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Regulation of human small nuclear RNA gene transcription by
the tumor suppressor protein p53

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Anastasia Alekseevna Gridasova

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REGULATION OF HUMAN SMALL NUCLEAR RNA GENE TRANSCRIPTION BY
THE TUMOR SUPPRESSOR PROTEIN P53

By

Anastasia Alekseevna Gridasova

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
DOCTOR OF PHILOSOPHY
Department of Biochemistry and Molecular Biology

2005

ABSTRACT

REGULATION OF HUMAN SMALL NUCLEAR RNA GENE TRANSCRIPTION BY
THE TUMOR SUPPRESSOR PROTEIN P53

By
Anastasia Alekseevna Gridasova

Activation of the tumor suppressor protein p53 or loss of the Cockayne syndrome
complementation group B (CSB) protein induces fragile site formation at RNA
polymerase II-transcribed U1 and U2 snRNA gene loci and at RNA polymerase III-
transcribed SS rRNA gene loci. Yu et a1. (2000) hypothesized that p53 interferes with
transcription elongation functions of CSB, resulting in accumulation of stalled RNA
polymerase and impaired chromatin condensation at these gene loci. However, a role for
p53 and CSB in transcription of these genes has not been investigated.

In this study I show that both p53 and CSB are involved in human U1 snRNA and
SS rRNA gene transcription. I found that p53 represses U1 snRNA and SS rRNA gene
transcription by RNA polymerases II and III, respectively. p53 also represses U6 snRNA
gene transcription by RNA polymerase 111. Both DNA binding competent and defective
forms of p53 revealed similar levels of snRNA promoter occupancy during transcription
repression, suggesting that sequence-specific DNA binding by p53 is not essential for
repression of snRNA gene transcription. I further demonstrated that CSB plays a positive
role in snRNA gene transcription by both polymerases II and III and a negative role in
transcription of those other classes of RNA polymerase III-transcribed genes that contain
an intragenic arrangement of promoter elements.

The functional interplay between p53 and CSB in snRNA gene transcription was

also investigated. Firstly, removing CSB from cell extracts modulates p53 transcription

activity in vitro. CSB immunodepletion potentiates the inhibitory effect of p53 on U1
snRNA gene transcription, but does not affect p53-mediated repression of U6 snRNA
gene transcription. Interestingly, at low amounts p53 activates and at higher amounts
represses SS rRNA gene transcription when transcription is performed with CSB depleted
extracts. Secondly, CSB association with snRNA gene promoters was diminished after
UV light treatment concomitant with increased p53 promoter association. As CSB was
described as an elongation factor for RNA polymerase 11, p53 may affect elongation by
interfering with CSB promoter association. Thirdly, transient transfection of p53 results
in snRNA gene transcription repression concomitant with accumulation of covalently
modified forms of RNA polymerase 111. These forms of RNA polymerase III are more
enriched in CSB cells, suggesting that p53 and CSB have opposing roles in post-
translational modifications of RNA polymerase III. I speculate that p53 represses
elongation by RNA polymerase III by facilitating post-translational modifications of the
polymerase. Together, these results suggest that p53 may modulate CSB transcriptional
activity and support the hypothesis that fragile sites at U1 and U2 snRNA and SS rRNA
gene loci may be caused by the inhibitory effect of p53 on CSB-mediated elongation by

RNA polymerases.

 

ACKNOWLEDGEMENTS

I would like to express my sincere gratitude to my mentor R. William Henry,

whose knowledge and guidance helped me during the years of my dissertation research.

I would also like to thank the members of my thesis committee, Dr. David Amosti,
Dr. Zachary Burton, Dr. John LaPres and Dr. Richard Schwartz for their guidance during
these years. I am also grateful to Dr. David Amosti and Dr. Zachary Burton and also Dr.
James Geiger and Dr. Min-Hao Kuo for helping me to secure my post-doctoral position.
Additionally, I would like to thank Dr. Min-Hao Kuo and Dr. Asha Acharya for an

interesting and productive collaboration.

I would like to thank the current and former members of the Henry lab: Craig
Hinkley, Zakir Ullah, Heather Hirsch, Liping Gu, Xianzhou Song, Melissa Bosma, and

especially Gauri J awdekar and Tharakeswari Selvakumar for all their help and friendship.

Also, I would like to thank Dr. Zeikus and the members of his laboratory,
especially Harini Krishnamurty for her friendship and Jake McKinlay for his support and
for being a very special person in my life. Additionally, I would like to thank Dr. Scott
Hill and his family (wife Peggy and daughter Heather) for encouraging me to come to
study in the US, welcoming me to Michigan, and helping me over these years. Finally, I

would like to thank my mom for her love and support.

iv

 

TABLE OF CONTENTS

LIST OF FIGURES ........................................................................................................ vii

KEY TO SYMBOLS AND ABBREVIATIONS ........................................................... ix
CHAPTER 1:

Introduction ....................................................................................................................... l

1. Human small nuclear RNA ............................................................................................ 1

1.1. Diverse functions in cells ............................................................................... .l

1.2. Promoter organization of human snRNA genes ................................... 2

1.3. The General Transcriptional Machinery ............................................ 5

1.4. Regulation of human snRNA gene transcription .................................. 8

2. Tumor suppressor protein p53 .................................................................. 8

2.1. Discovery of p53 .............................................................................................. 9

2.2. Cellular functions of the p53 tumor suppressor protein ................................... 9

2.2.1. Cell cycle control ......................................................... 10

2.2.2. Apoptosis ........................................................................................ 10

2.2.3. DNA repair ...................................................................................... 11

2.2.4. Cellular response to p53 and p53 protein partners .......................... 12

2.3. p53 as a transcriptional factor ........................................................................ 13

2.3.1. Role of p53 in RNA polymerase I, II and III transcription ............. 13

2.3.2. Possible mechanisms of the transcriptional control by p53 ............ 14

2.4. p53 stability, cellular stress and post-translational modifications ................. 16

2.5. Structure of the p53 protein ........................................................................... 19

2.5.1. Crystal structure of the p53 domains .............................................. 19

2.5.2. DNA binding activity ...................................................................... 22

2.6. The p53 family ............................................................................................... 23

2.6.1. p53/p63/p73 ............................................................... 23

2.6.2. alternative forms of p53 .................................................. 24

3. Cockayne Syndrome factor B protein ......................................................... 25

3.1. Functional interplay with p53 ...................................................... 25

3.2. Functions of CSB ..................................................................... 26

3.3.1. Role in DNA repair ....................................................... 26

3.3.2. Role in transcription regulation ......................................... 27

3.3. Structure of CSB ..................................................................... 28

3.4. Mutant CSB protein and Cockayne syndrome .................................... 31
CHAPTER 2:

The p53 tumor suppressor protein represses human snRNA gene
transcription by RNA polymerases II and [11 independently of

sequence-spesific DNA binding ...................................................................................... 47
Abstract ................................................................................................................. 47
Introduction ........................................................................................................... 48
Materials and Methods .......................................................................................... 51

Results ................................................................................................................... 57

Discussion ............................................................................................................. 91
References ............................................................................................................ 95

CHAPTER 3:

The Cockayne syndrome complementation group B protein modulates

transcription regulatory functions of the tumor suppressor protein p53 ................ 102
Abstract ............................................................................................................... 102
Introduction ......................................................................................................... 103
Materials and Methods ........................................................................................ 106
Results ................................................................................................................. 1 10
Discussion ........................................................................................................... 134
References ........................................................................................................... 140

CHAPTER 4:

Summary ........................................................................................................................ 144
Summary ............................................................................................................. 144
References ........................................................................................................... 149

APPENDIX:

Possible role of HDACs in p53-mediated snRNA gene transcription repression....151
Results and Discussion ....................................................................................... 151
Materials and Methods ........................................................................................ 162
References ........................................................................................................... 166

vi

 

Figure l-l.

Figure 1-2.

Figure 1-3.

Figure 1-4.

Figure 2-1.

Figure 2-2.

Figure 2-3.

Figure 2-4.

Figure 2-5.

Figure 2-6.

Figure 3-1.

Figure 3-2.

Figure 3-3.

Figure 3-4.

Figure 3-5.

LIST OF FIGURES

Schematic representation of RNA polymerase II- and III-transcribed
promoters ............................................................................ 4

Schematic representation of the general transcription machinery for

RNA polymerase II- and III- transcribed genes 7
Schematic representation of the structure of p5 3 and ANp53 proteins

and the consensus DNA binding sites .......................................... 21
Predicted motifs of CSB ......................................................... 30

p53 represses human snRNA gene transcription by both RNA
polymerases II and III in vitro ................................................... 59

UV light inhibits snRNA gene transcription and stimulates p53 binding
to human snRNA gene promoters .............................................. 63

Human Ul snRNA gene core promoters contain a high-affinity
p53 binding site .................................................................... 72

The high affinity p53 element in the U1 promoter is not essential
for p53 repression in vitro ........................................................ 77

The p53 C terminus is sufficient for transcriptional repression and
promoter association .............................................................. 83

Endogenous p53 associates with the general transcription
factor SNAPc ....................................................................... 88

Endogenous CSB associates with RNA polymerase II-transcribed
snRNA gene promoters and RNA polymerase III-transcribed

promoters ......................................................................... 112
Endogenous CSB associates with RNA polymerase III ................... 115
CSB regulates snRNA gene transcription .................................... 119

UV light exposure affects p53 and CSB occupancy on snRNA gene
promoters in vivo ................................................................. 123

p53 affects CSB and RNA polymerase III occupancy
on U6 snRNA gene promoters in vitro. 125

vii

Figure 3-6.

Figure 3-7.

Figure 3-8.

Figure A-l.

Figure A-2.

Figure A-3.

Figure A-4.

Loss of CSB accentuates the RNA polymerase II and III
transcriptional response to p53 ................................................. 128

CSB modulates p53-dependent modifications of
RNA polymerase IH ............................................................. 131

Model: Roles of p53 and CSB in RNA polymerase III elongation
and stability ....................................................................... 138

Endogenous HDACl and HDAC2 associate
with human snRNA gene promoters .......................................... 153

Sumoylated protein population is enriched on
U1 snRNA gene promoters afier UV light exposure ....................... 156

p53 sumoylation contributes to p53-mediated
snRNA gene transcription repression in vitro ................................ 159

Sumoylated p53 is enriched in acetylated population
of p53 in vivo ...................................................................... 161

viii

 

ASSP

ATM

Brfl -TFIIIB

Brfl-TFIIIB

CDK

CHI-I

ChIPs

CKII

CO-IP

CS

CSB

CTD

C-terminal

Da

DBD

ds

DSE

ELL

EMSA

GAPDH

HAT

KEY TO SYMBOLS AND ABBREVIATIONS

apoptotic-stimulating proteins of p53

ataxia telangiectasia mutated kinase

TFIIIB complex composed of TBP, Brfl and del

TFIIIB complex composed of TBP, 8er and del
cyclin-dependent kinase

cartilage-hair hypoplasia

chromatin immunoprecipitations

casein kinase II

co-immunoprecipitation

Cockayne syndrome

the Cockayne syndrome complementation group B protein
carboxy terminal domain of RNA polymerase II largest subunit
carboxy-terminal

Dalton

DNA binding domain

double stranded

distal sequence element

the Eleven Lysine-rich Leukemia protein

electrophoretic mobility shift assay
glyceraldehyde-3-phosphate dehydrogenase

histone acetyl transferase

ix

 

HDAC
Hdm2

IE

JMY
Mdm2

mRNA

NLS
NTB
N-terminal
PSE
P-TEFb
PTF

RB

RD
RNAP
RPA
rRNA
RT-PCR
SNAPC

snRNAs

histone deacetylase

the human double minute protein
intermediate element

immunoprecipitation

junction mediating and regulatory protein
the murine double minute 2 protein
messenger RNA

nucleotide excision repair

nuclear export signal

nuclear localization signal

nucleotide binding domain

amino-terminal

proximal sequence element

positive transcription elongation factor-b
proximal sequence element transcription factor
the retinoblastoma tumor suppressor protein
the regulatory domain

RNA polymerase

the replication protein A

ribosomal RNA

reverse transcriptase polymerase chain reaction
the snRN A activating protein complex

small nuclear RNAs

snRNPs

SS

SUMO

SV40

TAD

TAF

TBP

TCR

TD

tRN A

small nuclear ribonucleoprotein particles
single stranded

the small ubiquitin—like modifier

Simian virus 40 large T antigen

the transactivation domain
TBP-associated factor

TATA binding protein

transcription coupled repair

the tetramerization domain

transfer RNA

ultraviolet light

xi

CHAPTER 1

INTRODUCTION

1. Human small nuclear RNA
1.]. Diverse functions in cells

Small nuclear (sn) RNAs are short, stable, nontranslated RNAs that are
evolutionarily well conserved and found in the nuclei of all eukaryotic cells. These RNAs
are not polyadenylated and contain unusual 5’ cap structures. They exist in the cell
packaged with proteins as small nuclear ribonucleoprotein particles (snRNPs). Newly
synthesized snRNAs are immediately exported to the cytoplasm where they undergo
maturation and assembly into snRNPs prior to their reentry into the nucleus (55).

SnRNA perform essential functions in cells. As part of snRNPs, uridine rich
snRNAs are involved in messenger (m) RNA splicing (e.g. U1, U2, U4, U5 and U6) and
ribosomal (r) RNA processing (e.g. U3) (55). It is estimated that up to 15% of all point
mutations causing human genetic disease result in an mRNA splicing defect (70, 138).

In addition to their roles in RNA metabolism, some snRNAs also participate in
regulation of transcription initiation (e.g. U1 snRNA and BZ RNA) or transcription
elongation (e.g. 7 SK and components of the splicing apparatus) through association with
transcription factors (36, 39, 76, 108, 153). U1 snRNA associates with TFIII-I and
stimulates transcription initiation in vitro (76). In contrast, B2 RNA represses
transcription initiation when it binds to RNA polymerase 11 upon heat shock of mouse

cells (36). Repression of transcription elongation by 7SK RNA occurs through the

inhibition of the kinase activity of the positive transcription elongation factor (P-TEFb)
(108, 153).

Recent evidence suggests additional unexpected snRNA cellular functions in
innate immunity (58), human hereditary diseases (e.g. cartilage-hair hypoplasia (CHH)

disease) (116) and cancer (70, 138).

I . 2. Promoter organization of human snRNA genes

Human snRNA genes are transcribed by either RNA polymerase II (e.g. U1
snRNA gene) or by RNA polymerase III (e. g. U6 snRNA gene) and yet have very similar
promoter architecture (Figure 1—1). Both RNA polymerase II- and III- transcribed snRNA
genes contain the proximal sequence element (PSE) within the core promoter region.
This essential element is located at approximately —45 bp upstream of the start site of
transcription. In addition, the RNA polymerase III-transcribed snRNA genes contain a
TATA box located in the core promoter adjacent to the PSE. In humans, the TATA box
acts as dominant element for determining the specificity of RNA polymerase III-
transcribed snRNA gene promoters (56, 75, 88). Both the RNA polymerase II and III
snRNA gene promoters contain the distal sequence element (DSE), located in the
regulatory region around —220 (105). The DSE functions as an enhancer and is required
for maximum promoter activity.

As shown in Figure 1-1, human U6 snRNA genes are distinct from other RNA
polymerase III-transcribed genes. U6 snRNA genes belong to class 3 promoters, which
are defined by their extragenic RNA polymerase III promoters. In contrast, the class 1

and 2 RNA polymerase III-specific genes contain intragenic promoter elements and are

Figure 1-1. Schematic representation of RNA polymerase II- and III- transcribed
promoters. Class 1 and 2 RNA polymerase III-transcribed promoters are intragenic and
are exemplified by the SS rRNA and tRNA promoters, respectively. Class 1 genes
contain A and C boxes that are separated by an intermediate element (IE). Class 2
promoters consist of A and B boxes. Class 3 promoters are defined as extragenic RNA
polymerase III promoters and are exemplified by the human U6 snRNA promoter. The
architecture of the class 3 RNA polymerase III-transcribed snRNA gene promoters is
similar to other snRNA gene promoters that are transcribed by RNA polymerase 11 (e. g.
U1 snRNA gene). Both RNA polymerase II- and III— transcribed snRNA gene promoters
contain the proximal sequence element (PSE) and the distal sequence element (DSE). In

addition, class 3 RNA polymerase III-transcribed promoters have a TATA box.

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exemplified by the SS rRNA and tRNA promoters, respectively. Class 1 genes contain A
and C boxes that are separated by an intermediate element (IE). These sequence elements
constitute the internal control region that is required for transcription (55, 113). Class 2

promoters consist of an A and B box (40, 55).

1.3. The General Transcriptional Machinery

As illustrated in Figure 1-2, the PSE of class 3 RNA polymerase III-transcribed
and RNA polymerase II-transcribed snRNA genes is recognized by the snRNA activating
protein complex (SNAPC), also referred to as the PSE-binding transcription factor (PTF)
(119, 154). It is a multi-protein complex, composed of at least five proteins SNAP190,
SNAPSO, SNAP45, SNAP43 and SNAP19 (53). SNAPC is essential for the activity of
both RNA polymerase II- and III-transcribed snRNA promoters (54). The DSE for both
types of snRNA promoters is recognized by the POU domain Oct-1 protein (105, 136).
Other snRNA transcription factors are more promoter-specific. The TATA box serves as
a binding site for a TFIIIB-like complex (119) designated Brf2-TFIIIB, which is
composed of the TBP and TBP-associated factors Ber and del (126). TBP is also a
required factor for transcription of snRNA genes transcribed by RNA polymerase II (74).
Additionally, basal transcription from RNA polymerase II-transcribed snRNA gene
promoters requires TFIIA, TFIIB, TFIIF, and TFIIE (74).

RNA polymerase III-transcribed class 1 promoters recruit the transcription factor
TFIIIA that belongs to the zinc finger family of DNA binding proteins (98). The binding
of TFIIIA then allows the recruitment of TFIIIC (55). In contrast to class 1 promoters,

class 2 promoters recruit TFIIIC directly, without prior binding of TFIIIA (55). In both

Figure 1-2. Schematic representation of the general transcription machinery of the
RNA polymerase II- and III- transcribed genes. RNA polymerase III-transcribed class
1 promoters require the TFIIIC complex and TFIIIB complex. Class 1 genes have an
additional requirement for TFIIIA. The PSE and the DSE for both class 3 RNA
polymerase III-transcribed and RNA polymerase II-transcribed snRNA genes are
recognized by the multisubunit protein complex SNAPc and Oct-1 proteins, respectively.

Class 3 genes have an alternative TFIIIB complex, which binds to the TATA element.

58 rRNA
(Class 1)

tRNA
(Class 2)

U6 snRNA
(Class 3)

U1 snRNA

mRNA

 

O
Oct-1

 

A TFIIIC
"‘-

   

. \
SNAPc ATFIIIB I -

PSE TATA

 

 

class 1 and 2 RNA polymerase III promoters, the recruitment of TFIIIC is followed by
the recruitment of the TBP-containing complex designated Brfl -TFIIIB (99).
Brfl -TFIIIB is composed of the TATA box binding protein (TBP) and at least two

additional TBP-associated factors called Brfl (99, 100) and del (126).

1.4. Regulation of human snRNA gene transcription

In addition to the general transcription machinery snRNA gene transcription was
found to be regulated by the tumor suppressors and oncoproteins, suggesting that these
snRNAs may play important roles in controlling cellular homeostasis. The tumor
suppressor proteins RB and p53 were shown to repress snRNA gene transcription by
RNA polymerase III (14, 17, 43, 57). In addition p53 also represses RNA polymerase II-
transcribed snRNA genes (43). Interestingly, an oncoprotein CKII can both activate (61)
and repress (60) U6 snRNA gene transcription by RNA polymerase 111 during cell cycle
progression. CKII-dependent phosphorylation of RNA polymerase III stimulates U6
snRNA gene transcription (61). In contrast, CKII-dependent phosphorylation of the de1
subunit of the Brfl-TFIIIB complex during mitosis results in U6 snRNA gene
transcription repression (60). Recently, Gu et a1. (2005) demonstrated a positive role of

CKII for U1 snRNA gene transcription by RNA polymerase II (45).

2. Tumor suppressor protein p53

The role of the tumor suppressor protein p53 in snRNA gene transcription was
initially suggested based on their unusual transcriptional response to UV light treatment.

The U1 through U5 snRNA genes showed prolonged inhibition of RNA synthesis in

response to UV light treatment (34, 103), as well as to other DNA damaging agents (158),
also known to activate the tumor suppressor protein p53; thus, suggesting a role of p53 in
snRNA gene transcription. Indeed, my studies revealed that p53 is directly involved in
snRNA gene transcription repression by both RNA polymerases II and III (see chapter 2

and references 14, 17, 43).

2.]. Discovery of p53

The p53 protein was discovered in 1979 as a 53-kDa protein that is bound by the
large T-antigen of the sarcoma-associated virus SV40 (86). It was first described as an
oncoprotein, because overexpression of p53 appeared to cause oncogenic transformation
of cells (110). Later studies revealed that uncontrolled cellular proliferation results from
p53 inactivation and demonstrated that wild type p53 is a tumor suppressor protein (79).

The gene encoding p53 is lost or mutated in more than 50% of human cancers.
The other half of human tumors are thought to contain alterations in other components of
the p53 pathway (59). Furthermore, germline mutations in p53 are responsible for the
majority of cases of the inherited cancer family syndrome known as Li-Fraumeni
syndrome (92). Thus, p53 inactivation is considered to be an important step in

carcinogenesis.

2. 2. Cellular functions of the p53 tumor suppressor protein

Once p53 is activated by a stress signal, p53 can induce any of several different
cell fates, including cell cycle arrest, apoptosis and repair of damaged DNA. How this

decision is determined is not well understood, however, multiple factors contribute to the

cellular fate choice, including cell type and the specific stress activation. The choice of
response is determined by the subset of genes activated or inhibited by p53 as described

below (69).

