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6/01 c:/CIRCJDaIeDue.p65-p.15

REGULATION OF U6 SMALL NUCLEAR RNA TRANSCRIPTION BY THE
RETINOBLASTOMA TUMOR SUPPRESSOR PROTEIN

By

Heather Anne Hirsch

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

2003

ABSTRACT

REGULATION OF U6 SMALL NUCLEAR RNA TRANSCRIPTION BY THE
RETINOBLASTOMA TUMOR SUPPRESSOR PROTEIN

By
Heather Anne Hirsch
The Retinoblastoma tumor suppressor protein (RB) participates in many cellular
functions including cell cycle progression, apoptosis, differentiation, and growth control.
Key to the ability to participate in each of these processes is RB’s ability to regulate gene
expression. Postulated repression mechanisms suggest that RB directly blocks pre-
initiation complex assembly or recruits additional co-factors such as histone deacetylases
or ATP-dependent chromatin remodeling machines whose activities impair RNA
polymerase II access to promoters. RB also represses transcription of non-translated
genes that are transcribed by RNA polymerases I and III, potentially to control cell
growth. To further understand how RB regulates gene expression, we examined RB
repression of two classes of RNA polymerase III transcribed genes, the Adenovirus 2
VAI gene (similar to tRNA genes in promoter architecture and factor requirements) and

the U6 snRNA gene.

Herein we demonstrate that RB associates with the endogenous U6 snRNA promoter in
viva, an important step in the repression of a target gene. RB did not however, associate
with RNA polymerase II transcribed snRNA genes (U1 and U2) or repress transcription
of the U1 snRNA gene. We also demonstrate that the general transcription factors snRNA
activating protein complex (SNAPc) and TFIIIB are important for RB repression of

human U6 snRNA gene transcription by RNA polymerase III. RB interacts with these

basal factors that are required for RNA polymerase III transcription providing a potential
mechanism for RB recruitment to target genes. Together, SNAPc and TFIIIB act
cooperatively to recruit RB to a U6 snRNA promoter in vitro and TFIIIB acts as a
selectivity factor specifically recruiting RB to RNA polymerase III transcribed snRNA
genes. Additionally, we show that RB co-occupies the U6 promoter with RNA
polymerase III in vivo and RB repression in vitro does not preclude RNA polymerase III
recruitment. These results suggest a novel mechanism wherein RB represses transcription

at steps subsequent to RNA polymerase recruitment to gene promoters.

AKNOWLEDGMENTS

I would like to thank Dr. R. William Henry for his mentorship and guidance. I wish to
thank my thesis guidance committee: Dr. David Arnosti, Dr. Susan Conrad, Dr. Ronald
Patterson, and Dr. Steven Triezenberg. I would also like to thank members of the Henry
laboratory past and present, especially Dr. Craig S. Hinkley and Gauri Jawdekar. I would
also like to thank the graduate students from various programs for hours of scientific
discussion, numerous reagents, and emotional support especially Kirsten F ertuck,
Fransisco Herrera, Pete Davidson, Rich Grey, Saren Ottosen, Meghana Kulkami, and
Paolo Struffi. I also thank my family for years of undying love and support. Finally, I
would also like to thank my husband, Jim Mann, for being my unyielding pillar of

support and center of my sanity.

iv

TABLE OF CONTENTS

LIST OF FIGURES .............................................................................. viii
KEY TO SYMBOLS AND ABBREVIATIONS ............................................. x
CHAPTER 1:
Introduction .......................................................................................... 1
CHAPTER 2:

The retinoblastoma tumor suppressor protein targets distinct general transcription
factors to regulate RNA polymerase [11 gene expression

Abstract ................................................................................ 40
Introduction ........................................................................... 41
Materials and Methods ............................................................... 46
Results ................................................................................. 50
Discussion ............................................................................. 73
References ............................................................................. 82
CHAPTER 3:
Retinoblastoma protein and RNA polymerase III concurrently occupy a repressed human
U6 promoter
Abstract ................................................................................ 89
Introduction ........................................................................... 90
Materials and Methods ............................................................... 93
Results ................................................................................. 98
Discussion ........................................................................... 1 20
References ........................................................................... 125
CHAPTER 4:

The intact Large A/B pocket domain and C-terminus of R8 is required for efficient
repression of RNA polymerase III transcription

Abstract .............................................................................. 129
Introduction .......................................................................... 1 29
Materials and Methods ............................................................. 131
Results ................................................................................ 135
Discussion ........................................................................... 1 59
References ........................................................................... 165

CHAPTER 5:

Summary .......................................................................................... 167

APPENDIX A:

Recruitment of Basal Factors to the U6 snRNA promoter ................................... 179
Results and Discussion ............................................................. 180

Materials and Methods .............................................................. 191

References ........................................................................... 194
APPENDIX B:

Potential co-factors that regulate snRNA gene expression ................................... 195

Results and Discussion ............................................................. 195

Actin association with potentially active U6 snRNA promoters ....... 195

HMG-l association with snRNA promoters ................................. 198

p70: a potential co-repressor for RB? ....................................... 201

Materials and Methods .............................................................. 205

References ........................................................................... 208
APPENDIX C:

Subcellular localization of SNAPc and T F [113 components ................................. 209

Results and Discussion ............................................................. 209

Materials and Methods .............................................................. 212

References ........................................................................... 217

vi

 

Figure 1-1:

Figure I-2:

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:

Figure 3-6:

Figure4-l:

Figure 4-2:

LIST OF FIGURES

Representation of RNA polymerase III transcribed promoters.
Factor requirements for RNA polymerase III transcribed genes.
RB represses in vitro transcription by RNA polymerase III

Different factors reconstitute adenovirus VAI and human U6
snRNA gene transcription in GST-RB (379-928) treated
extracts.

Endogenous RB is associated with SNAPC.

Endogenous RB co-fractionates with SNAPc during
chromatographic purification.

RB interacts with two components of SNAPc

Model for RB repression of human U6 snRNA gene
transcription.

RB selectively occupies U6 snRNA promoters in vivo and
specifically represses U6 snRNA transcription in vitro.

RB interacts with components of TFIIIB complexes

TFIIIB acts as a selectivity factor to recruit RB specifically to
RNA polymerase III transcribed snRNA promoters

Contribution of individual components of TFIIIB to RB
recruitment to a U6 snRNA promoter:

RB co-occupies the same endogenous U6 snRNA promoter as
SNAPC, TFIIIB and RNA polymerase III.

RB and RNA polymerase III co-occupy the same repressed U6
snRN A promoter in vitro.

Amino acids 379-870 of RB contain the minimal region

RB occupancy of RNA polymerase III transcribed promoters in
vitro and in vivo

vii

53

57

63

67

72

79

101

105

108

111

115

118

137

140

Figure 4-3:
Figure 4-4:

Figure 4-5:

Figure 4-6:

Figure 4-7:

Figure 4-8:

Figure A-l:

Figure A-2:

Figure A-3:

Figure 8-1:

Figure B-Z:

Figure B-3:

SNAPc can recruit RB to promoter DNA.

Addition of or-RB antibodies precludes interaction with SNAPc.

Characterization of the region of RB necessary for
interaction with SNAPc bound to U6 promoter DNA.

Characterization of the region in RB that can interact
with RNA polymerase III basal machinery.

SNAP43 (234-368) is the minimal region required for
interaction with RB

RB repression of class 2 and class 3 RNA polymerase III
transcribed genes.

SNAPc assembly and binding to U6 snRNA promoter DNA in
vitro:

Mini-SNAPc can recruit Brf2 to a U6 snRNA promoter.
SNAPc and TFIIIB assembly at a U6 snRNA promoter in vitro.

Actin and SNAPc or RNA polymerase III co-occupy the
same U6 snRNA promoter in vivo.

HMG-l occupies snRNA promoters in vivo.

p70 associates with the U6 snRNA promoter in vivo and
interacts with RB.

Figure C- l : Subcellular Localization of components of SNAPc and TFIIIB.

