-SUMOylation of transcription factor Elk-1 in response to
either the MAPK activation or the phorbol ester treatment, where the concurrent Elk-
1 phosphorylation and de-SUMOylation accounts for the activation and de-
repression of its target gene transcription, respectively (Yang et al., 2003). Both the
phosphorylation and de-SUMOylation of Elk-1 contribute to its transcription
activation, even though it is not yet known whether its de-SUMOylation is dependent
on its phosphorylation. Nonetheless by observing that the phosphorylation-ablation
mutant of KAP1 did not significantly alter the activation of cell cycle checkpoint
upon DNA DSB, it seems that the phosphorylation and the de-SUMOylation of
KAP1 may function differentially upon DSB damages. Whether the function of
presumed KAP1 de-SUMOylation is confined to the transcriptional regulation awaits
further clarification.
98
In chapter four I demonstrated that in addition to p21, four other genes that
all harbor putative 15-bp ZBRK1-binding element are also under the regulation of
the SUMOylation of KAP1, where the exogenous expression of SUMO-1-KAP1
effectively brought down their inducible transcriptions by Dox. The selection of
these five genes was not random but came with a reason: two of them are involved in
the regulation of cell cycle control and the other three are activators of apoptosis, and
the transcription of all five genes can be induced by DNA damages in the p53-
dependent manner. In the first place it may seem contradictory that DNA damages
activate the transcription of both cell-cycle-control genes and apoptotic genes
synchronously as well as their transcriptional de-repression controlled by a change of
KAP1 SUMOylation, since the activation of cell cycle checkpoints turned on by the
expression of cell cycle regulators generally suppresses apoptotic activity. But
depending on the severity of the damage cells sustain, a decision of proceeding DNA
repair or undergoing apoptosis to avoid the replication of erroneous genetic
information will be made. The question: how this decision of cell fate is carried out?
The ZBRK1-KAP1 complex seems to be a plausible convergence point that
differentiates the fate of survival or death. KAP1 was previously reported to serve as
a scaffold protein capable of forming a 50nm subnuclear structure that can add its
mass up to 10,000kDa. It is also my speculation that KAP1 may also be a part of
senescence associated heterochromatin foci (SAHF), in which PIASy is an active
ingredient. Once the DNA damage is assessed and a decision is made, KAP1 is
activated in either 50nm supramolecular complex (SC), which will primarily activate
99
cell-cycle-regulator genes like p21 and gadd45, or in the SAHF where PIASy is
activated that leads to apoptosis possibly through a combined actions of the down-
regulation of Rb and the increase of p53 SUMOylation. It would be interesting to see
if the formation of SC and SAHF is linked to the SUMOylation status of KAP1.
100
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111
Abstract (if available)
Abstract
In pursuit of new SUMOylation targets that regulate the activities of cell cycle progression and mediate cellular apoptosis, a proteomic screening that combined affinity chromatography and tandem mass spectrometry was launched to identify novel targets from SUMO-1 stably-expressed HEK293 cells. With a series of discreet screens and analyses, this effort yields twenty-three SUMOylation candidates, which are found to carry out distinct cellular functions. KAP1 was verified as a bona fide SUMO-1 substrate and a following literature research pointed KAP1 to a novel role in regulating the transcription of a cluster of cell cycle regulator genes and pro-apoptotic genes via its interaction with a transcriptional factor ZBRK1 that bound a 15-bp DNA sequence motif.
Linked assets
University of Southern California Dissertations and Theses
Conceptually similar
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Post-translational modification crosstalk regulates KAP1 co-repressor functions in response to DNA damages
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Asset Metadata
Creator
Lee, Yung-Kang
(author)
Core Title
A regulatory transcription module of ZBRK1/KAP1 complex and its signaling network in regulating DNA damage-responsive genes expression
School
School of Pharmacy
Degree
Doctor of Philosophy
Degree Program
Molecular Pharmacology
Publication Date
10/12/2006
Defense Date
09/21/2006
Publisher
University of Southern California
(original),
University of Southern California. Libraries
(digital)
Tag
ATM ATR,DNA double strand break damage,Doxorubicin,histone acetylation methylation,KAP1 SUMOylation,OAI-PMH Harvest,SUMO-specific protease SENP
Language
English
Advisor
Ann, David K. (
committee chair
), Duncan, Roger F. (
committee member
), Li, Wei (
committee member
)
Creator Email
yungkanl@usc.edu
Permanent Link (DOI)
https://doi.org/10.25549/usctheses-m91
Unique identifier
UC1174997
Identifier
etd-Lee-20061012 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-18937 (legacy record id),usctheses-m91 (legacy record id)
Legacy Identifier
etd-Lee-20061012.pdf
Dmrecord
18937
Document Type
Dissertation
Rights
Lee, Yung-Kang
Type
texts
Source
University of Southern California
(contributing entity),
University of Southern California Dissertations and Theses
(collection)
Repository Name
Libraries, University of Southern California
Repository Location
Los Angeles, California
Repository Email
cisadmin@lib.usc.edu
Tags
ATM ATR
DNA double strand break damage
Doxorubicin
histone acetylation methylation
KAP1 SUMOylation
SUMO-specific protease SENP