2.2.1. Cell cycle control

p53 arrests the cell cycle in G1 and G2 phases to potentially give cells time to
repair damaged DNA that could otherwise lead to mutations and genomic instability. In
the DNA damage response, the most important transcriptional target for p53-induced G1

1 “Fl/CW, which is a potent inhibitor of several cyclin-dependent kinase (CDK)

arrest is p2
complexes. p53 activates p21 gene expression by binding to two response elements

located in the p21 promoter (33).

p53 regulates the G2/M transition at the level of the cyclin-dependent kinase Cdc2,
which is essential for entry into mitosis (114). Several of the transcriptional targets of p53
can inhibit Cdc2 levels and activity, including p21, 14-3-38 and GADD4S. p21 inhibits
Cdc2 directly, 14-3-36 anchors Cdc2 in the cytoplasm where it cannot induce mitosis and
Gadd45 prevents the association of Cdc2 with Cyclin B1. p53 also represses transcription
of cyclin B] and cdc2 genes, which further enforces G2 cell cycle arrest. In addition, p53
can bind to and inhibit CAK activity, which may cause a drop in the extent of

phosphorylation and activity of Cch (140).

2. 2. 2. Apoptosis

Apoptosis is an evolutionarily conserved process by which an organism removes

unwanted or damaged cells. Two major apoptotic pathways are defined in mammalian

10

cells: (i) the extrinsic and (ii) the intrinsic pathways (109). p53 is implicated in the
induction of both apoptotic signaling pathways (51). p53 can activate the extrinsic
apoptotic pathway through the induction of genes encoding death receptors (Fas/APOI,
KILLER/DRS, and PERP) (5, 106, 141, 150). p53 was also shown to activate
transcription of pro-apoptotic genes (Apaf-l, Bax, NOXA, p53AIP1, and PUMA), which
are involved in the intrinsic apoptotic pathway (66, 156).

In addition, there is a transcription-independent p53 function in apoptosis. In this
case, p53 binds anti-apoptotic proteins Bcl-2 and Bcl-xL in the mitochondria. These two
proteins are otherwise known to inhibit pro-apoptotic proteins Bax and Bak, which are
involved in the release of cytochrome c from mitochondria. It has been proposed that p53
binds directly to Bel-2 and Bcl-xL and hence liberates Bax and Bak, allowing the caspase

cascade to occur (18).

2.2.3. DNA repair

p53 is required for efficient nucleotide excision repair (NER) pathway by the
direct interaction with components of the repair machinery. p53 binds and modulates the
activities of the NER-associated helicases XPB and XPD (49, 147). Also, p53 associates
with and inhibits the replication protein A (RPA), which is needed for DNA replication,
homologous recombination, and NER. The inhibitory interaction is disrupted upon UV
radiation allowing RPA to participate in DNA repair (1). In addition, p53 has been shown
to interact with the Cockayne syndrome complementation group B (CSB) protein (147,
155). CSB is required for the transcription-coupled repair (TCR) pathway and may be a

DNA repair protein (142, 145). It has been proposed that CSB recruits the nucleotide

11

excision repair (NER) machinery to sites of stalled RNA polymerase to permit rapid
repair of the transcribed strand (11). In addition, p53 can recruit histone acetyl
transferases (HAT) complexes to chromatin and promote chromatin relaxation, which

results in increased chromatin accessibility for NER factors (118).

2. 2. 4. Cellular response to p53 and p53 protein partners

Whether a cell undergoes cell cycle arrest or apoptosis in response to p53 depends
on several factors. Firstly, the p53 activity itself can contribute to the choice of response.
The type and the magnitude of the cellular stress may control p53 functions by affecting
the level or activity of the p53 protein that is induced. Activation of apoptosis has been
associated with higher levels of p53 than those required for cell cycle arrest (16). It was
also suggested that promoters regulating expression of apoptotic genes bind p53 with a
lower affinity then the cell cycle arrest targets (16). Affinity of p53 to target promoters
might be regulated by covalent modifications of p53. For example, phosphorylation of
p53 on Ser46 is required for the induction of the apoptotic target gene p53AIP1 (p53-
regulated apoptosis inducing protein 1) (111). Secondly, the strength of p53 interaction
with a particular promoter might also be regulated by interactions with other cellular
factors. The ASPP (Apoptotic-stimulating proteins of p53) proteins bind to the
evolutionarily conserved DNA binding domain (DBD) of p53 and specifically increase
the transactivation of pro-apoptotic p53-responsive genes such as Bax and PIG3, but have
little effect on other p53 target genes that are involved in other functions such as cell
cycle arrest (122). Junction mediating and regulatory (JMY) protein and p53 family

members, p63 and p73, were also shown to be required for p53-induced apoptosis (38,

12

131). The new studies reveal an extra layer of complexity by showing that even when
both cell cycle arrest and apoptotic target genes are induced by p53, the resultant
response may depend on factors that cause selective inhibition of p53 target genes that

encode a survival function (129).

2. 3. p53 as a transcriptional factor
2.3.1. Role of p53 in regulation of the RNA polymerase I, II and III transcription

p53 has been shown to either activate or repress a variety of cellular promoters
transcribed by RNA polymerase 11. p53 activates RNA polymerase II-transcribed genes
by binding to its cognate DNA-binding sites in target gene promoters (33). In addition,
p53 represses certain RNA polymerase II-transcribed promoters, many of which lack a
p53 response element (130). A novel/altemative p53 DNA-binding site was identified at
the promoters of some p53-repressed genes (e. g. MDRI, Cyclin A, Cyclin BI). This novel
p53 DNA~binding site has defined consensus quarter-sites arranged in head-to—tail
orientation (64). p53 can also inhibit transcription from a variety of TATA-containing
RNA polymerase II-transcribed promoters. Direct interactions between p53 and TBP
(and/or TAFs) are thought to play a role in mediating p53 repression of certain RNA
polymerase II-dependent genes (37).

In addition to a role in regulating transcription from RNA polymerase II-
dependent promoters, p53 has been also shown to repress transcription of genes
transcribed by RNA polymerase I (157) and RNA polymerase III (14, 17). The major
product of these genes, rRNA, tRNA, and snRNA are essential for the translational

capacity of cells, and regulation of their synthesis is closely linked to cellular growth

13

 

rates. Thus, p53-mediated down-regulation of RNA polymerase I- and III-dependent
transcription may help restrict cell growth and, perhaps, tumor formation (27).
Consistently, synthesis of tRNA and SS rRNA is elevated significantly in fibroblasts
derived from p53 knockout mice (14) and inherited mutations in the p53 protein are often
associated with aberrant RNA polymerase III activity in primary fibroblasts from patients

with Li-Fraumeni syndrome (134).

2. 3. 2. Possible mechanisms for transcriptional control by p53
2. 3. 2. 1. p53 as a transcriptional activator

Recent studies revealed that p53-mediated transcription activation is tightly
associated with chromatin modifying activities of p53 (3, 118). During transcription
activation, promoter-bound p53 can recruit general transcription factors (6. g. TBP, TAFs)
or co-activators such as histone acetyl transferases p300/CBP or arginine
methyltransferases PRMTI and CARMI to chromatin (3, 47). Other co-activator
complexes have been shown to function in p53-dependent transcription regulation,
including SAGA (Spt-Ada-GcnS-acetylase) and NuA4/Tip60 complexes, whose common

subunit, the ATM-related protein TRRAP, can bind directly to p53 (4).

2.3.2.2. p53 as a transcriptional repressor

It has been shown that p53 can repress RNA polymerase II transcription by a
variety of mechanisms that regulate pre-initiation complex formation. Transcriptional
repression by p53 can be mediated by p53 binding to consensus DNA elements or in the

apparent absence of p53 binding to promoter DNA. For example, p53 can bind directly to

14

 

its consensus DNA sequences and either prevent binding of transcriptional activators or
the basal transcriptional machinery to promoters (14, 27, 157). Alternatively, p53 can
interact with a promoter-bound transcriptional activator and prevent the subsequent
recruitment of co-activators such as histone acetyl transferases (p300/CBP) (73). p53 may
also directly recruit co-repressors such as histone deacetylases (HDACs) to repress
transcription (65). p53-mediated transcriptional repression may also be achieved through
the physical interaction of p53 with the general machinery or activators rendering
proteins inactive for binding to the promoter and for transcriptional activation.

In addition to affecting pre-initiation complex assembly, p53 can regulate
transcription elongation (9). p53 interacts with and inhibits the activity of two
transcription elongation factors — the Eleven Lysine-rich Leukemia (ELL) protein and the
CSB protein (132, 155). ELL increases the overall rate of transcription elongation by
RNA polymerase 11 via suppression of transient pausing by the polymerase at many sites
along DNA, and it is thought that the CSB protein functions as an elongation factor to
promote transcription of genes encoding RNA products with significant secondary
structure, such as human U1 and U2 snRNA genes (132, 155). Additionally, p53
represses transcription elongation by interacting with and inhibiting the activity of the
CAK kinase — a component of the general transcription factor TFIIH (124). CAK plays a
role in RNA polymerase 11 promoter escape through phosphorylation of the C-terminal
domain (CTD) on RNA polymerase II (2).

Wild-type p53 can repress not only RNA polymerase II promoters but also RNA
polymerase I- and III-transcribed genes (14, 17, 27, 43, 135, 157). RNA polymerase I

transcription repression by p53 is probably achieved by inhibiting interactions between

15

general transcription factors SL1 and UBF and preventing the formation of a productive
initiation complex at rRNA gene promoters (157).

The mechanism for p53 regulation of RNA polymerase III transcription is
controversial. Several in vivo and in vitro studies demonstrated a direct role of p53 in
RNA polymerase IH transcription repression. It was proposed that p53 represses RNA
polymerase III transcription by interacting with the TBP component of TFIIIB and
preventing recruitment of TFIIIB to the promoters of repressed genes (14, 17, 27, 43,
135). An indirect role of p53 in RNA polymerase III transcription repression has also
been suggested. A kinetic analysis of RNA polymerase III transcription repression, using
p53 expressed from a stably integrated inducible p53 gene, revealed that RNA
polymerase III repression can be mediated indirectly through p53-dependent degradation

ofTFIIIB (31).

2. 4. p53 stability, cellular stress and p53 post-translational modifications

Under normal conditions p53 undergoes rapid turnover, and is thus maintained at
low steady state levels that restrict its impact on cell fate. Rapid p53 turnover in normal
cells is largely due to the murine double minute 2 (Mdm2) (or the human double minute 2
(Hdm2)) oncoprotein. Mdm2 binds to the N-terminal TAD region of p53 and represses
P53 activity via two mechanisms: by promoting p53 degradation and by blocking p53
transcriptional activation (52, 71, 102). Mdm2 is an E3 ubiquitin ligase that promotes the
covalent conjugation of ubiquitin residues to p53. Mdm2 can only mono-ubiquitylate p5 3
(73) The mono-ubiquitylation exposes a nuclear export signal (NES), which allows p53

to be exported from the nucleus into the cytoplasm. The poly-ubiquitylation of p53 is

16

achieved by histone acetyl transferase (HAT) p300, which has intrinsic ubiquitin ligase
activity (44). In addition, Pirh2 and Copl have recently been identified as other RING
finger proteins that bind p53 and mediate its poly-ubiquitylation (81). HAUSP has been
shown to deubiquitylate and stabilize p53 by removing its ubiquitin modifications (82).
The poly-ubiquitylated p53 is then subjected to proteasomal degradation by the 26S
proteasome. The binding of Mdm2 to p53 is inhibited by phosphorylation of p53 by
ATM-family and Chk-family kinases and phosphorylation of Mdm2 by ATM-family
kinases in response to DNA damage (15).

In unstressed cells, in addition to the low levels of the p53 protein, p53 exists in a
latent form, inactive for transcription. p53 activation is accomplished by post-
translational modifications in response to a variety of intrinsic and extrinsic cellular stress
signals. Different cellular stresses result in different patterns of post-translational
modifications of p53. The extensive covalent modifications affect p53 stability,
oligomerization, sub-cellular localization and association of p53 with cellular factors (12,
22, 47, 120, 151).

The multiple lysine residues at the extreme C-terrninal RD of p53 can be post-
translationally modified by multiple mechanisms. The five lysine residues of p53
(Lys370, -372, -373, -381, and 382) can be acetylated by CBP/p300 and Lys320 by
P/CAF (46, 120). The role of p53 acetylation has been studied extensively. Gu et al.
(1997) suggested that CBP/p300 mediated acetylation of p53 can increase p53 sequence-
Specific DNA-binding activity in vitro (46). However, subsequent studies showed that
acetylation does not increase the p53 DNA-binding activity when the protein is assayed

for binding to artificially reconstituted chromatin, but instead, p53 acetylation is

17

 

important for the recruitment of co-activators (35). Also, the same lysines can be
ubiquitylated by E3 ligases Mdm2, p300, Pihr2 and Copl (29, 52, 81). Several studies
suggested that acetylation and ubiquitylation of p53 at the C-terminus can regulate p53
stability. In contrast to ubiquitylation, p53 acetylation can stabilize p53 (63, 72). In
addition to acetylation and ubiquitylation, the C-terminal lysine residue Lys386 of p53
can be modified by conjugation to small ubiquitin-like protein SUMO-1, however the
functional consequence of p53 sumoylation remains controversial (117). Several studies
demonstrated both positive and negative roles of sumoylation for p53-mediated
transactivation of target genes (42, 117, 123). However, studies by Kwek et al. (2001) did
not confirm these observations (77). Interestingly, using a yeast-two-hybrid approach, Dr.
Min-Hao Kuo (Michigan State University) found that some Sumo-modifying E1 and E2
enzymes preferentially interact with p53 acetylated at K320. My subsequent studies
revealed that sumoylated p53 is enriched in the acetylated p53 population, suggesting that
p53 acetylation may be a prerequisite for subsequent p53 sumoylation. Recent studies
also showed that the ubiquitin-like protein Nedd8 can be covalently linked to p53 at
Lys370, -372, and —373. Neddylation of p53 negatively regulates its transcriptional
activity (151). Additionally, methylation of p53 at Lys372 by Set9 methyltransferase has
been identified and suggested to stabilize p53 and restrict it to the nucleus (22).
Phosphorylation has also been shown to regulate p53 transcriptional activity. For
exanmle, the rapid phosphorylation on the C-terrninus on Ser392 in response to UV light
(67) may stimulate sequence-specific DNA binding activity of p53 (62, 67). Also, an
ATM-dependent dephosphorylation of p53 at Ser376 creates a binding site for 14-3-3

proteins, which can activate sequence-specific DNA binding of p53 (148).

18

2. 5. Structure of the p53 protein
2.5.]. Crystal structure of the p53 domains

The human p53 protein contains 393 amino acids and has been divided
structurally and functionally into five domains (Figure 1-3A): (i) the transactivation
domain (TAD), (ii) the proline-rich domain, (iii) the DNA binding domain (DBD), (iv)
the tetramerization domain (TD) and (v) the regulatory domain (RD) (69). The TAD
(amino acids 1-42) is located in the N-terminal part of the protein. Next to a TAD is a
proline-rich area (63-97). The proline-rich SH3-target subdomain contains five copies of
the amino acid sequence PXXP, which contributes to the apoptotic functions of p53 (121,
146). Sequence-specific DNA binding is mediated through the DBD of p53 (amino acids
102-292). The C-terminal part of p53 is subdivided in the TD (amino acids 326-353) and
the RD (amino acids 363-393) (69).

Studies on the structural organization of p53 domains revealed that both TAD and
RD of p53 are natively unfolded (8, 28). The structure of the DBD and TD were
determined both by X-ray crystallography and NMR (19, 25, 68, 149). The DBD of p53
comprises nine anti-parallel B—strands, which form a B-barrel. B-strands are held close
together by three loop regions (L1, L2 and L3). Loops L2 and L3 are stabilized by a zinc
atom. In addition to three loops, the DBD of p53 also has a strand-loop-helix region
called the SLH motif. It connects the B-barrel with the ct-helix at the C-terrninus of the
DBD of p53. The C-terminal a-helix and loop L1 of p53 bind to the major groove of the
DNA. In addition, loop L3 contacts DNA in the minor groove (19). The majority of p53
mutations found in human tumors occur within DBD, including six hot spot mutations

most commonly found in human cancers. Two of these hot spot mutations (R248 within

19

Figure 1-3. Schematic representation of the structure of p53 and ANp53 proteins
and the consensus DNA binding sites. (A) Schematic representation of the tumor
suppressor protein p53 and ANp53: TAD = Transactivation domain, P = Proline-rich
region, DBD = DNA binding domain, TD = tetramerization domain, RD = regulatory
domain. (B) Schematic of p53 consensus DNA binding sites. The consensus p53 DNA
binding site consists of 2 half sites, each comprised of two copies of the sequence 5’-Pu-
Pu-Pu-C-A/T-3’ (Pu is purine), arranged head-to-head (HH) (for p53 acting as a
transcriptional activator) or head-to-tail (HT) (for p53 acting as a transcriptional

repressor), and separated by O to 14 nucleotides.

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21

L3 and R273 within the C-terminal or-helix) contact DNA directly. The other four (R175,
G245, R249 and R282) stabilize the surrounding protein structure (19, 149). The TD is
represented by a B-strand linked to an or-helix. A TD monomer has a V-shape. Two
monomers form a dimer through interactions between the B-strands and the or-helices
arranged in an anti-parallel fashion. The interactions that hold two monomers together as
dimers are mainly provided through hydrogen bonds in the B-strands and hydrophobic
interactions from both the B-strands and the or-helices. Two dimers are held together as a

tetrarner by a large hydrophobic surface of each or-helix (25).

2. 5. 2. DNA binding activity

As shown in figure 1-3B, the consensus p53 DNA binding site consists of 2 half
sites, each comprised of two copies of the sequence 5’-Pu-Pu-Pu-C-AfT-3’ (Pu is purine),
arranged head-to-head (for p53 acting as a transcriptional activator) or head-to-tail (for
p53 acting as a transcriptional repressor), and separated by 0 to 14 nucleotides (33, 64).
Some variations within consensus PuPuPuC(A/T) sequence are also permissible (115).
These specific cognate sites can bind tightly to the DBD of p53. It was suggested that one
p53 DBD dimer binds first to one half of the consensus DNA-binding site, increasing the
probability for the binding of the second p53 dimer to the adjacent half of the site (95, 96).

In addition to binding specifically to DNA at p53 consensus sites, p53 also binds
non-specifically to ssDNA, nicked DNA, damaged DNA with ds breaks, and DNA with
Holliday junctions (87). These DNA structures represent the intermediates of DNA
damage and repair. Binding to non-specific DNA was primarily mapped to the C-

terminal domain of p53 (112, 159). It was generally accepted that the C-terminus of p53

22

is a negative regulator of p53 sequence-specific DNA binding. Several groups have
demonstrated that various alterations of the C-terminal domain (deletion, post-
translational modifications and interaction with antibodies directed at a C-terminal
epitope) result in an increase of p53 DNA binding (46, 62, 120). However, more recent
studies have shown that p53 binds its target sites in vitro and in vivo in the absence of
DNA damage or extensive modifications of the C-terminus (6, 35) and the C-terminal-
deleted p53 is substantially less efficient at binding and transactivation of its targets in
vivo (94). It suggests that the C-terminus does not maintain p53 in a state that is inactive
for DNA binding, but rather that it is required for efficient binding of p53 to its target

promoters.

2. 6. T he p53 family
2.6.1. p53/p63/p73

Since the discovery of p53 in 1979, two more members have been added to the
p53 superfarnily, p63 and p73. The p53 family members share very significant homology
both at the genomic and at the protein levels (152). Each contains a TAD, a DBD, and
TD. In addition, p63 and p73 contain long C-termini. As a result of the alternative
splicing of their C-termini, three p63 isoforms (or to y) and seven p73 isofonns (or to n)
were identified. Additional complexity is also achieved because these isoforrns (called
the TA and AN isoforrns, respectively) are transcribed from the upstream promoter as
well as from a cryptic promoter within intron 3 (152). The highest level of homology
between the p53 family of proteins is reached in the DBD, which suggests that the three

proteins can bind to the same DNA sequence and regulate the same genes. Indeed, p63

23

and p73 bind to p53 consensus DNA elements and trans-activate certain p53 target genes
(38). p63 and p73 may also inhibit the transcriptional activity of p53 by competing for the
same binding sites on DNA (38). High conservation of the TD sequence between p53
family members results in formation of hetero-oligomers as well as homo-oligomers.
ANp63 and ANp73 forms lack TAD and may inhibit p53 in a dominant-negative fashion
through forming hetero-complexes with p53 (101).

Unlike p53, the genes encoding p63 and p73 are rarely mutated in human cancer.
The phenotype of the p63- and p73- deficient mice suggests that the primary biological
function of these proteins is to regulate development, which is in contrast to p53-null

mice, which are highly tumor prone but lack a developmental phenotype (101).

2. 6. 2. Alternative forms of p53

The human ANp53 and its murine counterpart p44 are naturally occun'ing
isoforrns of p53 (26, 91, 127). ANpS3 is encoded by the p53 locus, but uses an alternative
translation start site located in exon 4 at codon 40 in human RNA. The resultant 44 kDa
protein lacks the corresponding N—terminal amino acids (Figure 1-3A). Choice of start
site depends on an interaction between p53 and its cognate RNA, which requires the N-
terrninal domain of p53 and its DNA binding domain. The complex of the p53 protein
with newly synthesized RNA prevents the ribosome at ATG (codon 1) from being
activated and translating full-length p53. Instead, translation of ANp53 by the ribosome at
ATG (codon 41) occurs. The RNA — p53 protein complex is destabilized by Mdm2,

which competes with RNA for binding to N-terminus of p53 (127). Since the ANpS3

24

lacks the Mdm2 binding site, it is not a subject to Mdm2-mediated degradation. This
results in a prolonged half-life of ANpS 3.

ANp53 is severely compromised in ability to activate target genes due to complete
absence of N-terminal TAD that binds to the basal transcription machinery. Since ANpS3
is able to hetero-tetramerize with full length p53, the ratio of fiill-length to ANp53 can
determine cellular functions of p53. At low levels, ANp53 would be exclusively found in
tetramers with full—length. At high levels, however, excess short form would also be
present as homo-tetramers or even as non-tetrameric forms, such as monomers or dimer,
which could have several different effects (127). Homo- and hetero- tetramers of ANp53
would be severely compromised in their ability to activate target genes due to complete
absence of N-terminal TAD in ANpSB (97). Also, non-tetrarneric forms of ANp53 could
replace p53 in transcription-independent activities, such as mitochondrial cytochrome c

release, altering the ability of p53 to mediate apoptosis (97).