Figure C-2: Cell density affects subcellular localization of de1.

viii

145

148

151

154

158

164

182

186

190

197

200

203

211

214

ATP
de1
Brf- l
Brf-1_v2
Brf-2
Cch
CDK
ChIP
Co-IP
DHFR
DNA
EMSA
GAPDH
HAT
HDAC
HMT
HP 1
IC R
MCM
MEF

mS NAPc

KEY TO ABBREVIATIONS

Adenosine triphosphate

B double prime

TFIIB related protein 1

TFIIIB related protein 1 splice variant 2
TFIIB related protein 2

Cell division cycle 2 protein

Cyclin dependent kinase

Chromatin immunoprecipitation
Co-immunoprecipitation

Dihydrofolate reductase
Deoxyribonucleic acid

Electrophoretic mobility shift assay
Glyceraldehyde — 3 —- phosphate dehydrogenase
Histone acetyl transferase

Histone deacetylase

Histone methyltransferase
Heterochromatin protein 1

Internal control region
Mini-chromosome maintenance protein
Mouse embryonic fibroblast

Mini-SNAPC

ix

NTP
PcG
PCNA
PCR

RB

RT-PCR
RRMZ
SNAPc
SnRNA
SuVAR39H 1
TBP
TFIIA
TFIIB
TFIID
TFIIF
TFIIIA
TFIIIB
TFIIIC
TK

TS

Nucleotide triphosphate

Polycomb group complex

Proliferating cell nuclear antigen

Polymerase chain reaction

Retinoblastoma tumor suppressor protein
Ribonucleic acid

Reverse transcriptase polymerase chain reaction
ribonucleotide reductase M2

Small nuclear RNA activating protein complex
Small nuclear RNA

Suppressor of varigation

TATA binding protein

Transcription factor II A

Transcription factor II B

Transcription factor II D

Transcription factor II F

Transcription factor 111 A

Transcription factor 111 B

Transcription factor 111 C

Thymidine kinase

Thymidylate synthase

I r

I

‘1‘,

flit:

Chapter 1

Introduction

RNA Polymerase III

In eukaryotic organisms, nuclear genes are transcribed by three highly related RNA
polymerases: RNA polymerase I, II, and 111. Each of these polymerases is responsible for
transcription of a distinct subset of genes. RNA polymerase I synthesizes a single
tandemly arrayed set of non-translated structural RNAs that assemble into ribosomes.
RNA polymerase II transcribes a diverse set of genes including protein-encoding genes
and some non-translated uridine rich snRNA genes. RNA polymerase III produces an
assorted collection of non-translated structural and catalytic RNAs including small RNAs

that are usually shorter in length than 400 nucleotides (36, 1 13).

Structure of genes transcribed by RNA polymerase III

The genes transcribed by RNA polymerase [11 fall into four classes characterized by
promoter architecture (Figure I-l). Class 1 and 2 genes, represented by the SS ribosomal
RNA (SS rRNA) and transfer RNA (tRNA) genes respectively, contain required gene
internal promoter elements. Class 3 genes are characterized by external promoter
elements and are represented by the U6 snRNA gene and the 78K gene. Class 4 genes

exhibit a combination of the elements found in the first three classes.

Figure I-l: Representation of the three classes of genes transcribed by RNA
polymerase 111.

Class 1 genes such as SS rRNA genes contain gene internal promoter elements including
A and C boxes that are separed by an intermediate element (IE). Together, these elements
comprise the internal control region (ICR) that is required for gene expression. Class 2
genes such as the mammalian tRNA gene also contain gene internal cis-acting elements
referred to as the A and B boxes. Class 3 genes (U6 snRNA) are characterized by gene
external promoter elements including the distal sequence element (DSE), the proximal
sequence element (PSE) and a TATA element. Class 4 genes such as the vault RNA
genes or EBER gene from the Epstein Barr virus, contain a mixture of all three classes of
promoter elements.

ICR

 

 

 

l—P l'_‘——I
Class1 {HID—- ss rRNA
A IE C
Class 2 F’U—D—- tRNA
A B

Class 3 m/l—fl—Dr, us snRNA
DSE PSE TATA
fl—flv’fl—{I-Ij—H— Vault
DSE DSE PSE TATA A B

Class 4

DZ-H— seem

TATA A B

 

SS rRNA genes (class 1) contain an A box, an intermediate element (IE), and a C box
that is highly conserved through many species. These sequence elements combined
constitute the internal control region (ICR) that is required for transcription (5, 95-97).
For example, in Xenopus laevis, the A box is located at +50 to +64, the IE between +67
and + 72, and the C box is located from +80 to +97 (96). The spacing of these elements
is important, as effective transcription of SS rRNA genes is intolerant of changes in

spacing (96). In S. cerevisiae, only the C box is required for efficient expression of 58

genes (10).

Class 2 gene promoters, Ad2 VAI (adenovirus gene product similar to tRNA) and tRNA
genes, consist of an A box (e.g. X. laevis leucine tRNA +8 to +19) and B box (+52 to
+62) region downstream of the start site of transcription (35, 119). These intragenic
sequences are extremely well conserved from species to species perhaps in part because
these regions encode the T and D loop regions that are critical for tRNA function. In
contrast, the spacing between the A and B boxes is variable. However, recently it has
been shown that some extragenic sequences may be necessary for class 2 gene
transcription. For example, yeast U6 snRNA genes appear to have an extragenic TATA
like element (7). Similarly, some tRNA genes in D. melanogastor also appear to contain
TATA elements upstream of the start site of transcription that may be important for
efficient gene expression (127). Additionally, the —30 to —25 bp region of C elegans
tRNA genes may also contain sequences required for the transcription of these genes

(68).

Class 3 genes as represented by the mammalian U6 snRNA and 78K genes, are
characterized by gene external core promoter elements. The enhancer regions of these
genes contain a distal sequence element (DSE) that includes octamer sites, as well as sites
for other transcriptional activators such as Spl and Staf (69, 112). The DSE activates
transcription from the core promoter of Class 3 genes and can be located at variable
positions upstream of the core promoter from species to species. In mammalian systems
the DSE is located approximately 239 bp upstream of the start site of transcription (121,
150). The core promoter regions of class 3 genes consist of two essential elements: the
proximal sequence element (PSE) and a TATA element. These cis-acting elements are
located at approximately -46 and -20 bp upstream of the start site of transcription,

respectively. The spacing of these two elements is invariant in human genes.

Class 4 genes represent a mix of characteristic sequence elements found in the other three
classes of RNA polymerase 111 genes. These class 4 genes include vault genes (130, 132),
selenocysteine tRNA, EBER2 (Epstein-Barr viral product) (47, 48), MRP RNase
(involved in maturation of mitochondrial DNA replication), Y1 and Y3 (unknown
function). In the case of vault RNAs and selenocysteine tRNA, the promoters of these
genes contain gene external PSEs as well as gene internal control elements. Additionally,
many human vault RNA genes contain distal sequence elements similar to those found in
snRNA genes. The Epstein-Barr gene, EBER contains a TATA element as well as gene

internal A and B box.

RNA polymerase III Basal Transcription Machinery

Just as each class of RNA polymerase III transcribed gene contains characteristic
promoter sequences, each class of RNA polymerase III transcribed genes has distinct,
specific basal machinery requirements, as discussed in more detail in the following
sections (figure I-2). SS rRNA (class 1) genes require the binding of TFIIIA to the ICR
of the promoter followed by recruitment of TFIIIC. Interaction of TFIIIB with TFIIIC
allows TFIIIB to associate with the promoter, subsequently recruiting RNA polymerase
III. For preinitiation complex formation to occur at tRNA (class 2) genes, TFIIIC must
bind to both the A and B box of the promoters followed by interaction with TFIIIB.
Positioning of TFIIIB near the start site of transcription allows for RNA polymerase III
recruitment. U6 snRNA genes (class 3) require different factors. This set of genes
requires cooperative interactions between Oct-1, SNAPc (snRNA activating protein
complex), and a variant TFIIIB. At this time, the factor requirements for class 4 genes
are unclear. One possibility is that a blend of class 2 and class 3 transcription factors are

required, depending on specific promoter structure (47, 48, 130, 132).

TFIIIA:
TFIIIA is a basal factor required to recruit TFIIIC specifically to SS rRNA genes. A
founding member of C2H2 family of zinc finger proteins, Xenopus laevis TFIIIA was the

first eukaryotic transcription factor to be purified to homogeneity and to have its cDNA

Figure I-2: Factor requirements for RNA polymerase III transcribed genes.

Both class 1 and class 2 RNA polymerase III transcribed genes require the TFIIIC
complex and a TFIIIB complex consisting of del, TBP, and Brfl. Class 1 genes have
an additional requirement for TFIIIA which provides DNA binding specificity during
pre-initiation complex formation. Class 3 genes have very different factor requirements.
Transcriptional activators such as Oct-l bind to the DSE to activate transcription. A
multi—protein complex called SNAPc binds to the PSE to nucleate pre-initiation complex
assembly and is important for subsequent gene expression. An alternative TFIIIB
complex consisting of del, Brf2, and del associates with the TATA element and may
also play an important role in RNA polymerase III recruitment.

   
 

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cram TBP 819;",5
SSrRNA '
CHwZ
tRNA
In. ,..'L-.-.
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cloned (37). When TFIIIA binds to the promoter of the SS RNA gene, its nine zinc
fingers are aligned over the ICR with the C terminus of the protein near the 5’ end of the
promoter and its N-terminus at the 3’ end of the promoter. The C block of the promoter is
recognized by the three end most zinc fingers at the N terminus and the A block is
recognized by zinc fingers 7-9 (18, 33, 89). The middle three fingers adopt a completely
different conformation in order to span the intervening DNA. After binding to SS rRNA
genes, TFIIIA provides a platform for the recruitment of TFIIIC, which has little affinity

for the SS rRNA gene alone.

TFIIIC

TFIIIC is a required basal transcription factor that enables pre-initiation complex
formation to occur at all RNA polymerase III transcribed promoters in yeast and the 5S
and tRNA genes in mammalian systems. The mammalian U6 snRNA gene does not
require TFIIIC but rather another basal factor, SNAPc, which is discussed in detail
below. Productive recruitment of TFIIIC to SS rRNA promoters requires interaction with
TFIIIA, whereas recruitment to tRNA promoters and yeast U6 snRNA promoters is
mediated through sequence specific DNA contacts by TFIIIC. Yeast TFIIIC consists of
six subunits (36, 113) whereas human TFIIIC includes 5 subunits. The TFIIIC complex

can be separated into two domains separated by a flexible linker: TA which binds weakly

to the A box and 1:3 which binds with high affinity to the B box (78). The linker between
the domains confers a flexibility to the TFIIIC protein complex that allows for binding to
tRNA promoters that have varied distances between the A and B boxes. Stable

association of TFIIIC with promoters allows for recruitment and correct positioning of

TF 111B. TFIIIC has also been shown to contact RNA polymerase III itself (50) suggesting
that although TFIIIB alone is sufficient to recruit polymerase, contacts with TF [11C may

also be important.

SNAPc

Mammalian class 3 RNA polymerase III transcribed genes have slightly different factor
requirements, perhaps in part due to the presence of unique gene external promoter
elements. The QRNA activating protein complex (SNAPc) also called the proximal
element transcription factor (PTF) (2, 146, 147) is required for snRNA expression
including the U6 snRNA gene which is representative of Class 3 genes. SNAPc is a
multi-protein complex consisting of at least five subunits as designated by apparent
molecular mass: SNAP190 (PTFor) (32, 145), SNAPSO (PTFB) (2, 41), SNAP45 (PTFS)
(108, 147), SNAP43 (PTFy) (43, 147), and SNAP19 (42). SNAPc is specifically recruited
to the PSE of snRNA genes and is important for nucleation of pre-initiation complex
formation (41-43, 72, 109, 145). Further characterization of the SNAP complex
demonstrated that the minimal complex (mSNAPc) necessary for DNA binding and
transcriptional activation consists of SNAP190 (l-SOS), SNAP43, and SNAPSO (44, 74,
83). SNAPc interacts with TBP and cooperatively binds to DNA with TBP (82)

suggesting that SNAPc plays a role in recruitment of TFIIIB to U6 promoters.

TFIIIB

TFIIIB plays a central role in the establishment of a pre-initiation complex at the

promoters of RNA polymerase III transcribed genes. TFIIIB is recruited to promoters

lO

through interactions with TFIIIC (58-60) or SNAPc (13, 44). Subsequent to recruitment,
TFIIIB is responsible for bringing RNA polymerase to promoters to initiate transcription

(57, 133, 144).

Three proteins compose TFIIIB complexes: the TATA binding protein (TBP), TFIIB
related factor (Brf), and B” (de) (58-60, 64, 100, 117, 129). Most of the information
currently available for how TFIIIB complexes are assembled and how they function for
RNA polymerase III transcription comes from studies in S. cerevisiae. In yeast, TBP and
Brfl form a very stable complex (designated B’) from which the less tightly associated
B” is separable by chromatography (58). The Brfl subunit plays a central role in
maintaining TFIIIB complex integrity through extensive contacts with TBP and de1.
Less is known about the TFIIIB complexes that function in human systems. It appears
that multiple variant complexes may exist that function at different classes of RNA
polymerase III transcribed genes (81, 114, 125). Human Brfl and yeast Brf are highly
homologous, especially in the TFIIB related N-terminal half (133). Human del is a
very large protein that has some similarity (~40%) to yeast B” along a 400 amino acid

stretch that encompasses the indispensable SANT domain (1 14).

At the human U6 promoter, the TATA box directly recruits TBP, which is likely to be
important for recruiting additional BRF-related factor(s), and del (114). In contrast to
most RNA polymerase III-transcribed genes, human U6 snRNA gene transcription does
not require Brfl (79, 81). While the TFIIIB complex containing Brfl and TBP clearly

does not function for human U6 transcription (81), the exact nature of the TBP complex

11

that does function at human U6 promoters is currently unclear. Two proteins analogous to
Brfl have been shown to be important for U6 snRNA gene expression. Brf2 associates
with U6 promoters in vivo (114) and is necessary for gene expression as determined by
immunodepletion-reconstitution experiments (13, 114). Additionally, in vitro
transcription experiments performed using highly purified recombinant SNAPc, TBP,
del, Brf2 and chromatographic fractions enriched for RNA polymerase 111 support U6
snRNA transcription, suggesting that these proteins define the minimal set of factors
required for U6 transcription (13). However, a splice variant of Brfl called Brfl_v2 has
also been shown to participate in U6 gene expression in immunodepletion experiments
(79). Compared to Brfl, Ber has a conserved zinc finger and core domains and a
divergent C-terminus. Brfl_v2 lacks the zinc finger and lst repeat domain conserved in
Brfl and TFIIB. The role that each of these proteins plays in U6 snRNA gene expression
is unclear. However, both proteins may be in a complex that is required for U6 snRNA

transcription.

RNA polymerase III enzyme

RNA polymerase III has been well defined in S. cerevisiae and has been shown to have
17 subunits. RNA polymerase 111 contains ten subunits that are unique to RNA
polymerase III and are designated the C subunits. The two common subunits shared by
RNA polymerase I and III are designated the AC subunits. RNA polymerase III also
contains five subunits common to all three polymerases (ABC subunits) (46). Human
RNA polymerase III has been characterized both by chromatography (133, 134) and from

cell lines expressing epitope tagged subunits (51, 133, 134). All yeast subunits except

12

RPC37 have been disrupted and shown to be essential for viability (1 1). All of the human
RNA polymerase III subunits have now been characterized by mass spectrometry after
purification fi'om human cell lines contained double epitope tagged HsRPC4 (51). This
analysis found human orthologues to all of the yeast RNA polymerase III subunits except

for RPCIO, which may not have been detected due to small size.