3. Cockayne Syndrome factor B protein
3.]. Functional interplay with p53

Several studies demonstrated that infection of human cells by Adenovirus type 12
induces specifically four fragile sites including efficiently induced fi'agility of the U1 and
U2 snRNA gene loci and U1 snRNA pseudogenes in several different cell lines and
weakly induced fragility of the SS rRNA gene in primary human embryonic kidney cells
(30, 41, 84, 125). Formation of the same fragile sites were also observed after treatment
of cells with actinomycin D or cytosine arabinoside C (83, 90). All these factors are

known to activate p53 and do not induce fragility in cells lacking firnctional p53 (83). In

25

addition, these fragile sites can be induced by over-expression of the full length wild type
p53 or just the C-terminal domain of p53 alone (155). Together, these observations
suggest that p53 plays a direct role in fragile site formation at U1 and U2 snRNA and 5S
rRNA genes, and the C-terminus of p53 is required for p53-mediated induction of fragile
sites. Interestingly, loss of functional CSB in cells also results in fragile site formation at
the U1 snRNA, U2 snRNA and SS rRNA gene loci (155). The requirement of p53
activation or loss of CSB activity for fragile site formation at these loci suggests a
possible role for these factors in controlling transcription of these genes, even though U1
and U2 snRNA genes are transcribed by RNA polymerase II and SS rRNA genes are
transcribed by RNA polymerase III. Studies by Selby and Sancar (1997) suggested a role
of CSB for elongation of gene, encoding highly structured RNAs (such as U1 and U2
snRNAs) (128). Interestingly, CSB has been shown to interact with p53 within the C-
terrninal region (147, 155). It was suggested that p53 binding to CSB may interfere with
CSB activity; thus, mimicking loss of CSB (155). Yu et al. (2000) proposed that p53 may
repress RNA polymerase II transcribed snRNA genes by interfering with Cockayne
Syndrome group B (CSB)-mediated elongation, causing RNA polymerase II stalling at
U1 and U2 snRNA genes. Stalled RNA polymerase protein complexes may interfere with

local chromatin condensation, causing locus-specific chromosome fragility (155).

3.2. Functions of CSB
3.2.]. Role in DNA repair

The CSB protein was originally identified as a DNA repair protein and it was

cloned on the basis of its ability to complement the transcription coupled repair (TCR)

26

defect in CS cells (142, 145). It has been proposed that CSB recruits the NER apparatus
to sites of stalled RNA polymerase II to permit rapid repair (145). CSB is also critical for
the repair of nucleotide base damage induced by reactive oxygen species when such
lesions are located on the transcribed strand of active genes (80). CSB chromatin
remodeling activity may be required to open chromatin around lesions, thereby
stimulating repair. In addition, the CSB protein may also play a role in clearing the
stalled RNA polymerase II molecule from the lesion site so that repair can occur and
transcription resume (49). The observed interaction of CSB with RNA polymerases I and

11 both in vivo and in vitro might specifically target CSB to sites of blocked transcription

(11).

3.2.2. Role in transcription regulation

Various in vitro and in vivo experiments point to a possible role for CSB in
transcription. CSB was shown to associate with RNA polymerase I and 11 protein
complexes (11, 144). Gel mobility shift assays revealed that CSB interacts with a ternary
complex of DNA, RNA polymerase II, and nascent RNA (139) and in vitro transcription
experiments showed that CSB stimulates elongation by RNA polymerase II (128). It is
suggested that CSB functions as an elongation factor to promote transcription of genes
encoding RNA products with significant secondary structure, such as human U1 and U2
snRNA genes (155).

Additionally, CSB is also implicated in chromatin remodeling during transcription.
It was demonstrated that CSB remodels nucleosomes at the expense of ATP hydrolysis

and interacts with core histones in vitro (24). Also, CSB can alter DNA double helix

27

conformation upon binding by wrapping DNA using energy from ATP hydrolysis (7).
One possibility is that by changing DNA conformation CSB may disrupt the histone-
DNA interactions, as well as the interaction of stalled RNA polymerase with damaged

DNA.

3.3. Structure of CSB

The CSB gene encodes a 168 kDa protein, containing several conserved motifs
(142). As illustrated in figure 1-4, the central part of CSB is represented by the seven
consecutive ATPase motifs I, la, II, III, IV, V, and VI fi'om amino acids 527-950. These
motifs form a nucleotide-binding fold and are conserved among three superfamilies of
RNA and DNA helicases. The region of CSB encompassing these motifs is highly
homologous to proteins of the SNF2-like family (32). The N-terminus of CSB has an
acidic (A) domain (amino acids 356-394) (142). Between the acidic and SNF2 domains
is a glycine-rich (G) stretch and a highly hydrophilic region (H). C-terminal to the
ATPase motifs is a putative nucleotide-binding (NT B) domain. The protein also contains
a bipartite nuclear-localization signal (NLS). Studies by Christiansen et al. (2005)
demonstrated that CSB protein functions as a dimer. The homodimerization occurs via
the central ATPase domain of the CSB protein and is essential for ATP hydrolysis by
CSB (21).

Of the seven ATPase motifs, motifs I and 11 contain consensus NTP-binding
sequences and are involved in ATP binding by CSB (48). Mutant CSB protein with point

mutations in ATPase motifs I and II of CSB cannot complement UV sensitivity,

28

Figure 1-4. Predicted motifs of CSB: A = Acidic domain, G = glycine-rich stretch, NLS

= Nuclear localization sequence, NTB = Nucleotide binding motif.

29

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suggesting that ATP hydrolysis by CSB protein is required for TCR of DNA damage (20,
23). Studies on CSB ATPase motif Ia and III mutants revealed only partial
complementation of the sensitivity of CS cells towards UV and recovery of RNA
synthesis after UV irradiation (104). Studies of homologous proteins suggest the
involvement of motifs Ia and III in energy transduction between the ATPase site and the
nucleic acid binding site (48). The ATPase motif V and VI CSB mutants exhibit a similar
inhibition of ATPase activity and reduced ATP binding in vitro (20). These mutants also
have comparable defects in recovery of RNA synthesis after UV irradiation (143). So far,
there have been no reported structure-function experiments involving the putative NLS,
the glycine-rich stretch, the hydrophobic segment, or the ATPase motif IV. Also, no
functions have thus far been assigned to the acidic domain and putative NTP box (13,

137).

3.4. Mutant CSB protein and Cockayne syndrome

Transcription-coupled repair (TCR) is a cellular pathway for the removal of the
many DNA lesions that block and arrest transcription. TCR is responsible for the rapid
and preferential repair of damage in the transcribed strand of active genes (10). It occurs
in both prokaryotic and eukaryotic organisms. In humans, the absence of TCR is
associated with Cockayne Syndrome (CS). The majority of CS cases are caused by
defects in the Cockayne Syndrome complementation group B (CSB) protein (85).

CS is a premature aging syndrome with complex symptoms, including
developmental abnormalities, neurologic disfunction and a short average life span.

Cellular characteristics include hypersensitivity to UV light, and failure of RNA synthesis

31

to recover to normal rates following UV irradiation (93, 107, 145). The photosensitivity
of CS patients can be attributed to the TCR defect. It is assumed that accumulation of the
DNA damage causes growth arrest and apoptosis, which may explain the progressive
course of the disease. Interestingly, CS patients, despite their DNA repair deficiency, do
not have a predisposition to cancer. However, CSB deficiency itself has antineoplastic
effect in cancer predisposed mice (89). It has been suggested that precancerous cells in
CS patients are more efficiently eliminated by apoptosis than they are in healthy person
(50, 89). In contrast, neurological and developmental features of this disease can be a

result of the problems with transcription (50).

Could misregulation of U1 snRNA gene transcription contribute to development of the
CS phenotypes? It was shown that in addition to mutations in CSB and CSA genes the CS
phenotype can result from mutations in XPB, XPD, and XPG genes (133). The products
of these genes were shown to be a part or directly associate with the TFIIH protein
complex involved in both basal and activated transcription and NER (85), suggesting that
misfunctioning of TFIIH may be a common step in developing the CS phenotypes.
Interestingly, U1 snRNA was also shown to associate with TFIIH and stimulate
transcription initiation by TFIIH (76). Thus, misregulation of U1 snRNA in CSB
deficient cells may result in misfunctioning of TFIH-I similarly to the XBP, ED and HG
gene mutations and may contribute to development of the CS disease. Another interesting
possibility is that CS patients may additionally experience misregulation of other non-

coding RNAs, which may contribute to the pleotropic phenotype of the CS disease.

32

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10.

ll.

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46

CHAPTER TWO

THE P53 TUMOR SUPPRESSOR PROTEIN REPRESSES HUMAN SNRNA
GENE TRANSCRIPTION BY RNA POLYMERASES II AND 111
INDEPENDENTLY OF SEQUENCE-SPECIFIC DNA BINDINGi

Abstract

Human U1 and U6 snRNA genes are transcribed by RNA polymerases H and 111,
respectively. While the p53 tumor suppressor protein is a general repressor of RNA
polymerase III transcription, whether p53 regulates snRNA gene transcription by RNA
polymerase II is uncertain. The data presented herein indicate that p53 is an effective
repressor of snRNA gene transcription by both polymerases. Both U1 and U6
transcription in vitro is repressed by recombinant p53, and endogenous p53 occupancy at
these promoters is stimulated by UV light. In response to UV light, U1 and U6
transcription is strongly repressed. Human U1 genes, but not U6 genes, contain a high-
affinity p53 response element located within the core promoter region. Nonetheless, this
element is not required for p53 repression and mutant p53 molecules that do not bind
DNA can maintain repression, suggesting a reliance on protein interactions for p53
promoter recruitment. Recruitment may be mediated by the general transcription factors
TAT A-box binding protein and snRNA-activating protein complex, which interact well

with p53 and function for both RNA polymerase II and III transcription.

This work was published as the following manuscript: Anastasia A. Gridasova and R. William
Henry (2005) The p53 tumor suppressor protein represses human snRNA gene transcription by
RNA polymerases II and III independently of sequence-specific DNA binding. Molecular and
Cellular Biology, Apr. 2005, pp. 3247-3260.

47

Introduction

The p53 tumor suppressor protein plays a critical role in preventing unwarranted
cellular proliferation by activating transcription of key target genes that influence cell
grth and apoptosis (reviewed in references 28, 31, 35, and 64). Though p53 can enable
both pathways, the switch controlling which cellular outcome is enacted is uncertain
(reviewed in references 65 and 66), but both the p53 level and the nature of the DNA
damage can influence apoptotic response (8). Altogether, p53 activity serves to prevent
passage of mutations to daughter cells afier DNA damage.

Recent evidence suggests that p53 regulates transcription of genes that are not
obviously involved in controlling cell cycle arrest or apoptosis. Indeed, p53 can repress
RNA polymerase I (3, 72) and III (S, 9) transcription of genes encoding a variety of
nontranslated RNAs that play critical roles at numerous points during global gene
expression. RNA polymerase III activity is elevated in p53-/- knockout fibroblasts (5)
and in a variety of cancer-derived cell lines that lack p53 function (57). However, the
mechanism for p53 regulation of RNA polymerase III transcription is controversial. A
kinetic analysis of RNA polymerase III repression using p53 expressed from a stably
integrated inducible p53 gene suggested that RNA polymerase HI repression is mediated
indirectly through p53-dependent degradation of TFIIIB (11). In contrast, recombinant
p53 can repress in vitro transcription from a variety of RNA polymerase III-specific
promoters and can interact with components of the general transcription machinery
required for RNA polymerase III transcription (5, 9, 10, 58), indicating that p53 might

directly repress transcription by RNA polymerase III.

48

Within the group of genes transcribed by RNA polymerase IH, the human snRNA
gene family is intriguing because these genes contain similar sets of promoter elements,
and yet only some genes are transcribed by RNA polymerase III while others are
transcribed by RNA polymerase 11 (see references 19, 2,3, 24, and 42 for review).
Regardless of polymerase specificity, human snRNA genes contain a distal sequence
element in the upstream promoter region that serves as the recognition element for
activator proteins, including Oct-1, STAF, and Spl (33, 54). These factors activate
transcription from the core promoters that commonly contain a proximal sequence
element (PSE). The PSE is directly recognized by a general transcription factor called the
snRNA activating protein complex (SNAPC) (52), which is also known as the PSE
transcription factor (49). SNAPC is involved in human snRNA gene transcription by both
RNA polymerases II and 111 (20—22, 51, 69). RNA polymerase III-transcribed snRNA
genes also contain a TATA box that serves to recruit the TATA-box binding protein
(TBP) as part of an snRNA-specific TFIIIB complex (45, 55, 60).

The conservation of important promoter elements among human snRNA genes
suggests that transcription of these genes by RNA polymerases II and 111 may be
coordinately regulated. However, it is not known whether p53 can regulate human
snRNA gene transcription by RNA polymerase II. A role for p53 in this process is
suggested from two sources. Firstly, in response to UV light treatment, human U1 and U2
snRNA genes exhibit a delayed and prolonged reduction in transcription by RNA
polymerase II (14, 47, 48). In part, this reduction may be attributable to increased
hyperphosphorylation of the carboxy-terminal domain of the RNA polymerase 11 largest

subunit in response to UV light (27). However, in normal human diploid fibroblasts, the

49

balance of hyper- and hypophosphorylated RNA polymerase II is restored by 6 h after
UV light treatment (46), suggesting additional cellular mechanisms that enable snRNA
gene repression after UV light exposure. Potentially, p53 activation by DNA damage
might play a direct role in the prolonged repression of these genes.

Secondly, infection of human cells by adenovirus serotype 12 causes metaphase
fragility at four chromosomal sites, including the U1 snRNA (RNUl) and U2 snRNA
(RNU2) loci (1, 36), in a process that requires p53 (38, 39). It was postulated that fragile
site formation occurs during viral infection, because RNA polymerase II stalls at these
genes and interferes with chromosome condensation during metaphase (37). Interestingly,
p53 that harbors mutations in the DNA binding domain supports fragile site formation
(39), and overexpression of the C-terrninal domain of p53 alone, which lacks the DNA
binding domain, induces fragility during transient transfection (71). Together, these data
indicate that p53 is important for generation of fragile sites at the U1 and U2 snRNA
gene loci and may play a role in regulation of these genes in a fashion that does not
require sequence-specific binding of p53 to DNA.

In this study, the role of p53 in governing human snRNA gene transcription by
RNA polymerase II and III was examined. We show that recombinant p53 represses both
U1 and U6 transcription by RNA polymerase II and 111, respectively. Repression is
supported by the C-terminal region of p53 alone, indicating that sequence-specific DNA
binding by p53 is not critical for repression. Both the full-length and C terminus of p53
alone can associate with the U1 and U6 promoters during repression, and promoter
recruitment may be assisted through interactions with the general transcription factors

SNAPC and TBP, which are commonly required for transcription of both U1 and U6

50

snRNA genes. In vivo, p53 can bind to both U1 and U6 snRNA genes in untreated human
MCF-7 cells, and promoter occupancy is stimulated after UV light treatment. These

results firrther indicate that p53 contributes to snRNA gene regulation in response to

DNA damage.

Materials and Methods

Cell culture and UV irradiation

Human MCF-7 and HeLa cells were maintained in Dulbecco’s modified Eagle’s
medium supplemented with penicillinstreptomycin and 10% (MCF-7) or 5% (HeLa) fetal
bovine serum. Cells grown to 70 to 80% confluence were washed with phosphate-
buffered saline and irradiated with 50 J of UV light (254-nm peak)/m2 by using a UV
Stratalinker (Stratagene). After irradiation, growth medium was added and cells were
incubated at 37°C under 5% C02 for the indicated times. Additionally, HeLa cells were
grown to 50% confluence in ISO-mm plates and were then transiently transfected with
2.5 ug of the pRc/RSV or pRc/RSV-p53-Flag.wt plasmids by using Lipofectin reagent
(Invitrogen) for 6 h. Subsequently, the medium was replaced and cells were incubated for

48 h for further analysis in nuclear run-on assays.

Nuclear run-on assays

Nuclear run-on assays were performed as described elsewhere (6) in the presence
of [or-32P]UTP using approximately 107 nuclei that were isolated from MCF-7 or HeLa
cells before or 8 h after exposure to UV light. Additional assays were performed using

HeLa cells transiently transfected with pRc/RSV or pRc/RSV-p53-Flag.wt as described.

51

Labeled RNA was recovered and hybridized to a nitrocellulose membrane containing
approximately 7 ug of U1, U6, and 5S rRNA target gene DNAs or 10 ug of pUC119
plasmid, as a negative control. Target gene DNAs corresponding to the coding regions of
the indicated genes were generated by PCR and were immobilized on a nylon membrane
at levels calculated to be in excess relative to the corresponding snRNA population in the
nuclei. Hybridizations were performed for 16 h at 42°C in hybridization buffer containing
50% forrnamide. Membranes were then washed extensively in 2X SSC (1X SSC is 0.15
M NaCl plus 0.015 M sodium citrate) and 0.1% sodium dodecyl sulfate (SDS) at 50°C.
Hybridized RNA transcripts were visualized by autoradiography for 7 days. Similar
results were observed when signals were normalized to glyceraldehyde-3-phosphate

dehydrogenase (GAPDH) expression by varying the exposure time (data not shown).

RNA isolation and RT -PCR

RNA was isolated using the TRIzol reagent as recommended by the manufacturer
(Gibco-BRL). RNA preparations were quantified by UV spectrometry and examined for
integrity by agarose formaldehyde morpholinepropanesulfonic acid gel electrophoresis.
Reverse transcription-PCR (RT-PCR) was performed by a two-step procedure using U1-,
U6-, and GAPDH-specific primers. The primers used for amplification of each gene were
the following: U1 forward, 5’-ATACTTACCTGGCAGGGGAG-3’; U1 reverse, 5’-
CAGGGGGAAAGCGCGAACGCA-3’; U6 forward, 5’-GGAATCTAGAACATATACT
AAAATTGGAAC-3’; U6 reverse, 5’-GGAACTCGAGTTTGCGTGTCATCCTTGCGC-
3’; GAPDH forward, 5’-AGGTCATCCCTGAGCTGAAC-3’; and GAPDH reverse, 5’-

GCAATGCCAGCCCCAGCGTC-3’.

52

Expression and purification of recombinant proteins

Glutathione S-transferase (GST), GST-tagged full-length human p53, and a GST-
tagged C terminus of human p53 (amino acids 301 to 393) [p53 (301-393)] were
expressed in Escherichia coli BL21(DE3) codon+ cells (Stratagene) and were affinity
purified by binding to glutathione agarose beads (Sigma). GST and GST-p53 were then
eluted from beads in HEMGT-lSO buffer containing 50 mM glutathione for 4 h at 4°C or,
alternatively, untagged p53 was obtained by digestion with thrombin. Proteins were
further purified by chromatography using a Mono-Q (HRS/5) column (Pharrnacia) and
were concentrated by centrifugation using a Centricon YM-30 spin column (Millipore) in
HEMGT-80 buffer (20 mM HEPES [pH 7.9], 0.1 mM EDTA, 10 mM MgC12, 10%
glycerol [vol/vol], 0.1% Tween 20, 80 mM KCl) containing protease inhibitors and 1 mM

dithiothreitol.

In vitro transcription assays

In vitro transcription assays were performed as described previously (21, 43)
using 18, 2, 2, and 10 uL of HeLa cell nuclear extract for the U1 snRNA, U6 snRNA, 5S
rRNA, and adenovirus major late promoter (AdML) transcription reactions, respectively.
The pU1-4.0 (1 pg), pU6/Hae/RA.2 (250 ng), pHSSsa (250 ng), and M13-AdML (250 11g)
templates were used for the U1 snRNA, U6 snRNA, SS rRNA, and AdML transcription
reactions, respectively. Purified p53 or GST-tagged p53 proteins were added in the
amounts indicated in the figure legends. Transcription reactions were performed for 1 h at
30°C. Transcripts were separated by denaturing 6% polyacrylamide gel electrophoresis

(PAGE) and visualized by autoradiography.

53

Chromatin immunoprecipitation assay

Chromatin immunoprecipitation assays were performed as described previously
(25). Human MCF-7 cells were grown to 60 to 80% confluence and were then cross-
linked with 1% formaldehyde for 30 min at room temperature. After cell lysis and
sonication, immunoprecipitation reactions were performed overnight at 4°C using
chromatin from approximately 107 cells per reaction mixture and 1 ug of each antibody.
The anti-p53 antibodies used were the following: anti-p53 (21-25) (Ab-6; Oncogene),
anti-p53 (371-380) (Ab-1; Oncogene), anti-p53 (213-217) (Ab240; Pharmingen), anti-
acetyl-p53 (373-382) (Upstate), and anti-acetyl-p53 (320) (Upstate). Recovered
chromatin was suspended in 50 11L of H20, and PCR analysis was performed using 5 11L
of immunoprecipitated chromatin or input chromatin. The primers used for amplification
of each gene were the following: U1 forward, 5’-CACGAAGGAGTTCCCGTG-3’; U1
reverse, 5’-CCCTGCCAGGTAAGTATG-3’; U2 forward, 5’-AGGGCGTCMTAGCGC
TGTGG-3’; U2 reverse, 5’-TGCGCTCGCCTTCGCGCCCGCCG-3’; U6 forward, 5’-
GTACAAAATACGTGACGTAGAAAG-3’; U6 reverse, 5’-GGTGTTTCGTCCTTTCC
AC-3’; GAPDH forward, 5’AGGTCATCCCTGAGCTGAAC-3’; GAPDH reverse, 5’-
GCAATGCCAGCCCCAGCGTC-3’; U1 upstream forward, S’-GAACTTACTGGGATC
TGG-3’; U1 upstream reverse, 5’-GAGACAACTGAGCCACTTG-3’; p21 upstream
forward, 5’-CCGCTCGAGCCCTGTCGCAAGGATCC-3’; p21 upstream reverse, 5’-
GGGAGGAAGGGGATGGTAG-3’. PCR products were separated by 2% agarose

electrophoresis in Tris-borate-EDTA buffer and were stained with ethidium bromide.

54

Immunoprecipitations from in vitro transcription reactions

In vitro transcription assay mixtures containing U1 or U6 promoter plasmids and
equal molar amounts of pUCll9 were performed as described previously (25) in the
absence or presence of full-length GST-p53 (wild-type or R175H), GST-p53 (301-393),
or GST. Five microliters of each transcription reaction mixture was diluted to 500 1.1L and
was cross-linked in 1% formaldehyde for 10 min at room temperature, quenched with
125 mM glycine for 10 min at room temperature, and immunoprecipitated with
immunoglobulin G (IgG), anti-SNAP43 (CS48), or anti-p53 (Ab-1; Oncogene) antibodies.
Recovered plasmid DNA was analyzed by PCR using primers specific to the U1 and U6

promoter regions or to pUC119 as a negative control.

DNase I footprinting

Footprinting assays were generally performed as described elsewhere (4). Linear
DNA encompassing the human U1 promoter from -151 to +13 was generated by PCR
using primers that were end labeled with [y-32P]ATP by using T4 polynucleotide kinase
(New England BioLabs). U1 promoter probes were incubated with increasing amounts of
recombinant GST-p53 for 40 min at room temperature and were then digested with 0.04
U of DNase I (Roche) for 2 min at room temperature. The resultant fragments were
purified and separated by 8% denaturing PAGE. Footprints were visualized by
autoradiography and were mapped relative to sequencing ladders generated from the

same labeled primers used to generate the U1 promoter probes.

55

EMSA

Electrophoretic mobility shift assays (EMSA) were performed as described
elsewhere (7). The amounts of p53 used are indicated in the figure legends. Reaction
mixtures were incubated at room temperature for 20 min prior to addition of radiolabeled
probes. Unless otherwise noted, the U1 probe encompasses -312 to +13 and the U6 probe
encompasses -267 to +1. DNA binding reactions were carried out at room temperature for
20 min, and resulting DNA-protein complexes were separated on a 4% polyacrylamide
gel in 0.5X Tris-borate-EDTA running buffer at 150 V. Complexes were visualized by

autoradiography.

Coimmunoprecipitation and GS T pull-down experiments

GST pull-down assays were performed as previously described (25).
Coimmunoprecipitation assays were performed using 5 mg of total protein contained in
MCF-7 nuclear extracts from untreated or UV-treated cells and 2 pg of rabbit anti-
SNAP43 antibodies (CS48) (22). Western blot analyses of recovered proteins were
performed using anti-SNAP43 (CS48), anti-p53 (Ab-6; Oncogene), and anti-galectin-3
(Mac2) antibodies. The reciprocal immunoprecipitation reactions were performed using 2
pg of anti-p53 antibody (Ab-6) with approximately 1.6 or 5 mg of MCF-7 extract,

followed by anti-SNAP43 Western blot analysis.

56

Results

p53 represses human snRNA gene transcription by both RNA polymerases II and
111.

To determine whether p53 can repress U1 transcription by RNA polymerase II, as
has been previously shown for U6 transcription by RNA polymerase III (5, 9), the effect
of recombinant p53 on human U1 in vitro transcription was tested. The recombinant full-
length wild-type p53 and the GST proteins used for these experiments are shown in Fig.
1A. As shown in Fig. 1B, p53 effectively repressed correctly initiated U1 transcription
(labeled U1 5’) by RNA polymerase II, and this repressive effect was specific, because
concomitant RNA polymerase II transcription of an mRNA read-through transcript
derived from the same plasmid was unaffected in these reactions. As a positive control
for p53 activity, human U6 snRNA gene transcription was tested. Indeed, the same
amounts of p53 effectively repressed U6 snRNA transcription by RNA polymerase III,
while RNA polymerase II transcription from the AdML was unaffected. Therefore, p53
can repress human snRN A gene transcription by both RNA polymerases II and 111.

As a first step towards understanding the mechanism for p53 repression of U1 and
U6 transcription, a time course for p53 repression was performed (Fig. 1C). As a positive
control, p53 repression of SS rRNA gene transcription was also examined. To ensure
maximal repression, an excess of p53 was used, because 5S rRNA gene transcription
appears less sensitive to p53 repression (9) (data not shown). As was demonstrated
previously (5), p53 can repress SS rRNA transcription when added to reactions
concomitantly with nuclear extract (lane 2) or nuclear extract plus template DNA (lane 3)

prior to initiation of transcription by nucleotide addition. However, p53 did not repress

57

Figure 2-1. p53 represses human snRNA gene transcription by both RNA
polymerases II and III in vitro. (A) Recombinant full-length wild-type p53 and GST
proteins were separated by SDS—12.5% PAGE and were stained with Coomassie blue. (B)
In vitro transcription fi'om U1, U6, and AdML promoter constructs was tested using
HeLa nuclear extracts containing 0, 50, 200, and 800 11g of p53 (lanes 1 to 4) or 800 ng of
GST (lane 5). Fifty nanograms of p53 represents an approximate 2:1 molar ratio of
monomeric p53 to U1 promoter template DNA and an approximate 8:1 molar ratio to the
U6 and AdML promoter plasmids. p53 effectively repressed correctly initiated U1
transcription (U l 5 ) and U6 transcription (U6 5 ), but it didn’t affect read-through (RT)
transcription from the U1 reporter plasmid or transcription from the AdML promoter. (C)
U1, U6, and SS rRNA in vitro transcription reaction mixtures were supplemented with
800 ng of active or heat-inactivated p53 (lanes 2 to 4 and lanes 5 to 7, respectively) at
different times, as indicated. Transcription was allowed to proceed for an additional 60
min. Recombinant p53 repressed U6 gene transcription both prior to and after
preinitiation complex assembly but did not repress U1 and 5S rRNA gene transcription

after the formation of a preinitiation complex.

58

 

 

 

 

97.4— ‘9
66- w
45— one. “"""' _p53
31 '- quip
14.4- ‘-
1 2 3
Coomassie

 

 

 

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1 2 3 4 5
In vitro transcription

59

 

 

 

 

 

 

 

0' 15' 30'
+Extract +DNA +NTPs
— 0' 15' 30' o' 15' 30' Tlme

_ _ _ _ + + 4. HI p53
— + + + — — ._ P53
- RT
— U1 5'
— U6 5'
j— 58 5'

 

 

 

In vitro transcription

60

 

5S rRNA transcription when the nuclear extract was preincubated with template DNA
prior to p53 addition (lane 4). Presumably, p53 cannot repress SS rRNA gene
transcription after the formation of a preinitiation complex. For all time points, repression
was specific for functional p53, because repression was disabled by heat inactivation of
p53 (lanes 5 to 7). In contrast, p53 could effectively repress U6 transcription by RNA
polymerase III even after the nuclear extract had been preincubated with the template
DNA. This result suggests that the U6 preinitiation complex is not recalcitrant to p53
repression. Surprisingly, the pattern for p53 repression of U1 transcription by RNA
polymerase H was similar to that of SS rRNA rather than the U6 repression pattern. This
observation suggests that formation of a preinitiation complex could render U1 snRNA
genes refractory to p53 repression. This result also suggests that p53 represses U1 and U6
snRNA gene transcription by different mechanisms.

As UV light exposure activates p53, this treatment was used here to determine
whether p53 is involved in human snRNA transcriptional regulation in vivo. The majority
of in vivo studies presented herein were performed with human MCF-7 breast
adenocarcinoma cells, because these cells exhibit a robust increase in p53 levels in
response to UV light treatment (Fig. 2A, lanes 4 to 6) compared to human HeLa cervical
carcinoma cells, wherein p53 levels are low and remain unchanged after UV light
exposure (lanes 1 to 3).

To determine the effect of UV light on snRNA gene transcription, nuclear run-on
experiments were performed using nuclei harvested from HeLa and MCF-7 cells before
and 8 h after UV light exposure. As shown in Fig. 2B, UV light treatment of HeLa cells

did not substantially affect U1 transcription by RNA polymerase 11 compared to cells that

61

HeLa MCF7

 

 

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8 + UV 3 + uv

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f: 4hr 8hr : 4hr 8hr

 

 

 

 

 

1 2 3
anti-p53 Western

 

 

 

 

 

 

 

 

 

 

 

 

 

 

-UV +UV -UV +UV
0 hr 8 hr 0 hr 8 hr
,i — pUC119

_ __ .. ~— — U1 snRNA
“ u .. ~---- — U6 snRNA
1 2 3 4

Nuclear run-on Nuclear run-on

HeLa MCF-7

63

Figure 2-2. UV light inhibits snRNA gene transcription and stimulates p53 binding
to human snRNA gene promoters. (A) Whole-cell extracts from untreated and UV
light-treated HeLa (lanes 1 to 3) and MCF-7 (lanes 4 to 6) cells were analyzed by SDS—
12.5% PAGE and Western blot analysis of endogenous p53 and actin. MCF-7 cells
exhibited robust accumulation of endogenous p53 in response to UV light treatment,
whereas no change was observed in HeLa cells. (B) UV light represses transcription of
endogenous human U1 and U6 snRNA genes in MCF-7 cells, but not in HeLa cells.
Nuclear run-on assays measuring polymerase density at U1 snRNA, U6 snRNA, and SS
rRNA genes in nuclei from untreated HeLa cells (lane 1) or MCF-7 cells (lane 3) were
compared to results with nuclei harvested 8 h after UV light treatment (lanes 2 and 4).
After hybridization, membranes were exposed to film for 7 days. Similar trends were also
obtained when exposure times were varied to normalize to GAPDH gene transcription,
which was unaffected by UV light treatment in these assays (data not shown). (C)
Transiently transfected p53 represses U1 snRNA gene transcription in HeLa cells.
Nuclear run-on assays were performed on HeLa cells (lane 1) or HeLa cells transiently
transfected with either the empty vector pRC/RSV (lane 2) or pRC-RSV expressing wild-
type full-length Flag-tagged p53 (pRc/RSV-p53-Flag) (lane 3). Levels of p53 expression
were determined by Western blotting (bottom panel). (D) Endogenous p53 associates
with human snRNA gene promoters. Chromatin immunoprecipitation experiments were
performed using chromatin harvested from MCF-7 cells prior to or 8 h after UV light
treatment and using antibodies directed against SNAP43 (lane 3), various epitopes within
p53 (lanes 4 to 7), and nonspecific IgG (lane 8) as a negative control. Enrichment of U1
and U6 promoter regions was measured by PCR and was compared to the p21 promoter
(-1.4 kb site), as a positive control, and the U1 upstream region and GAPDH exon 2, as
negative controls. (E) Endogenous p53 was not detected at human snRNA gene
promoters in untreated or UV light-treated HeLa cells. Chromatin immunoprecipitation
experiments were performed using HeLa cell chromatin with the indicated antibodies. (F)
UV light causes a decrease in steady-state U1 snRNA levels. (Top panel) Total RNA was
isolated fiom untreated MCF-7 cells and was titrated (O, 0.1, 0.3, 1, 3, and 10 ng [lanes 2
through 7, respectively]) into RT-PCRs performed using U1 gene-specific primers. The
amount of U1 cDNA amplification is proportional to the amount of total RNA used for
RT-PCR. (Bottom panel) Steady-state levels of U1, U6, and GAPDH RNA were
measured by RT-PCR using 2 ng, 10 ng, and 1 ug of total RNA, respectively, harvested
before (lane 1) or after (lanes 2 to 7) UV light treatment.

62

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did not receive UV light exposure. Similarly, U6 snRNA and SS rRNA transcription by
RNA polymerase III was unaffected, suggesting that the transcription of these genes is
insensitive to UV light. These results for U1 transcription are in contrast with that
previously described wherein U1 transcription in nuclear run-on assays was markedly
reduced 2 h after UV light treatment of HeLa cells (48). In the present study, the 8-h
posttreatrnent time point was selected because RNA polymerase II is ubiquitylated and
degraded in response to UV light but normal levels are restored by 6 h after UV light
treatment (46). Additionally, a longer recovery period after UV light treatment was
desirable to allow sufficient time for DNA damage repair.

In contrast with HeLa cells, MCF-7 cells exhibited a marked reduction in U1
transcription 8 h after UV light treatment. Additional studies revealed that repression was
already established by 4 h posttreatrnent (data not shown). Interestingly, UV light elicited
different effects on RNA polymerase III-transcribed genes, causing reduced U6
transcription while stimulating SS rRNA transcription. In all cases, the signals detected
for these transcripts are specific, because no hybridization to pUC119 was detected in any
of these experiments. Therefore, U1 and U6 transcription exhibits cell type-specific
responses to UV light treatment, with UV light invoking a prolonged repressive effect in
MCF-7 cells but not in HeLa cells. In two independent replicates of this experiment, U1
and U6 transcription levels were reduced to 42 and 39%, respectively, of the untreated
sample levels, whereas SS RNA transcription rates were increased to 170%. Stimulated
SS rRNA gene transcription under these conditions was unexpected but does indicate that

the SS rRNA transcriptional response to DNA damage depends upon cellular p53 status.

66

To determine whether p53 contributes to regulation of endogenous snRNA genes,
p53 was overexpressed in HeLa cells and the effect on endogenous U1, U6, and SS rRNA
gene transcription was again measured by nuclear run-on assays. As shown in Fig. 2C,
increased p53 expression was correlated with diminished U1 transcription. In contrast,
U6 transcription was unaffected, whereas SS rRNA transcription was stimulated. The
reason for the unresponsiveness of U6 transcription to p53 expression is unknown, but
p53 may require additional UV light-stimulated modification for activity at this gene.
Interestingly, the expression pattern for U1 snRNA and SS rRNA transcription in
response to p53 expression is similar to that observed with UV light treatment of MCF-7

cells, consistent with the idea that p53 regulates these genes in response to DNA damage.

Endogenous p53 associates with human snRNA gene promoters.

Chromatin immunoprecipitation experiments were then performed to determine
whether p53 is directly involved in the regulation of endogenous snRNA genes in
response to UV light. As shown in Fig. 2D, substantial enrichment of U1 and U2 snRNA
promoter DNA was observed in anti-p53-immunoprecipitated samples (lanes 4 and 5)
with chromatin harvested from MCF-7 cells prior to UV light treatment, and these levels
were markedly enriched by using chromatin harvested from cells 8 h after UV light
treatment. In contrast, only low levels of U6 promoter DNA were enriched in the anti-
p53-immunoprecipitated samples prior to UV light treatment, but promoter recovery was
noticeably enhanced after treatment.

Previously, it was shown that p53 is acetylated within its C terminus in response

to DNA damage, which may stimulate DNA binding by p53 (18, 44) and increase

67

recruitment of coregulatory proteins (2). Furthermore, acetylation but not
phosphorylation of p53 within the C-terrninal domain contributes to fragile site formation
at the U2 snRNA gene loci, indicating that p53 acetylation may be important for p53
function at snRNA genes (71). Therefore, immunoprecipitation reactions were also
performed with antibodies that specifically recognize p53 acetylated at K320 or at K372
and K382 (Fig. 2D). Interestingly, significant levels of U1 and U2 promoter enrichment
were observed with antibodies that recognize acetylated p53 (lanes 5 and 6), but UV light
treatment either did not affect promoter enrichment or caused a modest reduction. In
contrast, no significant recovery of U6 promoter DNA was obtained with antibodies that
recognize acetylated p53. Enrichment of the p21 promoter (-1.4 kb site) in the anti-p53-
immunoprecipitated samples was low prior to UV light treatment, but recovery increased
significantly after treatment, as has been previously demonstrated (30). UV light
treatment also resulted in increased p21 promoter enrichment for those reactions
performed using anti-acetylated p53 antibodies. Together, these data indicate that p53
associates with the endogenous U1 and U2 snRNA gene promoters prior to genotypic
stress and UV light stimulates p53 association with these promoters, although the
apparent proportion of p53 that is acetylated decreases. Second, low levels of p53
associate with the U6 promoter prior to stress and UV light stimulates p53 promoter
association, but this p53 is not acetylated to a significant degree. These observations are
in contrast to those seen with the p21 promoter, where p53 association is low but the total
p53 level and proportion that is acetylated increase in response to UV light treatment.
The data in Fig. 2B show that neither U1 nor p21 promoter association by p53 was

observed in HeLa cells (lane 5) using an anti-p53 antibody that efficiently recovered

68

these DNA segments from MCF-7 cells (lane 6). As previously observed for MCF-7 cells,
UV light also did not affect SNAPC occupancy at the U1 promoter in HeLa cells (lane 3).
Human snRNA molecules are abundant and very stable ( 13, 17, 68) and, thus, it is
not clear what effect diminished U1 transcription would. have on overall U1 snRNA
levels. Therefore, RT-PCR assays were employed to determine the effect of UV light on
steady-state U1 and U6 snRNA levels. As shown in Fig. 2F (top panel), addition of
increasing amounts of total cellular RNA harvested from untreated MCF-7 cells resulted
in a linear amplification of U1 sequences (lanes 3 to 7), thus demonstrating that this assay
is suitable for measuring changes in steady-state U1 snRNA levels. Similar preliminary
experiments were performed to determine the range for linear amplification of both U6
snRNA and GAPDH mRNA (data not shown). Under these conditions, U1 steady-state
levels were noticeably reduced 8 h after UV light treatment, whereas U6 snRNA and
GAPDH mRNA levels remained relatively stable before and after treatment. In three
independent replicates of this experiment, the steady-state level of U1 snRNA at 8 h
posttreatrnent was 43% of levels in untreated cells (data not shown). The reduction in U1
steady-state levels in response to UV light could be attributable to increased degradation
of this RNA, decreased transcription from U1 snRNA genes, or a combination of both
factors. U1 snRNA is traditionally viewed as extremely stable, with a half-life greater
than 24 h and, thus, little change was expected in steady-state U1 levels by 8 h, even if
transcription were completely repressed. The kinetics of the decrease in U1 steady-state
levels and the results shown in Fig. 2B suggest that both a reduction in U1 transcription
and an increase in U1 snRNA degradation contribute to reduced U1 snRNA levels after

UV light treatment. Together, these results indicate that UV light initiates a complicated

69

network of control governing expression of human snRNA genes.

Human U1 snRNA gene core promoters contain a high affinity p53 binding site.

As a first step towards understanding the mechanism for p53 repression, EMSA
were performed to determine whether p53 could bind directly to human U1 snRNA gene
promoters. Indeed, recombinant p53 bound extremely well to a U1 probe encompassing
the region from -422 to +13 of the promoter (Fig. 3A, lanes 2 to 4). Competition
experiments suggest that p53 affinity for the U1 promoter is comparable to the p53
binding element contained within the GADD4S promoter (data not shown). Interestingly,
two different complexes formed on the U1 promoter probe, suggesting that the U1
promoter may contain two p53 binding elements. The protein-DNA complexes formed on
these probes are due to p53, because inclusion of anti-p53 antibodies in the DNA binding
reactions retarded the migration of these complexes (data not shown). To determine the
location of the hi gh-affinity p53 binding element, EMSA was also performed with probes
containing different regions of the promoter. Strong binding of p53 to DNA was observed
in reactions with equivalently labeled probes containing the -312 to +13 and -150 to +13
regions of the promoter. Consistently, low-affinity binding was observed in probes
containing the -422 to +152 and -312 to +152 promoter regions. Together, these data
indicate that the high-affinity p53 binding element is contained between -150 and +13 of
the U1 core promoter.

DNase I footprinting experiments were then performed to further map the location

of the high-affinity p53 binding element. As shown in Fig. 38, those reactions in which

70

Figure 2-3. Human U1 snRNA gene core promoters contain a high-affinity p53
binding site. (A) EMSA with increasing amounts of recombinant p53 (0, 80, 160, and
320 11g) were performed with double-stranded DNA probes encompassing various
regions of the core U1 promoter, as indicated. At higher p53 concentrations, two p53-
dependent complexes were formed on those probes that exhibited high-affinity p53
binding. (B) DNase I footprinting reactions were performed using labeled template stand
(lanes 1 to 5) or nontemplate stand (lanes 6 to 10) U1 promoter probes encompassing
-150 to +13 with increasing amounts of GST-p53 (0, 100, 200, and 400 ng). Digestion of
the probe DNA without added p53 is shown in lanes 1, S, 6, and 10. The relative
positions of the PSE and transcription start site are indicated. Two protected regions
within the template strand (labeled F1 and F2) overlap with the three protected regions

(labeled Fl’, F2’, and F 3’) from the nontemplate strand.

71

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72

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73

the template strand was end labeled exhibited two sites of protection (labeled F1 and F 2)
in the presence of GST-p53 (left panel), whereas three regions (F1’, F2’, and F3’) were
protected in reactions with the labeled nontemplate strand (right panel). The F1 ’ and F3’
regions correspond to the same region as the F l footprint (hereafter referred to as p53
footprint 1), whereas the F2 and F 2’ regions map to the same region (hereafter referred to
as p53 footprint 2). Interestingly, the regions protected by p53 flank the PSE, which is
required for DNA binding by SNAPC and is an essential promoter element for high level
expression of human snRNA genes. The juxtaposition of p53 footprintsl and 2 with the
PSE raises the possibility that p53 represses U1 transcription by occluding SNAPC
promoter binding. However, to date we have not observed any effect of p53 on promoter

recognition by SNAPc (data not shown).

p53 repression and sequence-specific DNA binding are separable activities.

As a transcription factor, p53 can either activate or repress transcription,
depending upon the structure of the target gene promoter. Notably, p53 can activate
transcription from gene promoters that contain consensus p53 binding elements (12). A
few examples have been described where p53 represses target genes that contain
consensus p53 binding elements (reviewed in reference 26), but it is generally believed
that p53 can repress transcription of other target genes whose promoters lack specific p53
recognition elements or contain a noncanonical p53 binding element (29). A comparison
of p53 binding elements from target genes that are activated by p53 revealed that a
consensus p53 binding site contains two half sites separated by O to 13 bp (50). Each half

site contains two quarter sites containing the sequence PuPuPuC(A/T) arranged in a head-

74

to-head orientation. Interestingly, the p53 footprint 1 region contains a sequence that is
similar to the consensus p53 binding element usually associated with transcriptional
activation by p53 (Fi g. 4A).