The Retinoblastoma Tumor Suppressor Protein

The Retinoblastoma tumor suppressor protein (RB) is a critical regulator of important
cellular processes including cell cycle progression (24, 25, 39, 40, 56, 80, 86, 101, 111,
135, 149), DNA replication (105), growth regulation (118, 140), differentiation (12, 38,
90, 99, 118, 126, 148), and apoptosis (49, 148). RB is frequently mutated in a variety of
human malignancies and tumors. Approximately 30% of all human cancers are deficient
for RB activity, and the RB signal transduction pathway is disrupted upstream in 50% of
human cancers. RB was originally identified as a result of frequent mutation in the rare
pediatric eye tumor (34, 67). Cancers that have been shown to be deficient in RB activity

include osteosarcomas, small cell lung cancers, bladder, prostate, and breast cancer (17).

RB belongs to a family of pocket proteins that also includes p107 and p130 (135). These
proteins were originally characterized by the presence of a large A/B pocket domain that
was identified by the ability to bind to viral oncoproteins, including SV40 Large T
antigen, Adenovirus Ela, and Papilloma virus E7 (27, 28). The Large A/B pocket extends

from amino acids 379-869 and is required for tumor suppression. This region can be

13

further subdivided into small A/B pocket (393-768) and the C pocket (768-869) (138).

The small A/B pocket is sufficient for repression of transcription (14).

Classically, RB has been studied in the context of cell cycle progression. RB mediates
the Gl/S transition of the cell cycle by regulating the expression of genes required to
enter into S phase. Additionally, RB is a phosphoprotein that is phosphorylated in a cell
cycle dependent manner. The hypophosphorylated form of the protein, considered the
active form of the protein, binds to and regulates target cellular factors. Phosphorylation
by cylin D/cdk4 (30, 61) during late Gl or by Cyclin E/cdk2 (22, 23, 26, 87) during early

Gl/S phase inactivates RB such that it can no longer bind to target factors.

RB plays an essential role in development

A powerful tool for dissecting the function of genes of interest is the technology that
allows for targeted gene disruption (knock out) or ectopic gene expression (knock in)
within a model organism. “Knock-out” or “knock-in” mice provide a wealth of valuable
information about the functions of RB family members in an in vivo and physiologically
relevant setting. Transgenic mice over-expressing RB show dwarfism by day 15
embryonic development (E15), indicating that RB regulation is required for proper
development (4). However the lack of RB also adversely affects development. Gene
targeted Rb-/- embryos die between E13.5 and E15.5 (16, 53, 65). Additionally, increased
apoptosis is observed in the nervous system as early as E115 and is particularly evident
in the hindbrain, spinal cord, and trigeminal and dorsal root ganglia. Ectopic mitoses are

also observed especially in the hindbrain. Rb-/- embryos exhibit defective hematopoiesis,

14

manifested as an increased number of immature nucleated erythrocytes. Introduction of a
RB transgene into knockout mice fully rescues developmental defects indicating the

importance of this gene in normal development (4).

Unlike human retinoblastoma patients, mouse RB chimeras develop pituitary gland
tumors rather than retinoblastomas (75, 143). A similar phenotype is observed in Rb+/-
mice, in that tumors develop in the brain and pituitary gland. These tumors exhibit loss of
heterozygosity of the remaining wild type allele, demonstrating that RB is a tumor
suppressor in mice as well as in humans. Although Rb+/- mice are cancer prone, they do
not accurately reiterate the tumor spectrum observed in human retinoblastoma patients.
This phenomenon may be explained by the fact that other RB family members could
compensate for the loss of RB in the eye. Mice homozygous for p107 and p130 knock
outs are viable, fertile and healthy (19, 66). p107-p130 double knock out mice
experience neonatal lethality and most Rb+/- p107-/- mice are growth retarded, and
increased mortality of these mice is observed in the first three weeks after birth. Although
Rb+/-p107—/- pups that survive to adulthood do not show increased cancer predisposition
compared to Rb+/— mice, they develop multiple dysplasic lesions of the retina. Unlike
RB, p107 and p130 are not required for embryonic development, and p107+/-, p130+/—,
p130-/-, p107-/- mice do not exhibit increased incidence of tumor development. Triple
knockout embryonic fibroblasts have normal growth characteristics but impaired
differentiation capacity (21, 110). Triple knockout mouse embryonic fibroblasts have a
shorter cell cycle and can spontaneously immortalize. These cells are also resistant to G1

arrest following DNA damage, contact inhibition or serum starvation.

15

Targets for RB regulation

Originally, genes proposed to be repressed by RB were known E2F responsive genes. It
had been shown that RB could convert a positive acting E2F site to a negatively acting
element in transient transfection experiments (137). Based on the data presented in this
study, genes that had E2F binding sites or were shown genetically or biochemically to be
dependent on E2F were postulated RB targets. Proposed target genes include S phase
cyclins and cdks, EZF family members, other pocket protein family members, genes that
function for DNA replication and nucleotide biosynthesis pathways (1, 56, 84, 86, 118,

136).

Many studies have been performed to identify the true physiological targets of RB. RT-
PCR and Northern blot analysis using mouse embryonic fibroblasts in which each
individual RB family member is knocked out were used to study relative amounts of
target gene expression in the absence of RB family members (52). This study showed a
de-repression of cyclin E and p107 at the GO/Gl check point in the absence of RB.
Additionally, b-myb, cch, E2F-1, thyrnidylate sythase (TS), ribonucleotide reductase
M2 (RRMZ), cyclin A2 and dihydroxyfolate reductase (DHFR) were shown to be de-
repressed at the 60/61 interface in the absence of both p107 and p130. Another group of
researchers employed DNA micro-array chips to study global changes in gene expression
under conditions in which a constitutively active RB was over-expressed in tissue culture
cells (77). This study identified 341 targets that were reproducibly repressed at least 1.7

fold in multiple cell lines. These targets fell into four categories including: (1) DNA

16

'-

 

II

2'.

replication, (2) DNA repair, (3) chromatin structure and transcription, and (4) G2/M
progression. While many of the genes identified in this study have previously been
shown to be E2F dependent, several novel targets identified have not been previously
linked to E2F. The micro-array analysis pinpointed several genes also identified
previously by RT-PCR analysis (52) including cyclin A, RRM2, thymidine kinase, TS,
cyclin E, cch and DHFR. Additionally, this screen also indicated that MCM6, MCM7,
DNA polymerase 5, topoisomerase IIor, PCNA, Cdk2, cyclin BI, and HMGZ were
repressed by the ectopically expressed RB. Together these two studies provide a pool of
candidate genes regulated by RB family members. However, these studies do not

distinguish between direct and indirect regulation of gene expression.

In order to determine which genes RB family members directly target, it is important to
study RB on the level of target gene promoter occupancy. Several groups have
undertaken this question by using chromatin immunoprecipitation (ChIP) to analyze RB
family member promoter occupancy. ChIP analysis provides a powerful tool for
understanding direct gene targets for proteins of interest because it relies on in vivo
endogenous proteins bound to genes in a physiologically relevant chromatin structure
rather than on over-expression and reporter gene assays. The results of these studies
however, are contradictory. Wells and co-workers (139) report that RB and RB family
members occupy the TK promoter during G0; DHFR, cdc2, and cyclin B during GO and
Gl/S; and b-myb in asynchronous cell populations (139). A second group, Takahashi
and co-workers (124), were unable to detect RB at any promoter studied. They instead

observed p130 at E2F1, cdc6, p107, b-myb, and cyclin A promoters during GO and

17

GO/Gl suggesting that p130 is the major player in cell cycle progression. One
explanation for this discrepancy may be that these two groups worked in two different
systems: mouse and human, respectively. RB family members may regulate cell cycle
progression and growth control differently in these two species. Additionally, the
experiments were performed from different cell types indicating that there may be tissue

specific regulation by RB family members.

Mechanisms for RB repression of RNA polymerase II transcribed genes

The function of RB as a tumor suppressor is linked to its ability to regulate gene
expression. Therefore, to fully understand the contribution of RB to cellular proliferation
observed during carcinogenesis, it is important to determine the mechanisms that RB uses
to regulate gene activity. Direct repression of transcription by RB implicates protein-
protein interactions between RB and transcriptional activators belonging to the E2F
family. E2F binds to enhancer regions of E2F responsive gene promoters to stimulate
transcription by RNA polymerase II. By binding to E2F at gene promoters, RB can
potentially directly block activation function of E2F by obscuring the trans-activation
domain that facilitates pre-initiation complex assembly, or RB can recruit co-regulatory

factors that alter chromatin structure surrounding these genes to repress transcription.