To investigate the contribution of the high-affinity p53 binding element for U1
promoter recognition and transcriptional repression, a scanning mutagenesis of the U1
core promoter was performed. The sequence of the U1 core promoter region and the
location of the p53 footprints are shown in Fig. 4A. This figure also shows the location
and identity of the mutations introduced into the U1 core promoter. The ability of
recombinant p53 to bind DNA probes harboring these mutations was tested by EMSA
(Fig. 4B). The majority of mutations throughout the U1 core promoter, including
mutations within the region corresponding to p53 footprint 2 (U 1-4.5 and Ul-4.6 probes),
had no significant effect on DNA binding by p53. A slight reduction for p53-DNA
complex 2 formation for the U1-4.5 and U1-4.6 probes seen in this figure was not
observed in replicates of this experiment. In contrast, mutations within the p53 footprint 1
region caused a marked reduction in p53 binding to the U1 promoter. The strongest effect
was seen with the Ul-4.9, U1-4.10, and U1-4.11 probes, whereas the U1-4.8 probe
exhibited only a modest reduction in p53 binding. Formation of both p53-DNA complex
1 and p53-DNA complex 2 was affected by these mutations. These data indicate that the
region adjacent to the U1 transcriptional start site contains the high-affinity p53 binding
element.

Next, the requirement of the high-affinity p53 binding element for p53 repression
was tested. As shown in Fig. 4C, recombinant p53 effectively repressed transcription

from all U1 reporter constructs tested. Neither single nor double sets of mutations within

75

Figure 2-4. The high-affinity p53 element in the U1 promoter is not essential for p53
repression in vitro. (A) Primary sequence of the U1 core promoter region and the
location of the p53 footprints. The plasmid pUl-4.0 contains a wild-type promoter
sequence from -151 to +13. Scanning mutagenesis across this region was performed, and
the introduced mutations and plasmid identity are indicated. Dots represent positions of
identity to this wild-type sequence. (B) Mutations in the p53 footprint 1 region disrupt
p53 binding. EMSA was performed using the various U1 promoter probes and either 200
or 400 ng of p53, as indicated (lanes 2 to 37). Lane 1 contains the wild-type U1 promoter
probe and no added p53. Mutations within the p53 footprint 1 region caused a marked
reduction in p53 binding to the U1 promoter (probes 4.8, 4.9, 4.10, and 4.11). (C)
Mutations within the p53 footprint 1 region do not affect p53 repression in vitro. Selected
mutations were incorporated into a U1 G-less reporter plasmid for in vitro U1
transcription assays (lanes 1 to 20) in the absence or presence of p53 (800 11g). Lane 21
shows transcription from the wild-type Ul reporter in the presence of GST (800 11g). The
read-through (RT) and correctly initiated transcripts (U l S ) are indicated. (D) p53
represses transcription of the wild-type and mutant U1 reporter constructs to similar
extents. (Left panel) An extensive titration of p53 (0, 20, 40, 80, and 160 ng) into U1
transcription assay mixtures was performed using the wild-type (U1 4.0; lanes 1 to 4) and
mutant (U 1 4.9 4.10; lanes 6 to 9) U1 reporter. Lanes 5 and 10 show transcription from
the wild-type U1 plasmid in the presence of 160 ng of GST, as a negative control. (Right
panel) Transcription levels for two independent experiments were normalized to the
signals from transcription reactions containing no added p53, and the average dose-

response curves are shown.

76

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79

the p53 footprint 1 region or within both p53 footprint 1 and 2 regions had a major effect
on p53 repression ability. In some cases, mutation of the U1 core promoter caused a
slight reduction in transcription in the absence of p53, which suggests that factors binding
to these regions may have a positive role in U1 transcription in the absence of added p53.

To more carefully examine the contribution of the high-affinity p53 binding element to
p53 transcriptional repression, a titration of p53 was performed on the wild-type U1

reporter (U 1-4.0) and mutant U1 reporter (U 1-4.9 + 4.10), which harbors two sets of
mutations within the high-affinity p53 binding element (Fig. 4D, left panel).

Transcription initiated from the U1 promoter was quantified, and levels were normalized
to levels in transcription reactions containing no added p53 for each U1 reporter construct.
The average Ul-specific response from two replicates of this experiment is shown in Fig.

4D (right panel). This analysis revealed that both U1 reporter constructs exhibited similar
repression responses to increasing amounts of p53. Together these data indicate that the
high-affinity p53 binding element contained within the human U1 core promoter region is

not required for in vitro transcriptional repression by p53.

The p53 C terminus contributes to both RNA polymerase II and III repression.

Our previous data suggested that p53 is an effective repressor of U6 transcription
by RNA polymerase 111. Therefore, p53 binding to the U6 promoter was next investigated
to determine the contribution of sequence-specific DNA binding by p53 to the repression
of RNA polymerase III transcription. As shown in Fig. SA, p53 binds well to the U1
promoter probe in EMSA, as expected, but approximately 10- to 20-fold less well to the

U6 promoter probes (lanes 6 to 8). The weak binding by p53 to the U6 promoter probe

80

 

suggests that p53 may not rely on direct promoter recognition to mediate RNA
polymerase HI repression. However, it is possible that p53 binds DNA specifically
elsewhere in the reporter constructs during repression.

To determine whether DNA binding by p53 is important for repression of U1 and
U6 transcription, a point mutation in the p53 DNA binding domain was introduced to
eliminate sequence-specific DNA binding. In particular, the arginine at position 175 was
changed to a histidine (R175H) to mimic a hot spot mutation commonly found in human
cancers. Additionally, the C-terrninal region of p53 (301-393) was tested for activity,
because this region is sufficient for chromosome fragile site formation at the multicopy
U1 and U2 snRNA gene loci during adenovirus infection (38, 71). First, the wild-type
and mutant GST-p53 proteins were tested for DNA binding activity in EMSA using
probes encompassing the U1 core promoter region. As shown in Fig. 5B, only the wild-
type recombinant GST-p53 bound effectively to the U1 promoter DNA while the GST-
pS3 (R175H) and GST-p53 (301-393) proteins were completely inactive in this assay.
Next, the ability of these proteins to regulate in vitro U1 and U6 transcription by RNA
polymerases II and 111, respectively, were tested (Fig. 5C). Interestingly, GST-p53
(R175H) repressed U1 transcription as effectively as wild-type GST-p53, whereas GST-
p53 (301-393) was approximately twofold less effective, but still capable of repression.
Both mutant GST-p53 proteins repressed U6 transcription, but GST-p53 (R175H) was
less effective than either wild-type GST-p53 or GST-p53 (301-393), which were
equivalently active in these assays. In these assays, the repression of RNA polymerase III
transcription by p53 (R175H) is in contrast with previous experiments, wherein the

introduction of the R-to-H mutation switched p53 from a repressor to an activator of

81

Figure 2-5. The p53 C terminus is sufficient for transcriptional repression and
promoter association. (A) The U6 core promoter does not contain a high-affinity p53
binding element. EMSA was performed with increasing amounts of recombinant p53 (0,
80, 160, and 320 ng) and equivalently labeled double-stranded DNA probes
encompassing the U1 core promoter (lanes 1 to 4) and U6 core promoter (lanes 5 to 8).
(B) Direct U1 promoter recognition requires the p53 DNA binding domain. EMSA were
performed with a wild-type U1 promoter probe ( 151 to 13) using 250, 500, and 1,000 11g
of full-length wild-type or mutant (R175H) GST-p53 (lanes 2 to 4 and 5 to 7,
respectively). Reactions containing truncated GST-p53 (301-393) or GST alone are
shown in lanes 8 to 10 and 11 to 13, respectively. Lane 1 contains no added protein. (C)
Wild-type and mutant GST-p53 repress transcription similarly. Approximately 100, 200,
and 400 ng of wild-type GST-p53 (lanes 2 to 4), GST-p53 (R175H) (lanes 5 to 7), GST-
p53 (301-393) (lanes 8 to 10), or 400 ng of GST (lane 11) were titrated into U1, U6, and
AdML in vitro transcription reaction mixtures. Both wild-type and mutant p53 molecules
repressed U1 and U6 transcription, whereas AdML transcription was unaffected. (D) The
p53 DNA binding domain is not required for U1 and U6 promoter association during
repression. A portion of untreated or p53 repressed U1 and U6 in vitro transcription
reaction mixtures (lanes 1, 4, 7, 10, and 11 in panel C) was cross-linked with
formaldehyde and subjected to immunoprecipitation with anti-SNAP43 (lanes 3 and 8),
IgG (lanes 4 and 9), or anti-p53 (lanes 5 and 10) antibody. Enrichment of the U1 and U6
reporter plasmids or negative control pUC119 DNA was compared by PCR using
promoter-specific or pUC-specific primers. Lanes 1, 2, 6, and 7 show the amplification
directly from the input DNA (10 and 1% in lanes 1 and 2 for U1 and lanes 6 and 7 for U6,
respectively) contained in the transcription assay mixtures prior to immunoprecipitation.
The results of PCRs using the pUC-specific primers under all conditions were
indistinguishable and are shown only for the reactions containing added wild-type GST-

p53.

82

 

 

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general RNA polymerase III transcription (57). These data herein indicate that DNA
binding by p53 is not essential for repression of U1 and U6 snRNA gene transcription by
RNA polymerases II and 111, respectively, and the C-terminal region encompassing
amino acids 301 to 393 is sufficient for p53 repression activity.

Next, recruitment of the mutant p53 proteins to the U1 and U6 promoters during
repression was examined. Portions of in vitro transcription reaction mixtures containing
wild-type or mutant GST-p53 proteins were cross-linked with formaldehyde prior to
immunoprecipitation by using antibodies directed against SNAP43 or p53. Transcription
reaction mixtures containing GST or no added proteins were also tested. The specific
recoveries of the U1 and U6 promoter-containing plasmids were compared to recovery of
an irrelevant plasmid (pUC119) included in the original transcription assay mixtures. As
shown in Fig. SD, significant U1 and U6 reporter plasmid recovery by anti-SNAP43
immunoprecipitation was obtained from the untreated transcription reaction mixture
(lanes 3 and 8). Neither wild-type nor mutant p53 affected reporter enrichment by anti-
SNAP43 immunoprecipitation, suggesting that p53 may not disrupt SNAPC promoter
binding during repression. However, it is difficult to eliminate the possibility that SNAPC
can bind to both actively transcribed and inactive templates, whereas p53 may be
specifically bound to only those active templates containing a complete preinitiation
complex. When the untreated U1 and U6 transcription assays were used for anti-p53
immunoprecipitation, only background levels of reporter plasmid enrichment were
observed (lanes 5 and 10). This result was expected, because these assays were
performed using HeLa cell nuclear extracts, which contain very low levels of endogenous

p53. However, significant recovery of the U1 and U6 promoter plasmids was observed in

85

 

reactions containing additional wild—type p53. Interestingly, U1 and U6 reporter DNA
enrichment was also observed in reactions containing the mutant p53 (R175H) and p53
(301-393) proteins. In all cases, recovery of the reporter-containing plasmids was
substantially greater than that observed for the irrelevant pUC119 plasmid (Fig. SD,
bottom panel, and data not shown). These results indicate that p53 can associate with the
U1 and U6 promoters during repression. The preferential association of the mutant p53
proteins with the promoter-containing plasmids suggests that during repression p53 is
actively recruited to U1 and U6 promoters independently of sequence-specific DNA
binding by p53. Furthermore, the C-terrninal domain of p53 is likely critical for promoter

recruitment.

p53 associates with general transcription factor SNAPC.

Promoter association by p53 in the absence of direct DNA binding suggests that
protein-protein interactions are critical for p53 recruitment. Furthermore, p53 was
capable of repressing both U1 and U6 transcription by RNA polymerases II and III,
raising the possibility that a factor commonly used for these genes could be targeted by
p53. Both the general transcription factors SNAPC and TBP are utilized during
transcription of these genes and are thus candidate p53 targets. Indeed, TBP has been
shown to interact strongly with p53 within the N-terrninal activation domain and the C-
terminal oligomerization domain (16, 41, 61). To determine whether p53 could
potentially target SNAPC, coimmunoprecipitation assays were performed using
antibodies directed against the SNAP43 subunit of SNAPC. The association of

endogenous p53 with SNAP43 was measured by Western analysis before and after UV

86

 

Figure 2-6. Endogenous p53 associates with the general transcription factor SNAPc.
(A) Irnmunoprecipitation with anti-SNAP43 antibodies was performed from whole-cell
extracts prepared from untreated and UV-treated MCF-7 cells at different time points
after UV light treatment. Western analysis revealed significant differences in recovered
p53 between the negative control IgG immunoprecipitations (lanes 8 to 10) and anti-
SNAP43 immunoprecipitation by the 4-h time point (lane 5). The amount of
coimmunoprecipitated p53 continued to increase up to 24 h after UV light treatment
(lanes 6 and 7), whereas the amount of precipitated SNAP43 did not change from 0 to 24
h after UV exposure. No galectin-3 was observed in any of the immunoprecipitations.
Lanes 1 and 2 contain 1% of the extract used for immunoprecipitation reactions. (B)
Irnmunoprecipitation with anti-p53 (Ab-6) antibodies was performed from whole-cell
extracts (5 mg, lanes 3, 4, and 6; 1.7 mg, lanes 5 and 7) prepared from untreated and UV-
treated MCF-7 cells 8 h after UV light treatment, as indicated. Lanes 1 and 2 contain 150
g of extract that was used as input for immunoprecipitation reactions. Western analysis
with anti-SNAP43 antibodies revealed increased SNAP43 association with p53 by 8 h
after UV light exposure. (C) p53 interacts with SNAP43, SNAP190, and TBP. GST pull-
down experiments were performed with 1 pg of recombinant full-length GST-p53 (lane
2), GST-p53 (301-393) (lane 3), GST (lane 5), or beads alone (lane 4) with individual
SNAPC subunits and TBP that were translated in vitro and labeled with [35S]methionine.

Lane 1 contains 5% of radiolabeled proteins added to each pull-down reaction mixture.

87

A

 

 

1% Input or-SNAP43 lP IgG IP
I7 II II 8'
a

-UV +uv -UV +uv————)-—— -UV +uv E
o 8 o o 4 e 24 o 8 2

SNAP43

11‘ 1.. ”~23 p53
\- - Galectin

1 2 3 4 5 6 7 8 9 10
Coimmunoprecipitation

 

 

lgGlP 0t-53IP
l——l F I
Input -UV +UV
If I f Iii 1
-UV +UV ‘ WCE

 

 

. SNAP43

 

 

 

1 2 3 4 5 6 7
Coimmunoprecipitation

88

 

 

 

 

 

 

 

 

 

 

 

 

*5 a a 3‘

8 e an? m

E 17. 17. a 3 ta

1}: <5 <5 9. 8 to
f * ' a"? — SNAP19O
cui— — SNAP50
3.., — SNAP45
' - SNAP43

,— SNAP19
— TBP

 

 

 

1 2 3 4 5
GST pull-down

89

light treatment. As shown in Fig. 6A, p53 was not detected in anti-SNAP43
immunoprecipitation reactions prior to UV treatment, but p53 recovery increased by 4 h
and continued to increase by 24 h after UV light treatment. No recovery of the splicing
factor galectin-3 was observed in these reactions. Reciprocal anti-p53
immunoprecipitation assays were performed using extracts prepared from MCF-7 cells
that were untreated or UV light treated (8 h posttreatment). As shown in Fig. 6B, UV
light treatment had little effect on the levels of SNAP43 present in the extract (lanes 1
and 2) but caused increased SNAP43 association with p53 (compare lanes 4 and 5 to 6
and 7). Together, these data indicate that UV light modulates the association between
endogenous p53 and SNAPC.

GST pull-down experiments were then performed to determine whether p53 could
interact with TBP and individual components of SNAPC (Fig. 6C). In these assays, full-
length GST-p53 interacted well with TBP and SNAP43 while SNAP190 interacted only
weakly in this assay. No significant interactions were observed for GST-p53 with
SNAP50, SNAP45, and SNAP19. Interestingly, truncating p53 to include only the C-
terminal domain abolished interactions with SNAP43 but stimulated interactions with
SNAP190. These observations suggest that p53 may interact with TBP and SNAPC to

correctly target human snRN A genes for repression.

9O

 

Discussion

The p53 tumor suppressor protein acts as a transcriptional activator in response to
cellular stress, but it is also competent for concomitant repression of other sets of target
genes. The data presented herein demonstrate that endogenous p53 associates with human
snRNA gene promoters and that UV light exposure stimulates p53 association with both
U1 and U6 snRNA genes. These in vivo data indicate that p53 is directly involved in the
regulation of human snRNA gene transcription by both RNA polymerases II and III. That
recombinant p53 effectively repressed both U1 and U6 snRNA gene transcription in vitro
suggests that these genes similarly could be repressed by p53 in response to DNA
damage in vivo. Indeed, cellular U1 and U6 snRNA gene transcription is down regulated
in p53-positive MCF-7 cells afier UV light exposure. In contrast, transcriptional
repression was not observed in p53- deficient HeLa cells after UV light exposure.

The data presented herein also suggest that p53 uses different mechanisms to
repress U1 and U6 transcription. First, U1 snRNA genes contain a high-affinity p53
binding element located with the core promoter region adjacent to the PSE. Second, U6
snRNA genes remain sensitive to p53 repression afier preinitiation complex assembly,
whereas U1 snRNA genes become refractory to p53 influence, suggesting that the
potential targets required for RNA polymerase II repression may be unavailable for p53
interaction after preinitiation complex assembly. Thus, it is tempting to speculate that p53
interferes with SNAPC or TBP promoter occupancy during repression, which for U1
genes could involve direct competition between p53 and SNAPC for adjacent promoter
elements. However, the high-affinity p53 binding element is clearly not required for U1

repression in vitro, and the levels of SNAPC detected on U1 and U6 snRNA gene

91

~ *—- —-.-

promoters by chromatin immunoprecipitation do not substantively change in response to
UV light, even though there is a substantial increase in p53 association. Consistently, we
have not observed any effect of full-length p53 on PSE binding by SNAPC in EMSA
(data not shown). We currently favor the hypothesis that p53 interferes with snRNA gene
transcription at steps occurring after promoter association by SNAPC.

As U1 and U6 snRNA gene transcription relies on different assemblies of
transcription factors, it is likely that to enact repression p53 targets different factors
specifically required for RNA polymerase II or III transcription. For RNA polymerase II
transcription, one candidate is TFIIH, which can interact with p53 (67, 70). TFIIH may
be utilized during transcription of human U1 snRNA genes, as removal of TFIIH from
extracts impairs U1 snRNA gene transcription (32). However, U1 transcription was not
reconstituted in the depleted extracts by addition of purified TFIIH, suggesting that
additional factors may complement the purified TFIIH for activity (32). A second
candidate coregulator is the Cockayne syndrome group B (CSB) protein, which also can
interact with p53 (67). CSB was originally suggested to play a role in DNA repair and,
more recently, was shown to interact with RNA polymerase II (63). CSB may be an
elongation factor (34, 56, 59, 62) which could mediate transcription of genes encoding
RNA products with significant secondary structure, such as human U1 and U2 snRNA
genes. As previously described for p53, mutations in CSB also cause chromosomal
fragile site formation at U1 and U2 snRNA genes (71), suggesting that CSB may play a
role in transcription of these genes by RNA polymerase II. It was hypothesized that
activated p53 interferes with CSB-mediated elongation to cause RNA polymerase II

stalling at U1 and U2 snRN A genes (71). The data presented herein support a role for p53

92

. _.. if:.§-i.§~‘=-

in mediating repression of U1 snRNA gene transcription by RNA polymerase II, but
whether CSB is involved in this process is not yet known.

During genomic threats, p53 is stabilized from destruction and activated by a
cascade of posttranslational modifications, including phosphorylation and acetylation.
Acetylation of p53 has been suggested to increase sequence-specific DNA binding by
p53 (18, 44) and to stimulate cofactor recruitment, such as the p300 histone
acetyltransferase, during activated transcription of p53 target genes (2, 15). Thus, it is
interesting that while acetylated p53 was detected at human U1 genes prior to UV light
exposure, the level of acetylated p53 at these genes was not stimulated by UV light even
though a substantial increase in the total p53 levels at these genes was observed.
Furthermore, little to no acetylated p53 was observed at a U6 snRNA gene promoter after
UV light treatment, indicating that most of the p53 associated with this gene is not
acetylated. One possibility is that p53 recruits a histone deacetylase to enact repression,
and this enzyme deacetylates p53. Another possibility is that p53 acetylation is not
compatible with transcriptional repression. For example, the sites of p53 acetylation are
all located in the C-terminal region (18, 40, 53), and this region is sufficient for
transcriptional repression of human U1 and U6 snRNA gene transcription. Presumably,
the p53 C terminus makes key protein-protein contacts to enact repression and, as we
have shown, this region interacts with SNAP190 and TBP. Acetylation of p53 may
disrupt these contacts and prevent p53 from being recruited to these snRNA promoters.
However, it is difficult to exclude the possibility that p53 acetylation is important for p53
repression where the sites of acetylation are intimately involved in protein-protein

contacts and are not available for antibody recognition during chromatin

93

 

immunoprecipitation. Nonetheless, p53 acetylation is unlikely to be essential for
repression, because the recombinant wild-type and mutant p53 used for the U1 and U6
repression assays were not substantially acetylated. The presence of acetylated p53 at the
U1 snRNA gene promoters in untreated MCF-7 cells suggests that activated p53 can bind
to these genes, possibly through direct recognition of the high-affinity p53 binding
element contained within the U1 snRNA gene core promoter even though this element is
not required for U1 repression in vitro. An intriguing possibility is that p53 may have
dual roles at human U1 snRNA genes, with direct promoter recognition and activation
functions prior to substantial DNA damage but repressive activity mediated via a

different promoter-targeting mechanism after DNA damage, such as that caused by UV

light.

Acknowledgements

The anti-galectin-3 antibody (Mac2) was a gift from John Wang (Michigan State
University). The pRc/RSV and pRc/RSV-pSB-Flagwt plasmids were a gift from Roland
Kwok (University of Michigan). We thank Liping Gu, Gauri Jawdekar, Min-Hao Kuo,
and Jim Geiger for critical assessment of the manuscript and appreciate the technical
assistance provided by Craig Hinkley, Brandon LaMere, Xianzhou Song, and
Tharakeswari Selvakumar. We acknowledge the National Cell Culture Center for
preparation of HeLa cells. This work was supported by an NIH grant (GM59805) to

R.W.H.

94

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monodisperse nuclear RNA. Biochim. Biophys. Acta 190:10—29.

Wong, M. W., R. W. Henry, B. Ma, R. Kobayashi, N. Klages, P. Matthias, M.
Strubin, and N. Hernandez. 1998. The large subunit of basal transcription factor
SNAPC is a Myb domain protein that interacts with Oct-1. Mol. Cell. Biol.
18:368—377.

Xiao, H., A. Pearson, B. Coulombe, R. Truant, S. Zhang, J. L. Regier, S. J.
Triezenberg, D. Reinberg, O. Flores, C. J. Ingles, et al. 1994. Binding of basal

100

 

 

71.

72.

transcription factor TFIIH to the acidic activation domains of VP16 and p53. Mol.
Cell. Biol. 14:7013—7024.

Yu, A., H. Y. Fan, D. Liao, A. D. Bailey, and A. M. Weiner. 2000. Activation
of p53 or loss of the Cockayne syndrome group B repair protein causes metaphase
fragility of human U1, U2, and SS genes. Mol. Cell 5:801—810.

Zhai, W., and L. Comai. 2000. Repression of RNA polymerase I transcription by
the tumor suppressor p53. Mol. Cell. Biol. 20:5930—5938.

101

 

 

CHAPTER THREE

THE COCKAYNE SYNDROME COMPLEMENTATION GROUP B PROTEIN
MODULATES TRANSCRIPTION REGULATORY FUNCTIONS OF THE

TUMOR SUPPRESSOR PROTEIN P53

Abstract

Activation of the tumor suppressor protein p53 or loss of the Cockayne syndrome

 

complementation group B (CSB) protein induces chromosomal fragile site formation at
the U1 snRNA, U2 snRNA, and SS rRNA gene loci, suggesting a possible role for these
factors in controlling transcription of these genes even though U1 and U2 snRNA genes
are transcribed by RNA polymerase II and SS rRNA genes are transcribed by RNA
polymerase III. p53 acts as a transcriptional repressor of both SS rRNA gene and U1
snRNA gene transcription. However, the relationship between CSB and these

transcription programs has not been established.

In this study I show that CSB associates with human SS rRNA and U1 snRNA
promoters and has Opposing roles in transcription of these genes. CSB represses SS rRNA
gene transcription but activates U1 snRNA gene transcription. Also, CSB was found to
be involved in general RNA polymerase III transcription, including U6 snRNA gene
transcription. Human U6 snRNA genes, even though they are transcribed by RNA

polymerase III, have promoter structures that are more similar to the RNA polymerase II-
trarjscribed U1 snRNA genes. Surprisingly, the role of CSB in U6 snRNA gene

transcription is more similar to Ul snRNA, rather than SS rRNA gene transcription.

102

Interestingly, CSB association with U1 and U6 snRNA gene promoters was diminished
after UV light treatment concomitant with increased p53 promoter association. Also,
removal of CSB from cell extracts enhances the inhibitory effect of p53 on U1 snRNA
gene transcription by RNA polymerase II, but does not affect p53-mediated repression of
U6 snRNA gene transcription by RNA polymerase 111. At low amounts p53 activates SS
rRNA transcription, but at higher amounts p53 represses SS rRNA gene transcription
better when CSB is depleted from in vitro transcription reactions. Together, these results
suggest that p53 modulates CSB transcription regulatory functions at U1 and U2 snRNA
genes and SS rRNA genes, which are susceptible for fiagile site formation during

metaphase.

Introduction

Chromosomal fragile sites are non-randomly located non-staining gaps in
metaphase chromosomes caused by incompletely condensed chromatin or, more rarely,
by chromosome breaks. Fragile sites have been proposed to be not only susceptible to
DNA instability in cancer cells, but also associated with genes that contribute to the
neoplastic process (27). Infection of human cells at low multiplicity by adenovirus
serotype 12 (Ad12), but not adenovirus 2 or 5, or transient expression of Ad12 ElB 55
kDa protein induces four sites of metaphase chromosome fragility that were found to co-
localize with four tandemly repeated multigene families: RNUI locus (contains U1
snRNA genes), RN U2 locus (contains U2 snRNA genes), RNSS locus (contains SS rRNA
genes), and PSUI locus (contains U1 pseudogenes) (9, 14, 24, 29, 37). It was

subsequently found that p53 was required for Ad12-induced chromosome fragility (24,

103

 

37). Surprisingly, the same chromosomal loci — RNUI, RNU2, and RNSS — are
constitutively fragile in Cockayne syndrome group B (CSB) cells that fail to express
functional CSB protein (3 7).

The involvement of p53 and CSB in fragile site formation suggests that both
factors are involved in the transcription of these genes, even though U1 and U2 snRNA
genes are transcribed by RNA polymerase II and SS rRNA genes are transcribed by RNA
polymerase III. Indeed, a role for p53 in transcription of these genes has been
demonstrated. p53 acts as a transcriptional repressor of SS rRNA gene transcription by
RNA polymerase III (4, 6) and U1 snRNA gene transcription by RNA polymerase II (15).
However, any role for CSB in transcription of these genes has not been investigated. CSB
belongs to the SWIZ/SNF2 family of proteins and has many of the activities expected for
a SWI2/SNF2 protein (10). It has DNA-stimulated ATPase activity and can remodel
nucleosomes at the expense of ATP hydrolysis (8). CSB was originally suggested to
function in transcription-coupled repair of RNA polymerase II-transcribed genes after
DNA damage (23, 33, 35). Recent studies also revealed a role of CSB in transcription. It
was demonstrated that CSB associates with RNA polymerase I and II protein complexes,
and in vitro transcription experiments showed that CSB stimulates elongation by RNA
polymerase II (2, 30, 34). The observed interaction of CSB with RNA polymerase I and
II in vivo and in vitro suggested that CSB might be specifically targeted to sites of
blocked transcription (2). In addition, the CSB protein may also play a role in clearing the
stalled RNA polymerase II molecule from the lesion site so that repair can occur and
transcription resume (l6). To-date, the relationship between CSB and RNA polymerase

III transcription has not been established.

104

RNA polymerase III-transcribed genes are divided into three classes based on the
structure of promoters (18). Class 1 and 2 genes are represented by the SS rRNA and
tRNA genes respectively and contain internal promoter elements. In contrast, class 3
genes, represented by the U6 snRNA, have external promoters. Even though class 3
genes are transcribed by RNA polymerase 111, they share similar promoter architectures
as well as some general transcription machinery requirements with RNA polymerase II-
transcribed snRN A genes.

In this current study I examined the role of CSB in RNA polymerase II and III
transcription and its functional interplay with p53. I found that CSB associates with U1
snRNA gene promoters and with a variety of RNA polymerase III-transcribed gene
promoters but CSB has different roles for RNA polymerase II and III transcription. CSB
plays a positive role in U1 snRNA gene transcription by RNA polymerase II and in U6
snRNA (class 3) gene transcription by RNA polymerase III, but plays a negative role in
transcription of other classes of RNA polymerase III-transcribed genes. In addition, I
present several pieces of data that suggest a modulatory role of p53 on CSB transcription

fimctions.

105

 

Materials and Methods

Cell culture and UV irradiation

Human MCF-7, HeLa and H1299 (p53-/-) cells were maintained in Dulbecco’s
modified Eagle’s medium supplemented with penicillin-streptomycin and 10% fetal
bovine serum. Cells grown to 70 to 80% confluence were washed with phosphate-
buffered saline and irradiated with 50 J of UV light (254-nm peak)/m2 by using a UV
Stratalinker (Stratagene). After irradiation, growth medium was added and cells were
incubated at 37°C under 5% CO; for the indicated times. Additionally, H 1299 cells were
grown to 50% confluence in ISO-mm plates and were then transiently transfected with 2
pg of the pRc/RSV or pRc/RSV-p53.wt or pRc/RSV-KRSA-p53 plasmids by using
Lipofectin reagent (Invitrogen) for 6 h. Subsequently, the medium was replaced and cells

were incubated for 48 h for further analysis.

Nuclear run-on assays

Nuclear run-on assays were performed as described elsewhere (5) in the presence
of [a-32P] UTP using approximately 107 nuclei that were isolated from MCF-7 cells.
Labeled RNA was recovered and hybridized to a nitrocellulose membrane containing
approximately 7 ug of U1, U6, and SS rRNA target gene DNAs or 10 ug of pUC119
plasmid DNA, as a negative control. Target gene DNAs corresponding to the coding
regions of the indicated genes were generated by PCR and were immobilized on a nylon
membrane at levels calculated to be in excess relative to the corresponding snRNA
Population in the nuclei. Hybridizations were performed for 16 h at 42°C in hybridization

buffer containing 50% formamide. Membranes were then washed extensively in 2X SSC

106

 

(1X SSC is 0.15 M NaCl plus 0.015 M sodium citrate) and 0.1% sodium dodecyl sulfate

(SDS) at 50°C. Hybridized RNA transcripts were visualized by autoradiography.

Expression and purification of recombinant proteins

Glutathione S—transferase (GST), GST-tagged full-length human p53 and GST-
tagged human CSB (528-1222) were expressed in Escherichia coli BL21 (DE3) codon+
cells (Stratagene) and were affinity purified by binding to glutathione agarose beads
(Sigma). GST and GST-CSB (528-1222) were then eluted from beads in HEMGT-ISO
buffer containing 50 mM glutathione for 4 h at 4°C. Untagged p53 was obtained by
digestion with thrombin. p53 was further purified by chromatography using a Mono-Q
(HRS/S) column (Phannacia) and were concentrated by centrifugation using a Centricon
YM-30 spin column (Millipore) in HEMGT-80 buffer (20 mM HEPES [pH 7.9], 0.1 mM
EDTA, 10 mM MgC12, 10% glycerol [vol/vol], 0.1% Tween 20, 80 mM KCl) containing

protease inhibitors and 1 mM dithiothreitol.

In vitro transcription assays

In vitro transcription assays were performed as described previously (17) using 18,
2, 2, and 10 uL of HeLa cell nuclear extract for the U1 snRNA, U6 snRNA, SS rRNA,
and adenovirus major late promoter (AdML) transcription reactions, respectively. The
pUl-4.0 (1 1.1g), pU6/Hae/RA.2 (250 ng), pHSSsa (250 ng), and M13-AdML (250 ng)
templates were used for the U1 snRNA, U6 snRNA, SS rRNA, and AdML transcription
reactions, respectively. Purified proteins were added in the amounts indicated in the

figure legends. Transcription was performed for 1 h at 30°C. Transcripts were separated

107

 

by denaturing 6% polyacrylamide gel electrophoresis and visualized by autoradiography.

Chromatin immunoprecipitation assay

Chromatin immunoprecipitation assays were performed as described previously
(19). Human MCF-7 cells were grown to 60 to 80% confluence and were then cross-
linked with 1% formaldehyde for 30 min at room temperature. After cell lysis and
sonication, immunoprecipitation reactions were performed overnight at 4°C using
chromatin from approximately 107 cells per reaction mixture and 1 pg of each antibody:
anti-TBP (SL2), pre-immune IgG (Sigma), anti-p53 (21-25) (Ab-6; Oncogene), anti-CSB
(N-l7, Santa Cruz), anti-RNA polymerase II (8WG16, Covance Research Products) and
anti-RNA polymerase III (TB2). Recovered chromatin was suspended in 50 pL of H20,
and PCR analysis was performed using 5 pl of immunoprecipitated chromatin or input
chromatin. The primers used for amplification of each gene were described previously
(15). PCR products were separated by 2% agarose electrophoresis in Tris-borate-EDTA

buffer and were stained with ethidium bromide.

Immunoprecipitations from in vitro transcription reactions

In vitro transcription assay mixtures containing U1 or U6 promoter plasmids and
equal molar amounts of pUC119 were performed as described previously (19) in the
absence or presence of hill-length wild type p53 or GST. Five microliters of each
transcription reaction mixture was diluted to 500 pL and was cross-linked in 1%
formaldehyde for 10 min at room temperature, quenched with 125 mM glycine for 10

min at room temperature, and immunoprecipitated with anti-TBP (SL2), pre-immune

108

 

(Sigma), anti-p53 (Ab-6; Oncogene), anti-CSB (N-l7, Santa Cruz) and anti-RNA
polymerase II (8WG16, Covance Research Products) or anti-RNA polymerase III (TB2)
antibodies. Recovered plasmid DNA was analyzed by PCR using primers specific to the

U1 and U6 promoter regions or to pUC119 as a negative control.

Immunoprecipitation experiments

Approximately 5 mg of HeLa cell nuclear extract was incubated with 1 pg of
antibodies directed against CSB (N-l7, Santa Cruz) or goat IgG (Sigma) 90 minutes at
room temperature. Reactions were diluted to 1 mL in HEMGT-IOO buffer and stable
complexes were affinity purified by incubation with Protein-G Fast Flow sepharose beads
(Upstate Biotechnology) for 90 minutes at room temperature. Beads were washed in
HEMGT-IOO buffer and boiled for 5 minutes in Laemmli Buffer. Bound proteins were
separated by 12.5% SDS-PAGE and transferred to nitrocellulose. Western blot analyses
of recovered proteins were performed using anti-RNA polymerase III (TB2), anti-RNA

polymerase II (8WG16, Covance Research Products) and anti-actin (Sigma) antibodies.

109

Results

Endogenous CSB occupies human U1 and U6 snRNA gene promoters.
The RN U1, RN U2, and RN55 loci are constitutively fragile in CSB deficient cells
that fail to express functional CSB protein (37) and it was postulated that loss of CSB
transcription functions is important for fragile site formation (37). To determine whether
CSB is involved in transcription of these genes, the association of endogenous CSB with
U1 snRNA gene promoter and with three different classes of RNA polymerase III-
transcribed promoters was examined using chromatin immunoprecipitation experiments.
As shown in figure 3-1, substantial recovery of U1 snRNA promoter DNA was observed
in anti-CSB-immunoprecipitated samples (lane 5) as compared to negative control IgG-
immunoprecipitated reactions (lane 4). Interestingly, enrichment of all three classes of

RNA polymerase III-transcribed promoters was also observed in anti-CSB-

immunoprecipitated samples. No CSB was detected on the negative control GAPDH
exon 2 region. The presence of CSB on RNA polymerase II-transcribed U1 snRNA gene
promoters and RNA polymerase III-transcribed SS rRNA gene is in agreement with the
observation that CSB is required for ‘fragile site’ formation on these genes. Together,

these data suggest a role of CSB in regulation of RNA polymerase II—transcribed U1

snRNA gene expression and RNA polymerase III gene transcription.

110

Figure 3-1. Endogenous CSB associates with RNA polymerase II-transcribed
snRNA gene promoters and RNA polymerase III-transcribed promoters. Chromatin
from MCF-7 cells was immunoprecipitated with anti-SNAP43, IgG (negative control),
anti-CSB, anti-TBP and anti-RNA polymerase II and III antibodies. CSB enrichment was
observed on RNA polymerase II-transcribed U1 snRNA gene and on the promoters of all
three classes of RNA polymerase III transcribed genes. No CSB was detected on the

GAPDH exon 2 region, which served as a negative control for immunoprecipitations.

111

 

 

 

 

 

 

 

 

 

 

 

 

 

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112

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Endogenous CSB associates with RNA polymerase 111 protein complexes.

The relationship of CSB to the RNA polymerase I and II transcription has been
examined previously. CSB was shown to associate with RNA polymerase I and 11 protein
complexes (2, 34); however, to-date a role of CSB in RNA polymerase HI transcription
has not been investigated. Thus, we examined the relationship of CSB to the RNA
polymerase III using confocal microscopy. The antibody staining patterns in these
immunofluorescent studies revealed that both CSB and RNA polymerase III are primarily
confined to the nucleus. The overlay of the confocal images showed that approximately
30% of RNA polymerase III co-localizes with CSB (Figure 3-2A).

To further test whether CSB associates with RNA polymerase III complex, HeLa
nuclear extract was subjected to immunoprecipitation with anti-CSB or negative control
IgG antibodies. As shown in figure 3-2B, RNA polymerase III was specifically enriched
in anti-CSB-immunoprecipitated samples, but not in negative control IgG-
immunoprecipitated samples. Also, no significant recovery of RNA polymerase II or
actin was observed in anti-CSB-immunoprecipitations. CSB was shown to be in the
complex with RNA polymerase II when immunoprecipitations are performed under less
stringent lower salt concentrations (2). Together, the co-localization of CSB and RNA
polymerase III in cells and the presence of these two proteins in a complex suggests a

role of CSB in RNA polymerase III transcription.

113

Figure 3-2. Endogenous CSB associates with RNA polymerase III. (A) HeLa cells
were immunostained using antibodies against RNA polymerase IH and CSB for
visualization by confocal microscopy. This experiment was performed by Chen Wang in
the laboratory of Dr. Sui Huang, Feinberg School of Medicine, Northwestern University.
(B) Co-immunoprecipitations with anti-CSB antibody (lane 3) or negative control IgG
antibody (lane 2) were performed from HeLa nuclear extract. Lane 1 contains 1% of the
HeLa nuclear extract that was used for each immunoprecipitation reaction. Western
analysis revealed significant differences in recovery of RNA polymerase III in the CSB
immunoprecipitation reaction but not in the negative control IgG immunoprecipitation.

No actin or RNA polymerase II were recovered in any of the tested conditions.

114

Hela cells
1 CSB for
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University.
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in the CSB

ecipitation.

 

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115

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CSB has positive and negative regulatory activities.

To examine the effect of CSB on RNA polymerase II and III transcription,
recombinant GST-CSB (5284222) protein, containing SNF2-like domains, was titrated
into RNA polymerase II-transcribed U1 snRNA and AdML in vitro transcription
reactions as well as RNA polymerase III-transcribed SS rRNA (class 1), AdVAI (class 2)
and U6 snRNA (class 3) reactions (Figure 3-3A). In these experiments addition of
recombinant GST-CSB (5284222) repressed RNA polymerase III-transcribed SS rRNA
(class 1) and AdVAI (class 2) gene expression. The repressive effect of CSB on these
RNA polymerase III-transcribed genes was specific because the same amounts of the
GST-CSB protein did not repress either RNA polymerase III-transcribed U6 snRNA

(class 3) gene expression or RNA polymerase II-transcribed U1 snRNA or AdML gene

transcription.

To further examine the role of CSB in transcription in vitro transcription reactions
were assembled in the presence of endogenous CSB or in its absence as a result of
immunodepletion of CSB from nuclear extracts (Figure 3-3B). The CSB depleted HeLa
nuclear extracts did not support expressions of the RNA polymerase II-transcribed U1
snRNA and AdML genes, which is in agreement with suggested positive role of CSB in
RNA polymerase II transcription (30). U6 gene expression by RNA polymerase III (class
3) was also modestly decreased in reactions performed with CSB depleted nuclear
extracts. In contrast to the effect of CSB immunodepletion on U6 snRNA (class 3) gene
eXpression, RNA polymerase III transcription of 5S rRNA (class 1) and AdVAI (class 2)
Were stimulated in the absence of CSB. This result is in agreement with the observed

repressive functions of exogenous CSB in transcription of class 1 and class 2 RNA

116

polymerase III-transcribed genes. Interestingly, AdVAI repression was not observed by
addition of CSB to nuclear extracts that were immunodepleted for endogenous CSB
(Figure 3-3C, compare lanes 5 and 6 to lanes 2 and 3), suggesting that CSB may require a

co-repressor for its transcription repression functions.

To determine whether CSB plays a role in RNA polymerase II and III gene
transcription in vivo, nuclear run-on experiments were performed using nuclei harvested
from human fibroblast cells with normal CSB status (NF) and fibroblast cells with non-
functional CSB originally isolated from CS patients (CSB) (Figure 3-3D). Interestingly,
U1 snRNA gene transcription was reduced in CSB cells and represented 78% of the U1
snRNA gene transcription in NF cells (p-value = 0.0027, 2-tailed t-test). Levels of U6
snRNA gene transcription in CSB cells were also reduced to 90% of that in NF cells (p-
value = 0.041, 2-tailed t-test). Reduced levels of endogenous U1 and U6 snRNA gene
transcription in CSB cells is consistent with the positive role of CSB in snRNA gene
transcription regulation. However, SS rRNA gene transcription was comparable between
NF and CSB cells. A possible explanation for the absence of detectable effect of CSB on
endogenous 5S rRNA gene transcription is that in contrast to endogenous CSB, the

recombinant protein lacks any covalent modifications that may be required for function in
certain contexts. Indeed, a study by Christiansen et al., (2003) showed that CSB is
phosphorylated in vivo and this can be reversed by UV irradiation (7). The modification
status of the CSB protein may determine its ability to interact with its co-factors. For
example, endogenous CSB, when phosphorylated, may not be able to interact with its co-
repressors. This speculation is in agreement with a conclusion from the experiment 3-3C,

Which suggests that CSB may require a co-repressor for AdVAI transcription repression.

117

Figure 3-3. CSB regulates snRNA gene transcription. (A) 100 and 400 ng of
recombinant GST-CSB (528-1222) were titrated to U1, AdML, SS, AdVAI and U6 in
vitro transcription reactions (lanes 2 and 3). Effect of GST—CSB on transcription was
compared to levels of transcription in untreated reactions (lane 1) and reactions
containing heat-inactivated (HI) GST-CSB. GST-CSB (528-1222) effectively repressed
SS and AdVAI transcription but it did not affect U1, AdML and U6 transcription. (B) U1,
AdML, SS, AdVAI and U6 in vitro transcription reactions were performed for 30 or 60
minutes with HeLa nuclear extracts containing endogenous CSB (lanes 1 and 2) or
immunodepleted for CSB with anti-CSB antibodies (lanes 3 and 4). As a control,
immunodepletion of HeLa nuclear extracts was also performed with IgG antibodies
(lanes 5 and 6). CSB immunodepleted nuclear extracts supported RNA polymerase II-
transcribed U1 snRNA and AdML gene transcription less efficiently as compared to the
level of transcription from untreated HeLa nuclear extracts or HeLa nuclear extracts
immunodepleted with IgG antibodies. RNA polymerase III-transcribed U6 snRNA gene
transcription was also reduced in CSB immunodepleted nuclear extracts. In contrast,
transcription of RNA polymerase III-transcribed class 1 (SS rRNA) and class 2 (AdVAI)
genes was stimulated in the absence of CSB. (C) 100 and 400 ng of GST-CSB (528-1222)
were titrated into AdVAI in vitro transcription reactions performed with HeLa nuclear
extracts containing endogenous CSB or immunodepleted for CSB. GST-CSB was not
able to repress AdVAI transcription in reactions where endogenous CSB complexes had
been removed. (D) Nuclear run-on experiments were performed with nuclei from normal
human fibroblasts (NF) and CSB-defective (CSB) human fibroblast cells. Transcription
of U1 and U6 snRNA genes was reduced in CSB cells as compared to NF cells. Levels of

SS rRNA gene transcription was comparable between these two cell lines.