Disruption of pre-initiation complex formation

One critical step in the activation of transcription for most RNA polymerase II

18

transcribed genes is the establishment of a stable preinitiation complex (PIC) (93).
Preinitiation complexes are thought to assemble in a step-wise fashion (93, 103, 104).
First the TBP containing factor, TFIID, binds to TATA elements, nucleating preinitiation
complex formation and recruiting additional basal factors. Next, TFIIA and TFIIB are
recruited to the start site of transcription, followed by association of TFIIF in complex
with RNA polymerase. Potentially, RB may repress transcription by interfering with one

of these steps.

Elegant studies using purified factors in an in vitro transcription assay show that RB does
in fact repress transcription through disruption of pre—initiation complex formation (107).
Specifically, RB inhibits the formation of the TFIID-TFIIA (DA) complex. However, if
the DA complex is allowed to bind to DNA prior to the addition of RB, it becomes
resistant to RB repression. Together, these results suggest that RB represses transcription
by blocking pre-initiation complex formation thereby not allowing for RNA polymerase

II association and subsequent gene expression.

RB and histone modification: HDA Cs

Covalent modification of histones and change chromatin structure is critical for efficient
gene regulation (3, 55). Histone tails in nucleosomes at the promoters of active genes are
generally acetylated on lysine residues. This acetylation neutralizes the positive charge of
the lysine that binds tightly to the surrounding negatively charged DNA. The resulting
unwrapping of the histone tails loosens the chromatin structure, allowing access to

transcription factors and RNA polymerase.

19

a
a
K.

 

 

M.‘

\‘u'n
1“

One potential mechanism that RB may use to repress transcription of target RNA
polymerase II transcribed genes is the recruitment of histone deacetylase (HDAC)
activities. HDACs are co-repressor complexes that enzymatically remove the acetyl
groups from the lysines of histone tails. Several groups have shown that RB interacts
with class I HDACs and that a deacetylase activity co-purifies with RB (6, 31, 73, 76, 99,
101, 149). Potentially, this HDAC activity could deacetylate histone tails at RB target
genes, causing a net compaction of surrounding chromatin. This alteration of chromatin
structure, resulting in a loss of accessibility to transcription factors and RNA polymerase,
would then effectively repress transcription of the gene. Nonetheless, HDAC activity
does not account for all of RB’s repression capability. The addition of HDAC inhibitors
only partially reverses RB repression, suggesting that RB has HDAC independent as well
as HDAC dependent repression mechanisms (6, 76). The HDAC independent repression
may include mechanisms such as masking of the E2F trans-activation domain or
preinitiation complex disruption. Additionally, there appears to be some promoter

selectivity for RB repression through histone deacetylases (73).

The true target of RB associated deacetylation activity is unclear. There is no direct in
vivo evidence that the HDACs recruited by RB repress transcription by deacetylation of
the histone tails in the promoters of target genes. While a corresponding lack of
acetylation has been observed, it may be an indirect effect perhaps caused by failure to
recruit the proper HAT complex. Another possibility is that the true target of RB-

associated deacetylase activity is not histone tails but rather other acetylated factors

20

important for gene expression. In fact, one group studying RB repression of RNA
polymerase I transcription demonstrated that acetylation of the required factor, UBF, was
important for active transcription of rRNA genes (9). Moreover, this group also showed
that deacetylation of UBF by RB associated HDACs results in loss of gene expression,
suggesting that HDACs may have substrates in addition to histone tails. Altogether,
studies to date indicate that HDAC activity is important for the regulation of select

promoters even if the mechanism of repression is still as yet unclear.

RB and chromatin remodeling: S WI/SNF

Chromatin remodeling is another fundamental process in the regulation of gene
expression. Many chromatin-remodeling complexes reposition nucleosomes that create
repressive barriers within gene promoters. Repositioning these nucleosomes alters the
local chromatin structure and increases promoter access to factors necessary for active
transcription of the gene (85). Chromatin remodeling is also another key process that RB
may influence in order to repress transcription. Several groups have demonstrated that
RB can interact with mammalian orthologues of the Drosophila Brahma protein: hBRM
and Brg-l (120, 122, 128, 149). These paralogous human proteins are the large ATPase
subunits of the human SWI/SNF complexes. SWI/SNF is an ATP-dependent chromatin
remodeling complex that is required for the activation of many RNA polymerase II
transcribed genes (94). SWI/SNF appears to be required for RB regulation of cell cycle
progression (149). In contrast to the activation activity normally associated with
Chromatin remodeling, when SWI/SNF is recruited to target genes by RB, the overall

effect is negative, perhaps by repositioning nucleosomes in a fashion that would obscure

21

the promoter. Interestingly, RB may recruit both SWI/SNF and HDACs to the same
promoter to repress transcription through a combination of histone modification and
chromatin remodeling. RB appears to regulate exit from G] phase of the cell cycle
through the recruitment of both HDACs and SWI/SNF, whereas exit from S phase is
regulated through RB recruitment of SWI/SNF alone. Again these results suggest

multiple mechanisms of RB repression that may be dependent on promoter selectivity.

RB and co-repressor complexes: Polycomb

Additionally, RB may regulate gene expression and consequently cell cycle through
recruitment of Polycomb (PcG) complexes. Members of the Polycomb family have roles
in the negative regulation of Hox genes during development (98, 115) and also serve as
critical regulators of cell cycle progression (54, 70). Polycomb complexes bind to
Polycomb response elements (PRE) in target genes to establish a silenced chromatin state
(98) and also contribute to transcriptional memory such that once silenced in a cell, a

gene will also be silenced in progeny cells (8).

RB has been shown to associate with a PcG complex containing HPC2, Ringl, and Bmi-
1 in an interaction mediated by CtBP (20). Specifically, this HDAC-independent
mechanism appears to arrest cells in the GZ phase of the cell cycle by repressing the
expression of cyclin A and cdc2. This PcG complex also associates with the cyclin A
promoter under conditions of cell cycle arrest. The authors of this study suggest that this

mechanism of repression may occur under conditions of irreversible grth arrest

22

whereas HDAC-dependent RB repression may occur in the case of reversible cell cycle

arrest.

RB and histone methylation: HP] — Su Var39

RB may also regulate gene expression by recruitment of HPl and SUV39H1 (88, 131).
SUV39H1 specifically methylates lysine 9 on histone H3 tails. (91, 102). The resulting
methylation on K9 of H3 tails induces the formation of a high affinity-binding site on the
chromatin for proteins of the heterochromatin protein 1 (HPl) family, which are involved

in heterochromatin silencing (29).

RB associates with SUV39H1 in co-immunoprecipitation experiments and directly
interacts with HP] (88). A histone methyltransferase activity also co-purifies with RB.
Furthermore, HPI and RB are both recruited to a methylated H3 peptide, suggesting that
HP] can recognize RB and methylated histone tails simultaneously. In vivo chromatin IP
data demonstrates that HPl is present at the cyclin E promoter in RB+/+ cells and that the
H3 tails at these promoters are methylated, whereas HPl does not associate with this
promoter in the absence of RB and the H3 tails exhibit lower methylation. Collectively
the results suggest that RB recruits the SUV39H1 enzyme to target promoters where it
methylates K9H3, providing a binding site for HP]. Since methylation cannot occur at a
lysine residue that is acetylated (102), the deacetylase activity associated with RB may be
required as a preceding step to SUV39H1 mediated methylation. Potentially the role for
HPl in this repression is not to promote the formation of heterochromatin but rather to

mask the methylated lysine 9 in histone H3 tails such that it cannot be de-methylated and

23

subsequently acetylated. These results indicate that the SUV39Hl-HP1 complex is not
only involved in heterochromatic silencing but also has a role in repression of

euchromatic genes by RB and perhaps other co-repressor proteins.