118

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120

p53 antagonizes CSB promoter occupancy.

As activation of p53 or loss of CSB function both cause fragility of the RNU 1,
RNU2, and SS loci, the functional interplay between p53 and CSB in U1 snRNA gene
transcription was examined because both proteins were shown to associate with U1
snRNA gene promoters. Human U6 snRNA gene promoter occupancy by these proteins
was also examined in this study because U6 and U1 snRNA genes have similar promoter
structures but U6 snRNA genes are transcribed by RNA polymerase 111 instead of RNA
polymerase II. Promoter occupancy by p53 and CSB was examined in MCF-7 and HeLa
cells in response to UV treatment, which causes activation of the endogenous p53 in
MCF-7 cells, but not in HeLa cells. As shown in figure 3-4, CSB associates with U1 and
U6 snRNA gene promoters in unstressed MCF-7 cells; however, less CSB was detected
on the U1 and U6 snRNA gene promoters in MCF-7 cells harvested 4 and 8 hours after
UV light exposure (lane 5). In contrast, p53 occupancy increased on the U1 and U6
snRNA gene promoters in MCF-7 cells after UV light treatment (lane 4). In contrast with
MCF-7 cells, U1 and U6 snRNA gene promoter occupancy by CSB was unaffected in
HeLa cells after UV light treatment. Interestingly, no change was noted in TBP promoter
occupancy in both cell types in response to UV light treatment. Thus, UV light exposure
decreases CSB occupancy on RNA polymerase II- and III- transcribed snRNA gene
promoters in a process that requires functional p53.

The connection between p53 accumulation and CSB loss on snRNA gene
promoters was examined in vitro using U6 snRNA gene promoters during p53-mediated
transcription repression. Portions of U6 in vitro transcription reaction mixtures (Figure 3-

5A) containing wild-type p53 or GST were cross-linked with formaldehyde prior to

121

Figure 3-4. UV light exposure affects p53 and CSB occupancy on snRNA gene
promoters in vivo. Chromatin from untreated or UV light treated MCF7 and HeLa cells
was immunoprecipitated with TBP antibodies (lanes 2 and 7), negative control IgG
antibodies (lanes 3 and 8), antibodies against the C-tenninus of p53 (lanes 4 and 9), and
CSB antibodies (lanes 5 and 10). Treatment of MCF7 cells with UV light caused
accumulation of p53 on the U1 and U6 snRNA gene promoters concomitant with
decreased detectable CSB association. In contrast, CSB association with these snRNA

promoters was unchanged in HeLa cells that lack functional p53.

122

 

 

 

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Figure 3-5. p53 affects CSB and RNA polymerase III occupancy on U6 snRNA gene
promoters in vitro. (A) U6 snRN A in vitro transcription reaction. 400 ng of recombinant
p53, but not GST, effectively repressed U6 gene transcription. (B) Portions of the U6
transcription reactions shown in Figure 3-SA were cross-linked with formaldehyde and
were subjected to immunoprecipitation with anti-TBP (positive control), IgG (negative
control), anti-p53, anti-CSB and anti-RNA polymerase III antibodies. U6 reporter gene
DNA was enriched in anti-CSB and anti-RNA polymerase III immunoprecipitation
reactions from untreated or GST-treated extracts, whereas p53 addition caused decreased
U6 reporter gene recovery by anti-CSB and anti-RNA polymerase III
immunoprecipitations. PCR reactions performed directly from the U6 transcription
reactions revealed similar amplification of the U6 reporter plasmid (lane 1). The specific
recoveries of the U6 promoter-containing plasmid were compared to recovery of an
irrelevant pUC119 promoter-less plasmid included in the original transcription assay

mixtures.

124

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125

Untreated

p53 treated

GST treated

GST treated

immunoprecipitations by antibodies directed against TBP, IgG, p53, CSB or RNA
polymerase 111 (Figure 3-5B). A ranscription reaction untreated with any proteins was
also tested. The specific recovery of the U6 promoter-containing plasmids were
compared to recovery of an irrelevant plasmid (pUC119) included in the transcription
assays. As shown in figure 3—SB, significant U6 reporter plasmid recovery by anti-CSB
immunoprecipitation was obtained from the untreated or GST treated transcription
reaction mixtures, but not from p53 treated reactions, suggesting that p53 may disrupt
CSB promoter association. Interestingly, association of RNA polymerase III with U6
promoters was also reduced in p53 treated reactions. In contrast to the effect of p53 on
CSB and RNA polymerase promoter occupancy, no change was noted in TBP occupancy.

Together, these data suggest that p53 antagonizes CSB promoter occupancy.

CSB modulates transcription regulatory functions of p53.

To test whether CSB is required for p53-mediated U1, SS rRNA and U6
transcription repression, increasing amounts of recombinant p53 were titrated into in vitro
transcription reactions performed using untreated HeLa nuclear extracts or extracts
immunodepleted of CSB using anti-CSB antibodies (Figure 3-6). These experiments
revealed that p53 repressed U1 snRNA gene transcription better in the absence of CSB.
In contrast, the presence or absence of CSB in transcription mixtures did not significantly
affect U6 snRNA gene transcription repression by p53. Interestingly, SS rRNA gene
transcription was activated by low amounts of p53 (5 and 10 ng) in the absence of CSB.

However, higher amounts of p53 (40, 80 and 160 ng) exhibited stronger repression of SS

126

Figure 3-6. Loss of CSB accentuates the RNA polymerase II and III transcriptional
response to p53. Approximately 0, 2.5, 5, 10, 20, 40, 80 and 160 ng of p53 were added
to U1, 5S and U6 transcription reactions that were performed in vitro using either HeLa
nuclear extracts containing CSB or HeLa nuclear extracts immunodepleted for CSB. For
comparison, the transcription levels for both types of nuclear extracts with no added p53
were set to one. In these experiments, p53 repressed U1 and U6 transcription better in the
absence of CSB. In contrast, 5S rRNA gene transcription was enhanced by low amounts

of p53 but was further repressed by higher amounts of p53 in extracts lacking CSB.

127

 

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128

snRNA (RNAP ll)

Class 1 (RNAP Ill)

Class 3 (RNAP lll)

rRNA gene transcription in the absence of CSB as compared to the extent of p53-
mediated SS rRNA gene transcription repression when CSB was present. Together, these
data suggest a modulatory role of CSB on p53 transcription repression functions on these

promoters.

CSB modulates p53-dependent modifications of RNA polymerase III.

It has been reported that RNA polymerase 11 large subunit (LS) becomes
ubiquitylated and proteolytically degraded following UV light treatment in normal but
not in CSB deficient fibroblasts. The degradation of elongation incompetent RNA
polymerase at sites of DNA damage may be important for the efficient recovery of
transcription as cells recover from stress (3). As with RNA polymerase II, CSB may also
affect RNA polymerase III stability. To understand the role of p53 - CSB functional
interplay for RNA polymerase III stability, I compared RNA polymerase HI levels in
cells with and without functional CSB or p53 under normal conditions and in response to
UV light treatment (Figure 3-7A). Western blot analysis of RNA polymerase III levels in
CSB deficient cells revealed multiple slower migrating forms of the polymerase III,
which may represent post-translational covalently modified forms of RNA polymerase.
These modified forms of RNA polymerase III were less pronounced in MCF-7 cells
containing functional CSB and were undetectable in HeLa cells. The proportion of the
slower migrating forms of RNA polymerase III to the putative unmodified RNA
P01ymerase III was the highest in CSB deficient cells (Figure 3-7B), suggesting a

negative role of CSB in formation of these forms of RNA polymerase III. Interestingly,

129

 

 

Figure 3-7. CSB modulates p53-dependent modifications of RNA polymerase III. (A)
The Cockayne syndrome complementation group B (CSB) protein-defective human
fibroblast cells (CSB), HeLa and MCF7 cells were treated with UV light and harvested at
indicated times afier UV exposure. Western blot analysis revealed slower migrating
forms of RNA polymerase III, which were more prominent in untreated CSB cells as
compared to MCF-7 cells containing functional CSB. These RNA polymerase III forms
were underrepresented in HeLa cells with defective p53. UV light triggered p53
accumulation in CSB and MCF-7 cells and concomitant reduction of modified forms of
RNA polymerase III in these cells. (B) Western blot analysis of whole cell extracts from
MCF-7 (lanes 1 through 3) and CSB (lanes 4 through 6) cells revealed the enrichment of
modified forms of RNA polymerase III in CSB deficient cells as compared to MCF-7
cells. (C) Transient transfection of wild type or acetylation-incompetent KRSA mutant
p53 expression plasmids into H1299 (p53-/-) cells causes formation of the higher
molecular weight form of RNA polymerase 111. After exposure of H1299 cells to UV
light, transient transfection of wild type p53, but not acetylation-incompetent KRSA p53,

resulted in formation of the slower migrating form of RNA polymerase III.

130

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132

 

HeLa cells with non-functional p53 had the smallest fraction of modified forms of RNA
polymerase III, suggesting a positive role of p53 in the formation of modified forms of
RNA polymerase III. Intriguingly, UV light treatment of cells resulted in reduction of
modified forms of RNA polymerase III in CSB and MCF-7 cells, suggesting that these
RNA polymerase III modifications may serve as a ‘code’ for subsequent modification in
response to stress stimuli (e.g. UV light), which may result in intermediate forms of RNA
polymerase III, that are more vulnerable for degradation.

To determine whether p53 contributes to RNA polymerase III post-translational
modifications, wild type p53 or acetylation-incompetent mutant p53 (KRSA p53) were
over-expressed in human osteosarcoma H1299 (p53-/-) cells and levels of RNA
polymerase III were evaluated by Western blot analysis (Figure 3-7C). The KRSA p53
was chosen because in contrast to the wild type p53, p53 with mutations that prevent its
acetylation was not being able to cause fragile sites induction on U1 and U2 snRNA and
SS rRNA gene loci (37), suggesting that p53 acetylation status may be an important
determinant for p53 in this process. Upon transient transfection of wild type or KRSA
mutant p53 expression plasmids in H1299 cells a slower migrating form of RNA
polymerase III was observed. This form of RNA polymerase III was not present in cells
transfected with a negative control empty vector. Interestingly, in UV light treated cells
over-expression of wild-type p53, but not acetylation-incompetent KRSA p53, resulted in
RNA polymerase III modification, which suggests that the acetylation of p53 may be

important for RNA polymerase III modifications in UV light treated cells.

133

Discussion

A role of CSB in U1 snRNA gene transcription by RNA polymerase II and SS
rRNA gene transcription by RNA polymerase III was suggested based on observation
that loss of functional CSB in cells causes metaphase fragility of these genes (37). The
same study revealed that fragility in the same genes can be induced by over-expression of
p53 (24, 37). The data presented herein directly demonstrate a role of CSB in regulation
of RNA polymerase II- and III transcription. In contrast to the positive role of CSB in
RNA polymerase II-transcribed snRNA genes, CSB has a negative role in RNA
polymerase III-transcribed class 1 and 2 genes. In contrast to other RNA polymerase III-
transcribed genes, class 3 (U6 snRNA) was not repressed by CSB, but was modestly
activated by CSB similarly to RNA polymerase II-transcribed snRNA genes. Class 3
RNA polymerase III promoters are structurally similar to RNA polymerase II-transcribed
snRNA gene promoters (18). Furthermore, class 3 RNA polymerase 111 genes share some
of the general transcription factors with snRNA genes transcribed by RNA polymerase II
(18). Thus, CSB may function as a transcriptional activator or repressor depending on
promoter architecture and transcription machinery.

A positive role of CSB in RNA polymerase II transcription is suggested from the
observation that CSB can modestly stimulate the rate of transcription by RNA

polymerase II and thus, may function as an elongation factor (30). The ability of CSB to

remodel nucleosomes may also contribute to its functions as a transcriptional activator (8).

An interesting feature of the CSB protein is its ability to bind and wrap DNA (1). This
could affect the distance between regulatory and core promoter elements and either

activate or repress gene transcription. Indeed, snRNA gene transcription activation

134

 

requires the cooperative binding of Oct-1 and SNAPC (13, 38). This cooperativity is
mediated by a positioned nucleosome that resides between the DSE and PSE and brings
these two snRNA gene promoter elements in proximity (3 8). The ability of CSB to bind
and wrap DNA could also modulate distance between DSE and PSE, thus mimicking the
effect of positioned nucleosome on snRNA genes. Also, a negative role of CSB in RNA
polymerase III transcription may be the result of CSB—mediated degradation of RNA
polymerase III. It has been reported that RNA polymerase 11 large subunit (LS) becomes
ubiquitylated and proteolytically degraded following UV light treatment in normal but
not in CSB deficient fibroblasts, suggesting that CSB play role in RNA polymerase II
degradation. Following UV-irradiation, the degradation of elongation incompetent RNA
polymerase at sites of DNA damage may be important for the efficient recovery of
transcription as cells recover from stress (3). As with RNA polymerase II, CSB may also
affect RNA polymerase III stability. When levels of RNA polymerase III were analyzed
in different human cell with different status of CSB and p53 proteins, we observed that
CSB deficient cells have lower levels of unmodified RNA polymerase (Figure 3-7B). We
also detected slower migrating and likely covalently modified forms of RNA polymerase
III in cells (Figure 3-7A). Interestingly, a larger portion and variety of these modified
RNA polymerase 111 forms was observed in CSB defective fibroblasts (Figure 3-7A and
3-7B), suggesting that presence of functional CSB in HeLa and MCF-7 cells interferes
with the accumulation of modified RNA polymerase III in cells. We speculate that these
modified RNA polymerase 111 forms may represent elongation incompetent forms of

RNA polymerase III. The absence of CSB-triggered release or degradation of elongation

13S

incompetent RNA polymerase 111 may result in the accumulation of these forms of RNA
polymerase in CSB deficient cells.

Based on the present study I suggest a model for the roles of p53 and CSB in
RNA polymerase III modifications and degradation (Figure 3-8). In this model I
hypothesize that slower migrating forms of RNA polymerase III represent post-
translationally modified elongation incompetent forms of RNA polymerase III. p53 acts
as a general repressor of RNA polymerase III transcription by affecting RNA polymerase
III transcription elongation or re-initiation, because accumulation of elongation
incompetent forms of RNA polymerase III directly correlates with presence of p53 in
cells. In contrast to p53, the presence of functional CSB in cells antagonizes
accumulation of elongation incompetent forms of RNA polymerase III, suggesting that
CSB either helps RNA polymerase III to resume elongation or plays a role in removing
elongation incompetent RNA polymerase from the gene, so it can undergo degradation.
Cellular stress (e.g. UV light) triggers activation of multiple cellular signaling cascades,
which may cause additional modifications of RNA polymerase III, affecting RNA
polymerase III gene association and stability. In the future it will be important to
determine what types of RNA polymerase III post-translational modifications exist and
are induced by p53 in cells.

In summary, 1 found that in addition to the role of CSB in RNA polymerase I and
II transcription, CSB is also involved in RNA polymerase III transcription. Interestingly,
CSB can function both as activator or repressor of RNA polymerase III-transcribed genes
depending on promoter architecture. I also discovered a novel role of CSB as a modulator

of p53 fimctions. The functional antagonism between p53 and CSB most likely occurs at

136

Figure 3-8. Model: Roles of p53 and CSB in RNA polymerase III elongation and
stability. p53 plays a role in RNA polymerase III modifications, which associates with
elongation incompetent RNA polymerase. In contrast, CSB helps RNA polymerase IH to
resume elongation. In response to UV light exposure elongation incompetent RNA
polymerase 111 could be a substrate for additional modifications triggered by UV light
activated signaling cascades. This results in dissociation of RNA polymerase III from

gene and its subsequent degradation.

137

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the level of RNA polymerase III stability. As p53 triggers RNA polymerase to an
elongation incompetent mode, CSB works antagonistically by helping RNA polymerase
III to resume elongation or targeting the elongation incompetent RNA polymerase III for
destruction.

Cockayne syndrome (CS) is a rare autosomal disease that caused by mutations in
CSB. Interestingly, mutations in CSB cause at least four different diseases: Cockayne
syndrome (CS), UV-sensitive syndrome (UVSS), DeSanctis-Cacchione syndrome (DS-C)
and cerebro—oculo-facio-skeletal syndrome (COFS) (25, 31), suggesting that CSB may be
involved in different cellular functions possibly via its general role in transcription by all
three RNA polymerases. The CSB role in transcription of non-coding RNAs by both
RNA polymerase II and III is intriguing. Non-coding RNAs have been shown to be
involved in controlling a variety of cellular functions via their role in transcription and
RNA metabolisms (l 1, 12, 22, 26, 36) and misregulations of some non-coding RNAs
were recently linked to different human diseases such as human hereditary cartilage-hair
hyp0plasia disease (28) and cancer (21, 32). Multiple transcription targets for CSB helps

to explain multiple phenotypes of CS patients.

139

 

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143

CHAPTER FOUR

SUMMARY

Non-translated snRNAs perform essential cellular functions by controlling RNA
synthesis and RNA metabolism (8). The involvement of snRNAs in controlling diverse
cellular functions suggests that these genes should be tightly regulated under normal
conditions and in response to cellular stress. Indeed, misregulation of some snRNAs have
been recently linked to several human diseases, such as cartilage-hair hypoplasia (CHH)
disease (15) and cancer (13, 17). In addition, both tumor suppressor proteins (e. g. p53 (3,
4) and RB (6)) and oncoproteins (e. g. Myc (6) and CKII (7,10, 11)) have been linked to
snRNA gene transcription, suggesting that regulated snRNA production plays a role in
controlling cellular homeostasis.

Depending on the promoter architecture, snRNA genes are transcribed by either
RNA polymerase II or by RNA polymerase III (8). Regardless of RNA polymerase
specificity, snRNA genes have similar promoter structure and share some of the general
transcription factors (reviewed in 8), suggesting that these genes may be similarly

regulated in response to cellular stress stimuli.

The overall goal of my research project was to understand the role of the tumor
suppressor protein p53 in human snRNA gene transcription. The tumor suppressor
protein p53 is a multifunctional transcription factor known to control cellular
proliferation and genomic stability by activating or repressing transcription of target

genes by all three classes of RNA polymerases (3-5, 16, 21). It was previously

144

demonstrated that p53 represses snRNA gene transcription by RNA polymerase III (3, 4);
however, the importance of p53 for transcription of RNA polymerase II-transcribed
snRNA genes was not investigated.

My studies revealed that in addition to the role of p53 in snRNA gene
transcription by RNA polymerase III, p53 also represses snRNA gene transcription by
RNA polymerase 11. Thus, p53 is a general repressor of snRNA gene transcription by
both polymerases. I found that p53 associates with human U1 and U6 snRNA genes
during transcription repression and represses endogenous snRNA gene transcription upon
transient transfection or in response to UV-light, suggesting a role of p53 in snRNA gene

transcription regulation in response to DNA damage.

It has been shown that p53 can repress transcriptiOn of its target genes by a
variety of mechanisms that regulate pre—initiation complex formation, elongation by RNA
polymerase and at the level of chromatin (reviewed in 9). It was proposed that p53-
mediated repression of RNA polymerase III transcription occurs via TBP-mediated p53
interaction with TFIIIB, which interferes with TFIIIB binding and subsequent
recruitment of RNA polymerase III to RNA polymerase III promoters (3, 5). In contrast,
similar levels of TBP at snRNA gene promoters before and during p53-mediated
transcription repression were observed, suggesting that p53 interferes with snRNA gene
transcription at steps occurring afier TBP or TFIIIB recruitment for RNA polymerase II-
and III-transcribed genes respectively. Indeed, RNA polymerase III was not detected at
U6 snRNA gene promoters during repression by p53, suggesting that p53 interferes with
RNA polymerase III recruitment to snRNA gene promoters. Whether p53 also affects the

recruitment of RNA polymerase II to snRN A gene promoters remains to be investigated.

145

 

Interestingly, upon p53 accumulation on snRNA gene promoters the concomitant
enrichment of HDACs was observed, suggesting that p53 repression of snRNA gene
transcription may in part occur by recruitment of the chromatin remodeling activities of
HDACs. The HDAC-mediated p53 transcription repression has been described for some
genes (12, 14) and the role of HDACs in deacetylation of core histones as well as p53
itself was suggested (12). What are the determinants for p53 decision to recruit HDAC
for transcription repression? Recently, SUMO modification of several transcription
factors has been linked to HDACs recruitment and transcription repression (19).
Interestingly, I observed accumulation of sumoylated forms of p53 in cells after UV light
treatment. When the proportion of sumoylated protein population was analyzed on
snRNA gene promoters, the direct correlation between levels of total p53, sumoylated
proteins and accumulation of HDACs on snRNA gene promoters was detected,
suggesting that promoter-bound sumoylated p53 may recruit HDACs during transcription
repression of snRNA genes.

As part of our investigation of the mechanism of p53-mediated snRNA gene
transcription repression, I tested a hypothesis that p53 represses RNA polymerase II-
transcribed snRNA genes by interfering with CSB-mediated elongation causing stalling
of RNA polymerase II (20). Our studies reveal a direct role of CSB not only in snRNA
gene transcription by RNA polymerase II, but also in transcription by RNA polymerase
III, including class 3 RNA polymerase III-transcribed snRNA genes. Thus, CSB may be
targeted by p53 during snRNA gene transcription repression by both polymerases. Indeed,
our data suggests that CSB modulates p53 transcription repression functions and, in the

absence of CSB, p53 exhibits stronger repression of U1 snRNA and SS rRNA gene

146

transcription. In addition to the role of CSB as an elongation factor for RNA polymerase
II-transcribed genes, CSB has also been reported to play a role in ubiquitylation and
degradation of RNA polymerase 11 large subunit (LS) following UV light treatment (2);
however, whether CSB also plays a role in RNA polymerase III stability was uncertain.
Degradation of elongation incompetent forms of RNA polymerases at sites of DNA
damage may be important for the efficient recovery of transcription as cells recover from
stress. Interestingly, when we compared RNA polymerase III levels in human cells with
different status of CSB, we observed that levels of RNA polymerase III are lower in CSB
deficient cells, suggesting that similarly to RNA polymerase II, CSB may also affect
RNA polymerase III stability. However, we found that CSB defective fibroblasts have a
larger portion of slower migrating and possibly covalently modified forms of RNA
polymerase 111. As CSB was described as an elongation factor for RNA polymerase II,
we speculate that these modified forms of RNA polymerase III in CSB deficient
fibroblasts represent elongation incompetent forms of RNA polymerase III. Interestingly,
as p53 accumulation in cells correlates with RNA polymerase III transcription repression,
it also results in enrichment of slower migrating forms of RNA polymerase III in cells.
Thus, I hypothesized that p53 represses elongation by RNA polymerase through its post-
translational modifications. At this point it is not clear what kinds of covalent
modifications of RNA polymerase III exist in cells and which modifications serve as
‘markers’ for elongation incompetent RNA polymerase 111. As of now, these
modifications may represent either phosphorylated, ubiquitylated, sumoylated or
neddylated forms of RNA polymerase 111. So far, only ubiquitin-modification of proteins

are considered to be a marker for protein degradation by the 26S proteasome (18).

147

Though both mono- and poly—ubiquitylation of proteins were described, poly-
ubiquitylated proteins are more likely be targeted to proteasomal degradation (18). The
function for protein mono-ubiquitylation has not been yet well characterized. It was
proposed that mono-ubiquitylation is a prerequisite for poly-ubiquitylation and several
ubiquitin-ligases are only capable to mono-ubiquitylate their substrates (1). We speculate
that p53 plays a role in mono-ubiquitylation of RNA polymerase III, which results in
elongation incompetent forms of RNA polymerase III. What is the mechanism for p53-
mediated RNA polymerase III modification? We do not yet have an answer to this
question. It is possible that p53 itself serves as an E3 ubiquitin ligase for RNA
polymerase III modification; however, to-date neither p53 interactions with polymerases
nor p53 activity as an E3 ubiquitin ligase were reported. An other possibility is that p53
may recruit ubiquitin li gases (e. g. Mdm2 and/or others) during transcription repression.
Together, our data suggests that p53-mediated snRNA gene transcription
repression is complex multilevel process, which may be important to assure snRNA gene

repression by p53 in response to cellular stress.

148

References

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Alarcon-Vargas, D., and Z. Ronai. 2002. p5 3-Mdm2--the affair that never ends.
Carcinogenesis 23:541-7.

Bregman, D. B., R. Halaban, A. J. van Gool, K. A. Henning, E. C. Friedberg,
and S. L. Warren. 1996. UV-induced ubiquitination of RNA polymerase II: a

novel modification deficient in Cockayne syndrome cells. Proc Natl Acad Sci U S
A 93: 1 1586-90.

Cairns, C. A., and R. J. White. 1998. p53 is a general repressor of RNA
polymerase III transcription. EmboJ 17:3112-23.

Chesnokov, I., W. M. Chu, M. R. Botchan, and C. W. Schmid. 1996. p53
inhibits RNA polymerase III-directed transcription in a promoter-dependent
manner. Mol Cell Biol 16:7084-8.

Crighton, 1)., A. Woiwode, c. Zhang, N. Mandavia, .r. P. Morton, L. J.
Warnock, J. Milner, R. J. White, and D. L. Johnson. 2003. p53 represses RNA

polymerase III transcription by targeting TBP and inhibiting promoter occupancy
by TFIIIB. Embo J 22:2810-20.

Felton-Edkins, Z. A., N. S. Kenneth, T. R. Brown, N. L. Daly, N. Gomez-
Roman, C. Grandori, R. N. Eisenman, and R. J. White. 2003. Direct

regulation of RNA polymerase III transcription by RB, p53 and c-Myc. Cell
Cycle 2:181-4.

Gu, L., W. J. Esselman, and R. W. Henry. 2005. Cooperation between small
nuclear RNA-activating protein complex (SNAPC) and TATA-box-binding
protein antagonizes protein kinase CK2 inhibition of DNA binding by SNAPC. J
Biol Chem 280:27697-704.

Hernandez, N. 1992. Transcription of vertebrate snRNA genes and related genes,
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He, J., and S. Benchimol. 2003. Transcriptional repression mediated by the p53
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Hu, P., K. Samudre, S. Wu, Y. Sun, and N. Hernandez. 2004. CK2

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Hu, P., S. Wu, and N. Hernandez. 2003. A minimal RNA polymerase III
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Juan, L. J., W. J. Shia, M. H. Chen, W. M. Yang, E. Seto, Y. S. Lin, and C.
W. Wu. 2000. Histone deacetylases specifically down-regulate p53-dependent
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Krawczak, M., J. Reiss, and D. N. Cooper. 1992. The mutational spectrum of
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Murphy, M., J. Ahn, K. K. Walker, W. H. Hoffman, R. M. Evans, A. J.
Levine, and D. L. George. 1999. Transcriptional repression by wild-type p53

utilizes histone deacetylases, mediated by interaction with mSin3a. Genes Dev
13:2490-501.

Ridanpaa, M., H. van Eenennaam, K. Pelin, R. Chadwick, C. Johnson, B.
Yuan, W. vanVenrooij, G. Pruijn, R. Salmela, S. Rockas, O. Makitie, I.
Kaitila, and A. de la Chapelle. 2001. Mutations in the RNA component of
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Stein, T., D. Crighton, L. J. Warnock, J. Milner, and R. J. White. 2002.
Several regions of p53 are involved in repression of RNA polymerase III
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Susani, L., A. Pangrazio, C. Sobacchi, A. Taranta, G. Mortier, R.
Savarirayan, A. Villa, P. Orchard, 1’. Vezzoni, A. Albertini, A. Frattini, and
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Yang, S. H., and A. D. Sharrocks. 2004. SUMO promotes HDAC-mediated
transcriptional repression. Mol Cell 13:611-7.

Yu, A., H. Y. Fan, D. Liao, A. D. Bailey, and A. M. Weiner. 2000. Activation
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150

APPENDIX

POSSIBLE ROLE OF HDACs IN P53-MEDIATED snRNA GENE

TRANSCRIPTION REPRESSION

p53 has been shown to repress transcription of its target genes by mechanisms
that regulate pre-initiation complex formation and elongation by RNA polymerase. In
addition, transcriptional repression by p53 can be also achieved through p53-mediated

alteration of chromatin (reviewed in 6).

The ability of p53 to repress transcription at the level of chromatin was linked to
recruitment of histone deacetylases (HDACs) to target genes (7, 8). p53 was shown to
associate with HDACs via direct interaction of p53 with Sin3a protein of the Sin3/HDAC
co-repressor complex (8). Recruitment of HDACs by p53 is believed to result in p53-
mediated repression by at least two different mechanisms: core histone deacetylation and
p53 deacetylation (7). HDAC-mediated histone deacetylation results in chromatin
condensation and reduced promoter accessibility to the transcriptional machinery and/or
co-activators (l 1). Consistent with the positive role of p53 acetylation for transactivation
activity of p53 (1), p53 deacetylation by HDACs results in p53-mediated transrepression

of target gene transcription (7, 8).

To test whether HDACs are involved in snRNA gene transcription repression,
snRNA gene promoter occupancy by HDACs was analyzed using immunoprecipitation
experiments (Figure A-l). As shown in figure A-lA, p53 and HDACl and 2 occupancy

on the endogenous U1 snRNA gene promoter was concomitantly increased in response to

151

Figure A-l: Endogenous HDAC] and HDAC2 associate with human snRNA gene
promoters. (A) Chromatin from untreated or UV light treated MCF—7 cells was
immunoprecipitated with SNAP43 antibodies, antibodies against total p53 (21-25) or
K320 acetylated p53, and HDACl, HDAC2 or negative control IgG antibodies (lanes 3
through 8, respectively). Lanes 1 and 2 represent input titration. p53, I-IDACl and
HDAC2 occupancy on U1 snRNA promoter (but not on negative control U1 upstream
region) was increased after UV light treatment. No change in U1 snRNA promoter
occupancy by acetylated p53 was observed after UV light exposure. (B) A portion of
untreated or p53 or GST treated U6 in vitro transcription reaction mixtures was cross-
linked with formaldehyde and subjected to immunoprecipitation with anti-TBP, IgG,
anti-p53, anti-RNA polymerase III, anti-HDACI and anti-HDAC2 antibodies (lanes 2
through 7, respectively). Enrichment of the U6 reporter plasmids or negative control
pUC119 DNA was compared by PCR using promoter-specific or pUC-specific primers.

Lane 1 represents 10% input DNA.

152

rrRVA gene
7 cells was
(2135,) or
lies (lanesi
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3 8
g e m 9'. < o o
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~ F‘- -UV
- -—~ +UV
1 2 3 4 5 6 7 8
Chromatin IP
6 v- N
n. O O
O. m < < <
a 'T <5 ‘.1 ‘F 3.3 3.:
E 5 2’ 5 5 5 5
m - . ""
U6 promoter _ ........ ..... _. I... ._.....
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pUC119 —
1 2 3 4 5 6 7

Cross-linking IP

153

U1 Upstream
Control

Untreated

p53 treated
GST treated

GST treated

UV light treatment, suggesting that HDACs are involved in snRNA gene transcription in
response to UV light treatment and that p53 may recruit HDACs to U1 snRNA gene
promoters during transcription repression. Specific enrichment of HDACl and 2 was also
observed on exogenous U6 snRNA gene promoters during p53-mediated repression using
in vitro transcription assays (Figure A-lB, lanes 6 and 7 of the p53 treated reactions);
however, no recovery of U6 snRNA gene promoters was observed in anti-HDAC] and 2
immunoprecipitated samples from untreated or GST treated transcription reactions.
Together, these data suggest that p53 may recruit HDAC 1 and 2 during snRNA gene

transcription repression.

What are the determinants for p53-mediated recruitment of HDACs during
transcription repression? Recently, it has been reported that sumoylation of proteins may
promote HDAC recruitment and transcription repression (12). It has been shown that p53
can be sumoylated at Lys3 86 (9) and both positive and negative roles of sumoylation for
p53-mediated transactivation of target genes were described (2, 9, 10). Thus, we asked
whether p53 sumoylation mediates HDACs recruitment and transcription repression by
p53. Interestingly, as UV light exposure results in accumulation of total p53 as well as
sumo-modified forms of p53 (Figure A-2A), the concomitant enrichment of p53 and
sumoylated protein population was observed on the endogenous U1 snRNA gene
promoters in response to UV light treatment in chromatin immunoprecipitation
experiments (Figure A-2B). Since there are no commercially available antibodies against
sumoylated p53, we can only speculate that U1 snRNA gene promoter occupancy by
sumoylated p53 was also enriched in response to UV light exposure and contribute to the

signal from the total sumoylated protein population recognized by anti-Sumo-l

154

 

Figure A-2: Sumoylated protein population is enriched on U1 snRNA gene
promoters after UV exposure. (A) UV light exposure of MCF-7 cells results in
accumulation of total p53 as well as sumo-modified forms of p53 as measured by
Western blotting. Whole cell extracts from untransfected and pCDNA-HA-Sumo-l, -2,
and -3 cotransfected MCF-7 cell harvested prior and 8 hours after UV light treatment
were analyzed by 12.5% SDS-PAGE and Western blot analysis of HA-tagged
sumoylated proteins (lanes 1 and 2) and p53 (lanes 3 through 6). Co-migrating bands that
were detected by both anti-HA and anti-p53 antibodies were labeled as SUMO-p53. (B)
Chromatin from untreated or UV light treated MCF-7 cells was immunoprecipitated with
antibodies against total and K320 acetylated p53, antibodies against Sumo-1 or negative
control IgG antibodies (lanes 3 through 6, respectively). Lanes 1 and 2 represent input
titration. p53 and sumoylated proteins were concomitantly enriched at endogenous U1
snRNA gene promoters after UV light treatment. No change in U1 snRNA promoter

occupancy by acetylated p53 was observed after UV light exposure.

155

SDRVA gene
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ting bands Lit
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idogenous 11

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- + - + - +'Sumo-HA

 

 

 

 

 

 

 

 

 

 

 

190..
120-
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H -UV
....... .. .. .. .. +UV]U1 SnRNA
L: -UV]U1 Upstream
+UV Control
1

Chromatin IP

156

 

antibodies (lane 5). Thus, we observed direct correlation between levels of p53,
sumoylated proteins and HDACl and 2 on U1 snRNA promoters after UV light treatment
(Figures A-1 and A-2B), suggesting that sumo-modified p53 may recruit HDACs for

repression of snRNA gene transcription.

I further tested whether Sumo-modification of p53 is required for p53-mediated
snRNA gene transcription repression by comparing the ability of wild type GST-p53 and
GST-p53 (K386R) to repress U1 and U6 snRNA gene transcription (Figure A-3). Both
GST-p53 and GST-p53 (K386R) were expressed in E. coli and supposedly lack any post-
translational modifications. However, during in vitro transcription assays these proteins
could undergo covalent modifications by enzymes present in the HeLa nuclear extract.
Thus, during in vitro transcription wild type GST-p53 could become sumoylated; in
contrast, GST-p53 (K386R) should lack the sumo-modification. These experiments
revealed that sumoylation-deficient mutant GST-p53 (K3 86R) repressed both U1 snRNA
gene transcription by RNA polymerase II and U6 snRNA gene transcription by RNA
polymerase III approximately two fold less efficiently than wild type GST-p53,
suggesting that sumoylation of p53 may contribute to p53-mediated snRNA gene
transcription repression. A possible explanation of the modest effect of p53 K386R
mutation on snRNA gene transcription repression is that in vitro transcription assays were
performed using naked DNA, but not a chromatinized template. If p53 sumoylation is
indeed important for HDAC recruitment, HDAC-mediated repression of snRNA genes
may require a chromatin context. Indeed, we did not observe any effect of the HDAC
inhibitor sodium butyratc on p53-mediated snRNA gene transcription repression using

non-chromatinized DNA templates (data not shown). In addition, the proportion of wild

157

Figure A-3: p53 sumoylation contributes to p53-mediated snRNA gene transcription
repression in vitro. Recombinant wild type GST-p53 (lanes 2 through 4) and GST-p53
(K386R) (lanes 6 through 8) were titrated into U1, U6 and AdML in vitro transcription
reactions. 0.5, 1 and 2 pg of proteins were used in U1 and AdML in vitro transcription
assays and 0.15, 0.3 and 0.6 pg of proteins were added into U6 in vitro transcription
mixtures. Heat-inactivated GST-p53, GST-p53 (K386R) and GST were also tested in
these transcription reactions (lanes 5, 9 and 10, respectively). Lane 1 shows level of

transcription when no proteins were added.

158

 

   

 

 

 

 

 

 

 

 

iptiorr

I'll-’13 +GST-p53 +GST-p53 (K386R)

I I ..

“Latina I W HI ('7)

‘ ‘ 1°

_ +

intinn .

‘ urn- ~-- - . -~- - u..- —-- Orr- ~ ~- ---o *- _U1 5
~{3.1011 u..— .— “ .... . «n.- 1“ ——- «— r *’ —'. w — U6 5I
tedir: m-—-——_.~— —AdM|_
V61 01 1 2 3 4 5 6 7 8 9 10

In vitro transcription

159

 

Figure A-4: Sumoylated p53 is enriched in acetylated population of p53 in vivo.
Whole cells extracts from UV light treated MCF-7 cells expressing HA-Sumo-l, -2 and —
3 proteins were immunoprecipitated with either full length p53 antibodies (lane 4),
antibodies against p53 acetylated at K320 (lane 5) or negative control IgG antibodies
(lane 3). Presence of p53 and Sumo-p53 was detected by immunoblotting with another

p53 (Ab-6) (lefi panel) and anti-HA (right panel) antibodies.

160

p53 in 1111.
10-1, -2 and-
lies (lane 4)
;G antibodies

with another

or-p53 Westem

161

 

oe-HA Westem

«Sumo-p53

type p53 that gets sumoylated during in vitro transcription is low and majority of the wild
type GST-p53 was not sumoylated, but may repress transcription via sumoylation-
HDAC-independent mechanism. Thus, it would be useful to know what proportion of the

wild type p53 (if any) gets sumoylated during in vitro transcription assays.

Interestingly, I observed that sumoylated forms of p53 are enriched in acetylated
p53 population, suggesting that acetylation may be a prerequisite for a subsequent
sumoylation of p53. As shown in figure A-4, despite the fact that more p53 was
immunoprecipitated with antibodies against total cellular p53 (lane 4, left panel) as
compared to acetylated p53 (lane 5, left panel), levels of sumoylated p53 in these

immunoprecipitated materials were comparable.

Materials and Methods

Cell culture and UV irradiation

Human MCF-7 cells were maintained in Dulbecco’s modified Eagle’s medium
supplemented with penicillin-streptomycin and 10% fetal bovine serum. Cells grown to
70 to 80% confluence were washed with phosphate-buffered saline and irradiated with 50
J of UV light (254-nm peak)/m2 by using a UV Stratalinker (Stratagene). After irradiation,
growth medium was added and cells were incubated at 37°C under 5% C02 for the
indicated times. Additionally, MCF-7 cells were grown to 50% confluence in ISO-mm
plates and were then transiently co-transfected with 5 pg of pCDNA3-HA-Sumo-l,
pCDNA3-HA-Sumo-2 and pCDNA3-HA-Sumo-3 plasmids (gift from Dr. Kwok,
University of Michigan) using 3 pl Lipofectin reagent (Invitrogen) per transfection.

Transfections were performed for 6 h. Subsequently, the medium was replaced and cells

162

were incubated for 48 h for further analysis.

Expression and purification of recombinant proteins

Glutathione S—transferase (GST), GST-tagged. full-length human p53 and GST
were expressed in Escherichia coli BL21 (DE3) codon+ cells (Stratagene) and were
affinity purified by binding to glutathione agarose beads (Sigma). GST-p53 and GST
proteins were then eluted from beads in HEMGT-ISO buffer containing 50 mM
glutathione for 4 h at 4°C and concentrated by centrifugation using a Centricon YM-3O
spin column (Millipore) in HEMGT-80 buffer (20 mM HEPES [pH 7.9], 0.1 mM EDTA,
10 mM MgC12, 10% glycerol [vol/vol], 0.1% Tween 20, 80 mM KCl) containing

protease inhibitors and 1 mM dithiothreitol.

In vitro transcription assays

In vitro transcription assays were performed as described previously (4) using 18,
2, and 10 pL of HeLa cell nuclear extract for the U1 snRNA, U6 snRNA, and adenovirus
major late promoter (AdML) transcription reactions, respectively. The pUl-4.0 (1 pg),
pU6/Hae/RA.2 (250 ng), and M13-AdML (250 ng) templates were used for the U1
snRNA, U6 snRNA, and AdML transcription reactions, respectively. Purified proteins
were added in the amounts indicated in the figure legends. Transcription was performed
for l h at 30°C. Transcripts were separated by denaturing 6% polyacrylamide gel

electrophoresis and visualized by autoradiography.

163

Chromatin immunoprecipitation assay

Chromatin immunoprecipitation, assays were performed as described previously
(5). Human MCF-7 cells were grown to 60 to 80% confluence and were then cross-linked
with 1% formaldehyde for 30 min at room temperature. After cell lysis and sonication,
immunoprecipitation reactions were performed overnight at 4°C using chromatin from
approximately 107 cells per reaction mixture and 1 pg of each antibody: anti-SNAP43
(CS48), anti-p53 (21-25) (Ab-6; Oncogene), anti-acetyl-p53 (320) (Upstate), anti-
HDACl (Santa Cruz), anti-HDAC2 (Santa Cruz), or pre-immune IgG (Sigma) antibodies.
Anti-Sumo-l antibodies were purchased from Zymed. Recovered chromatin was
suspended in 50 pL of H20, and PCR analysis was performed using 5 pL of
immunoprecipitated chromatin or input chromatin. The primers used for amplification of
each gene were described previously (3). PCR products were separated by 2% agarose

electrophoresis in Tris-borate-EDTA buffer and were stained with ethidium bromide.

Immunoprecipitations from in vitro transcription reactions

In vitro transcription assay mixtures containing U1 or U6 promoter plasmids and
equal molar amounts of pUC119 were performed as described previously (5) in the
absence or presence of full-length wild type p53 or GST. 5 pL of each transcription
reaction mixture was diluted to 500 pL and was cross-linked in 1% formaldehyde for 10
min at room temperature, quenched with 125 mM glycine for 10 min at room temperature,
and immunoprecipitated with anti-TBP (SL2), pre-immune IgG (Sigma), anti-p53 (Ab-6;
Oncogene), anti-RNA polymerase III (TB2) and anti-HDACI (Santa Cruz) or anti-

HDAC2 (Santa Cruz) antibodies. Recovered plasmid DNA was analyzed by PCR using

164

primers specific to the U1 and U6 promoter regions or to pUC119 as a negative control.

Immunoprecipitation experiments

Whole cell extracts from pCDNA-HA-Sumo-l, -2, and -3 cotransfected MCF—7
cell harvested 8 hours after UV light treatment were diluted to 1 mL using HEMGT-ISO
buffer containing protease inhibitors and incubated with 1 pg of antibodies directed
against goat IgG (Sigma), full length p53 (Upstate) or anti-acetyl-p53 (320) (Upstate) 90
minutes at room temperature. Stable complexes were then affinity purified by incubation
with Protein-G Fast Flow sepharose beads (Upstate Biotechnology) for 90 minutes at
room temperature. Beads were washed in HEMGT-l 50 buffer and boiled for 5 minutes in
Laemmli Buffer. Bound proteins were separated by 12.5% SDS-PAGE and transferred to
nitrocellulose. Western blot analyses of recovered proteins were performed using anti-

p53 (21-25) (Ab-6; Oncogene) and anti-HA antibodies.

165

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Ho, J., and S. Benchimol. 2003. Transcriptional repression mediated by the p53
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Juan, L. J., W. J. Shia, M. H. Chen, W. M. Yang, E. Seto, Y. S. Lin, and C.
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Schmidt, D., and S. Muller. 2002. Members of the PIAS family act as SUMO
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167

   

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