RB and DNA methylation: DMN T 1

DNA methylation at CpG dinucleotides is essential for embryonic development and is
catalyzed by three DNA methyltransferases, DMNTl, DMNT3a and DMNT3b, which
have differential capacities for maintenance and de novo methylation (71, 92). During
chromatographic purification of the DMNTl methyltransferase activity, RB was shown
to co-purify in a complex with DMNTI (106) that also includes E2F and HDAC]. The
methyltransferase activity that is isolated by immunoprecipitation for RB and DNMTl
appears to work co—operatively with RB and HDACl to repress transcription. Potentially,
the DNA binding domains of the associated E2F proteins provide the necessary
specificity to recruit the repression activities to target genes. The results of this study
establish a link between DNA methylation, sequence specific DNA binding activity,
histone deacetylation as well as 3 grth regulatory pathway this is disrupted in the

majority of cancer cells.

RB regulation of RNA polymerase III

In addition to regulating genes expressed by RNA polymerase 11, RB also regulates genes
transcribed by RNA polymerase III. RB globally represses RNA polymerase III
transcripts both in vivo and in vitro, including RNA polymerase III transcribed snRNA

genes (15, 45, 62, 116, 123, 142). Interestingly, nuclear run-on assays in RB-/- mouse

24

embryonic fibroblasts suggest that while global RNAP III activity is elevated in these
cells, RNAP II transcription is largely unaffected (142). Regulation of RNA polymerase
III transcribed genes may be important for controlling the growth potential of the cell.
Genes transcribed by RNA polymerase III encode small RNAs that act as metabolic
machinery and thus contribute to the biosynthetic capacity of the cell. Thus, cells lacking
RB would be expected to have increased proliferation potential as a result of increased
available biosynthetic machinery, and this potential may contribute to unregulated growth

during tumor formation.

How RB regulates RNA polymerase III activity in the cell is not clear. RNA polymerase
III transcriptional activity is under cell cycle control, with higher levels observed in the
late G l, S and G2 phases of the cell cycle than in GO and early G1. The increase in RNA
p01ytnerase III activity correlates with an increase in phosphorylated RB (inactive form)
during the G1 phase of the cell cycle, suggesting a link between RB regulation and RNA
pOIanerase III activity (116, 141). Over-expression of RB during transient transfection
assays in RB-/- SaOS-2 osteosarcoma cells results in the repression of tRNA genes as

well as Ad2 VAI genes.

One proposed mechanism for RB repression of SS rRNA and tRNA transcription by
RNA polymerase III is the disruption of interactions between basal transcription factors
I'tl‘luired for pre-initiation complex formation. RB has been shown to associate with both
TFIIIB (63) and TFIIIC2 (15). Specifically, RB associates with the Brfl and TBP

Sllbunits of TFIIIB in co-immunoprecipitation experiments and co-purifies with TFIIIB

25

biochernically. RB can also interact with TFIIIC2 in GST pull-down experiments
performed in HeLa cell nuclear extracts (15). RB however does not appear to interact
with TFIIIA (63) or RNA polymerase III. Sutcliffe and collaborators (123) demonstrated
that RB interaction with TFIIIB precludes TFIIIB interaction with TFIIIC. Since TFIIIB
can no longer associate with promoters through interactions with TFIIIC, RNA
polymerase III is not recruited to the start site of transcription, effectively abolishing gene

expression.

Less is known about how RB regulates the expression of class 3 RNA polymerase III
transcribed genes. RB represses transcription of the human U6 gene by RNA polymerase
111 both in vivo and in vitro. However, U6 snRNA genes differ significantly from other
genes transcribed by RNA polymerase III. Importantly, these genes contain snRNA-
specific promoter elements and do not employ the same TFIIIB and TF 111C complexes
implicated in repression of tRNA genes. One suggestion is that the factors recognizing
these sequence elements are important for regulating U6 transcription specifically with

respect to RB.

Because the promoter elements are simple and well defined and the factor requirements
are known, RNA polymerase 111 genes provide an excellent model system for
understanding regulation of eukaryotic gene expression. We therefore wished to exploit
this model system to better understand how the Retinoblastoma tumor suppressor protein

functions to repress transcription.

26

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39

Chapter 2

The retinoblastoma tumor suppressor protein targets distinct general

transcription factors to regulate RNA polymerase III gene expression'.

Abstract

The retinoblastoma protein (RB) represses RNA polymerase III transcription effectively
both in vivo and in vitro. Here we demonstrate that the general transcription factors
SNAPc and TBP are important for RB repression of human U6 snRNA gene transcription
by RNA polymerase III. RB is associated with SNAPc as detected by both co-
immunoprecipitation of endogenous RB with SNAPc and co-fractionation of RB and
SNAPc during chromatographic purification. RB also interacts with two SNAPc subunits,
SNAP43 and SNAPSO. TBP or a combination of TBP plus SNAPc restores efficient U6
transcription from RB-treated, extracts indicating that TBP is also involved in RB
regulation. In contrast, the TBP-containing complex TFIIIB restores adenovirus VAI but
not human U6 transcription in RB-treated extracts suggesting that TFIIIB is important for
RB regulation of tRNA-like genes. These results suggest that different classes of RNA
polymerase III-transcribed genes have distinct general transcription factor requirements

for repression by RB.

 

This work was published as the following manuscript: Hirsch, H. A., Gu, L., and Henry, R. W.
(2000). The Retinoblastoma tumor suppressor protein targets distinct general transcription factors to
regulate RNA polymerase III gene expression. Mol Cell Biol 20, 9182-91.

40

Introduction

RB is a tumor suppressor that controls cell growth by influencing cell-cycle progression
(8, 12, 44), differentiation (5, 15, 44), and apoptosis (23, 68). Mutations in the gene
encoding RB are associated with diverse human cancers (21, 22, 27, 45). RB function is
also compromised in other human malignancies through disruption of upstream control
pathways or downstream targets of RB (reviewed in 58). The function of RB as a tumor
suppressor is linked to its ability to regulate gene expression. Therefore, to fully
understand the contribution of RB to cellular proliferation observed during
carcinogenesis, it is important to determine the mechanisms that RB uses to regulate gene

activity.

An understanding of RB firnction in gene regulation was revealed through its role as a
modulator of E2F transcription factor activity (16, 24, 25, 59). However, RB controls
additional cellular firnctions beyond regulating E2F activity. The intracellular
concentration of RB exceeds the concentration of E2F (58) and interactions between RB
and other transcription factors have been described (10, 34, 51). Thus, further activities
performed by RB involve regulation of other genes besides E2F-responsive genes.
Interestingly, RB is not limited to regulating mRNA production by RNA polymerase II
but also inhibits the synthesis of ribosomal RNAs by RNA polymerase I (4) and of 5S
rRNA, tRNA, and U6 snRNA by RNA polymerase III (63). It was proposed that loss of

control of these genes is an important step in tumor progression because the products of

41

genes transcribed by RNA polymerases 1 and III are important determinants of
biosynthetic capacity (reviewed in 61). Repressed synthesis of non-translated RNAs is
expected to inhibit cell proliferation presenting a significant hurdle to unregulated cell
growth. Therefore, control of RNA polymerase I and III transcriptional activity may

represent an essential component of growth regulation by RB.

How RB regulates RNA polymerase III activity in the cell is not clear. RNA polymerase
III transcriptional activity is under cell cycle control with higher levels observed in the
late G1, S and G2 phases of the cell cycle than G0 and early G1 (62). The increase in
RNA polymerase III activity correlates with an increase in phosphorylated RB during the
G1 phase of the cell cycle. This is important because the firnction of RB is controlled by
phosphorylation (6, 38). Hypo-phosphorylated RB can interact with potential target
proteins to regulate their activity whereas hyper-phosphorylated RB cannot interact and
therefore is inactive (58). RNA polymerase III activity is maximal during the cell cycle
when RB is inactive. This implies that hypo-phosphorylated RB may target factors that
function in RNA polymerase III transcription. The correlation between RB levels and
RNA polymerase III activity has been further demonstrated in vivo by transient
transfection assays of adenovirus (Ad) VAI gene transcription. Transcription of this gene
by RNA polymerase III is elevated in a human osteosarcoma cell line (SAOS2) that is
RB-deficient compared to an osteosarcoma cell line (UZOS) that contains functional RB.
Over-expressing RB in SAOSZ cells represses RNA polymerase III transcription whereas
RNA polymerase II transcription from the HIV LTR is unaffected. Furthermore, in

nuclear run-off assays, RNA polymerase III-specific transcription is diminished in nuclei

42

isolated from wildtype mouse embryonic fibroblasts compared to nuclei isolated from
mouse RB-/- embryonic fibroblasts whereas wholesale RNA polymerase II activity is
unchanged in RB+/+ and RB-/- embryonic fibroblasts (63). These experiments suggest

that RNA polymerase III activity in vivo is regulated by RB.

We have focused on understanding the contribution of RB to repression of RNA
polymerase III activity. Genes transcribed by RNA polymerase 111 can be subdivided into
four classes. Class 1 and class 2 genes contain gene-intemal promoter elements
exemplified by the 5S rRNA and tRNA genes, respectively. Class 3 genes contain gene
external promoter elements exemplified by the human U6 snRNA genes. A fourth class
exemplified by the Vault RNA genes contain both external and internal promoter
elements (54). RNA polymerase III-transcribed genes also have distinct general
transcription factor requirements consistent with their different promoter architectures. SS
rRNA genes require TFIIIA, TFIIIB, and TFIIIC, whereas tRNA gene transcription only
requires a subset of these factors, TFIIIB and TFIIIC, for full activity (50, 52, 64). In
contrast, human U6 gene transcription requires the snRNA activating protein complex or
SNAPc (48), which is also known as PTF (42). While TFIIIC is not required, the
requirement for TFIIIB is controversial (39, 57). In addition to these general transcription
factors, other transcription activator proteins including Oct-1 (3), Spl (30), and STAF

(43, 49) positively regulate U6 snRNA gene transcription.

The mechanism that RB utilizes to repress RNA polymerase III transcription is not

known. However, potential targets for regulating RNA polymerase III activity include

43

TFIIIB and the TFIIIC2 form of TFIIIC (7, 29). Human TFIIIB consists of the TATA
box binding protein (TBP) and a tightly associated factor called TFIIB-related factor or
BRF (39, 57). By analogy with yeast TFIIIB (26), a loosely associated factor referred to
as B" may also be a component of human TFIIIB. BRF and TBP associate with RB
during chromatographic fractionation of cellular extracts and during co-
immunoprecipitation experiments (29). TFIIIC2 is a multi-protein complex containing
five proteins (67). RB can also interact with TFIIIC2 in GST-pulldown experiments from
HeLa nuclear extracts (7). Together, these data indicate that RB can interact with the
RNA polymerase 111 general transcription machinery, and this may be important for RB

repression of RNA polymerase III-specific gene transcription.

It is not known whether RB targets similar factors to regulate all classes of RNA
polymerase III transcribed genes. In contrast to the clear requirement of TFIIIB and
TFIIIC2 for RNA polymerase III transcription of genes containing gene-internal
promoter elements, neither TFIIIC (55) nor a TFIIIB complex of BRF and TBP is
required for human U6 snRNA gene transcription in vitro (17, 39). Potentially, a different
form of TFIIIB may function both for U6 gene transcription and regulation by RB. Other
alternative spliced forms of BRF have been identified and one form referred to as hBRF 2
functions for human U6 transcription in vitro (37). It is also possible that RB targets other
factors to regulate human U6 snRNA genes. One potential target is SNAPc. SNAPc is a

multi-protein complex composed of at least five proteins SNAP19 (19), SNAP43/PTFy

(20, 66), SNAP45/PTF5 (47, 66), SNAPSO/PTFB (l, 18) and SNAP190/PTFOt (65). In

44

addition, SNAPc associates with TBP (20). SNAPc binds to the proximal sequence
element (PSE) contained in the core promoter regions of human U6 snRNA genes and
interacts with TBP bound to the TATA box. Together SNAPc and TBP act cooperatively
to facilitate transcription by RNA polymerase III (40). The binding of these factors to the
promoter is a crucial early step in pre-initiation complex assembly at these genes and
therefore SNAPc and TBP are attractive targets for regulating human snRNA gene

transcription.

Our results suggest that RB regulates different RNA polymerase III-transcribed genes by
targeting different components of the general transcription machinery. The general
transcription factor TFIIIB, composed of BRF and TBP, functionally restores Ad VAI
gene transcription but not human U6 snRNA gene transcription in RB-treated extracts. In
contrast, a combination of the general transcription factors SNAPc and TBP act
cooperatively to reconstitute U6 snRNA gene transcription after RB treatment indicating
that these factors are also important for RB regulation of RNA polymerase III activity.
Depleting extracts with GST-RB (379-928) resulted in a reduction in SNAP43 levels
consistent with the idea that RB is targeting SNAPc. In HeLa cell nuclear extracts, a sub-
population of RB is associated with SNAPc and this association may be direct because
RB interacts effectively with two components of SNAPc. These data indicate that the
general transcription factors SNAPc and TFIIIB provide an important targeting
mechanism governing RB function for different classes of RNA polymerase III-

transcribed genes.

45

”'3

C")

Materials and Methods

Expression and purification of recombinant proteins: The region of RB corresponding
to amino acids 379 to 928 was amplified by polymerase chain reaction and cloned into a
pETl lc-based expression vector to generate pGST-RB (379-928). This contains an N-
terrninal GST tag fused in frame with RB (379-928). Both SNAP43 (1-368) and SNAPSO
(1-411) were constructed in a similar manner to generate pGST-SNAP43 (1-368) and
pGST-SNAPSO (1-411). GST-fusion proteins were expressed in E. coli BL21 DE3 and
extracts were prepared by sonication. Proteins were purified by binding to glutathione
agarose beads (Sigma) followed by extensive washing in HEMGT-150 buffer (20 mM
Hepes pH 7.9; 0.5 mM EDTA; 10 mM MgC12; 10% glycerol; 0.1% Tween-20)
containing protease inhibitors (0.1 mM PMSF, 1 mM sodium bis-sulfate, 1 mM
benzamidine, 1 uM pepstatin A) and 1 mM DTT. Bound proteins were then used directly
for GST pulldown assays. For in vitro transcription experiments, GST-RB (379-928) and
GST were eluted from beads in HEMGT-ISO buffer containing 50 mM glutathione for 1
hr at 4°C. Eluted proteins were dialyzed against Dignam buffer D (9) and concentrated by
centrifugation through YMlO centricon columns (Millipore) to give a final concentration
of at least 200 ng/ul. Protein expression levels, efficiency of binding to glutathione
agarose beads, and protein concentrations were monitored by SDS-PAGE and staining

with Coomassie blue.

46

.11,

kr.’

1,1,)

.T'

In vitro transcription assays: In vitro transcription of the adenovirus VAI and human
U6 snRNA genes were performed essentially as described (31, 32, 48). For repression
assays depicted in Figure 1, HeLa cell nuclear extracts were pre-incubated with purified
recombinant proteins at 30°C for 30 minutes. The amounts of each protein used are
indicated in the figure legend. Twenty microliter transcription reactions were initiated by
addition of 0.25 ug DNA templates (pBSM13+VAI; pU6/Hae/RA.2), rNTPs, and
transcription buffer. Transcription reactions were performed for 1 hour at 30° C.
Transcripts were separated by denaturing PAGE and visualized by autoradiography or by

phosphoimager analysis (Molecular Dynamics).

RB afl‘mity depletion assays: For human U6 snRNA gene repression assays shown in
Figures 2A, 8 uL HeLa cell nuclear extract (7.5 ug/ uL) was pre-incubated with 1, 2, or 3
uL of purified GST-RB (379-928) or GST (each at 1 ug/ uL) for 30 min at 30°C. GST-
RB (379-928) and GST were removed by affinity purification with glutathione sepharose
beads (Pharmacia) added at a 1:1 ratio of beads to extract. Samples were then incubated

at 4°C for 4 hours and centrifuged to remove associated proteins. One half of each
supernatant (4.5, 5.0, and 5.5 uL of the l, 2, and 3 ug treated samples, respectively) was
used for in vitro transcription assays as described above. For the experiment shown in
Figure 2B, the affinity depletion reactions were scaled up to include 32 uL nuclear
extract (7.5 ug/ LIL) plus 10 ug GST-RB (379-928) (200 ng/ uL). For Ad VAI gene

repression assays shown in Figure 2C, 24 uL HeLa cell nuclear extract (7.5 ug/ uL) was

47

pre-incubated with approximately 14 ug purified GST-RB (379-928) (200 ng/ uL).
Similar pre-incubation reactions were performed with either Dignam buffer D (mock
depleted) or GST protein at equivalent amounts as used for GST-RB (379-928). After
affinity depletion, supematants were used immediately in adenovirus VAI and human U6
transcription reactions as described previously. Transcription reactions were also
supplemented with chromatographic fractions containing SNAPc (Mono-Q peak fraction;
approx. 0.3 mg/ml protein; ref. 20) or TFIIIB (PII-B; approx. 0.6 mg/ml; ref. 32). For the
experiment presented in Figure 28, 10 ng of recombinant human TBP (Promega) was
also added as indicated. For the experiment presented in Figure 2D, 20 uL HeLa nuclear
extract was treated with 6 ug of purified GST-RB (379-928) or GST as described above.
15 uL of each supernatant and proteins bound to the beads after extensive washing were
separated by 12.5 % SDS-PAGE and tested by western blot analysis using rabbit anti-

SNAP43 antiserum (CS48; ref. 20) or mouse monoclonal antibodies (SL2; ref. 33).

RB/SNAPc co-immunoprecipitation: Approximately 300 uL HeLa cell nuclear extract
was incubated with 2 ug mouse anti-RB (clone G3-245; Phanningen) or anti-
Haemaglutinin (12CA5) antibodies overnight at 4°C. Samples were diluted with 1 mL of
HEMGT-ISO containing protease inhibitors and 20 uL Protein-G agarose beads (Gibco-
BRL) were added to each reaction. Samples were further incubated at 4°C for 4 hours.
Antibody beads were washed in HEMGT-lSO containing protease inhibitors (3 x 1 mL),
bound proteins were eluted by boiling in 1x Laernmli buffer prior to size fractionation by

12.5% SDS-PAGE. SNAP43 was detected by western blot analysis using antibodies

48

specific to SNAP43 (C848; ref. 20). To perform the reciprocal immunoprecipitations,
approximately 300 uL of HeLa cell nuclear extract was incubated with 50 11L protein-A
agarose beads (Boehringer Mannheim) pre-coupled with either rabbit anti-SNAP43 or
pre-immune antibodies. Reactions were incubated for 2 hr at 4°C with mixing. Beads
were washed extensively in Dignam buffer D (100 mM KCl) containing protease
inhibitors and bound proteins were then competitively eluted in 200 uL Dignam buffer D
containing either a specific peptide (CSH375: ref. 20) or non-specific peptide each at 1
mg/mL. Eluted samples were precipitated with trichloroacetic acid. Precipitates were re-
dissolved in 1x Laernmli buffer and size fractionated by 12.5% SDS-PAGE. Full length
RB was detected by western blot analysis using mouse monoclonal antibodies directed

against an epitope contained within amino acids 300-380 (G3-245; Pharmingen).

Protein Chromatography and EMSA analysis: Nuclear extracts were prepared from
HeLa cells by the method of Dignam et al. (1983). SNAPc- and TFIIIB-containing
fractions were generated essentially as described previously (20, 32, 48). The SNAPc
fractions used are from the Mono-Q step of purification. The TFIIIB fractions used are
from the P1 l-B step of purification. PSE-specific DNA binding by SNAPc was assayed

by EMSA as described (48).

GST-pulldown assays: Individual SNAPc subunits and full-length RB were individually

expressed in vitro using rabbit reticulocyte lysates (TNT-Promega) and proteins were

labeled with 35S-methionine. GST-pulldown reactions were performed using 20 uL

49

glutathione agarose beads containing approximately 1 ug of GST-RB (379-928), GST-

SNAPSO (1-411), GST-SNAP43 (1-368) and GST or beads alone. These were

individually incubated with 10 [IL of 35S-labelled proteins for 2 hours at 4°C in 1 mL

HEMGT-ISO containing protease inhibitors and 1 mM DTT. The specific combinations
of proteins used are indicated in the figure legend. Beads were washed extensively in
HEMGT-ISO and bound proteins were separated by 17% SDS-PAGE. Proteins were
stained with Coomassie blue to ensure equivalent loading of GST tagged proteins in each

sample. Associated radioactive proteins were detected by autoradiography.

EMSA experiments: See appendix A

Results

RB represses RNA polymerase III transcription

RB is an important regulator of cellular growth and its ability to perform this function can
partially be attributed to regulation of RNA polymerase III activity. RB contains 928
amino acids and can be divided into at least three regions: the N-terminal region from
amino acids 1-378, the NB region from 393-772, and the C region from 768-869. Most
functions ascribed to RB including tumor suppressor activity and interactions with

regulatory target proteins require either the NB and/or C regions (56, 60).

To determine the function of RB in regulating RNA polymerase III activity, recombinant

RB containing the A/B and C regions was tested for its ability to repress in vitro

50

transcription by RNA polymerase III. Specifically, in vitro transcription assays of the Ad
VAI gene and a human U6 snRNA gene were performed to compare RB regulation of
RNA polymerase III transcription for genes containing gene-internal (class 2) and gene-
extemal (class 3) promoter elements. A schematic representation of the core-promoters of
the genes used for this study is shown in Figure 2-1A. The Ad VAI gene contains gene-
intemal A and B box control elements that are also characteristic of human tRNA genes.
The core-promoter regions of human U6 snRNA genes contain a PSE and a TATA box.
In addition, the U6 gene contains a DSE that recruits Oct-l to activate U6 transcription.
The GST-RB (379-928) and GST proteins typically used for these experiments are shown
in Figure 2-lB. GST-RB (379-928) and GST were each expressed in E. coli and were
purified to homogeneity by affinity purification using glutathione agarose beads. In each
case, the full-length protein is the most prevalent species observed. To determine the
effect of these proteins on RNA polymerase III transcription, increasing amounts of
purified GST-RB (379-928) and GST were added to HeLa cell nuclear extracts and these
were tested for ability to support Ad VAI transcription. As shown in Figure 2-1C, GST-
RB (379-928) inhibited transcription of the Ad VAI gene (top panel: lanes 2-5) compared
to levels observed for the untreated extract (lane 1). This repression appears to be specific
because addition of equivalent amounts of the GST control protein had no significant
effect on Ad VAI transcription (lanes 6-9). The repression observed for RB is not limited

to "classical" RNA polymerase III transcribed genes containing gene internal promoter

51

Figure 2-1: RB represses in vitro transcription by RNA polymerase III.
(A) Schematic representation of the adenovirus VAI and human U6 snRNA promoters.

(B) Analysis of GST-RB (379-928) and GST proteins used in transcription reactions.
GST—RB (379-928) (lane 2) and GST (lane 3) were expressed in E. coli and purified by
affinity chromatography using glutathione agarose beads and competitive elution with
glutathione. After dialysis against Dignam buffer D proteins were separated by SDS-
PAGE and visualized by staining with Coomassie blue. Lane 1 contains a protein size
standard.

(C) GST-RB (379-928) represses adenovirus VAI transcription by RNA polymerase III.

Approximately 2 uL of HeLa cell nuclear extract (approx. 7.5 ug/uL) was incubated with
200, 400, 800, and 1200 ng of GST-RB (379-928) (lanes 2-5) or GST protein (lanes 6-9)
at 30°C for 30 minutes. In vitro transcription of the Ad VAI gene (top panel) was then

initiated by addition of template, cold rNTPs, [a32P]-CTP, and transcription buffer. Lane

1 shows the level of transcription with the untreated extract. Sample handling was
monitored by a non-specific RNA handling control transcript (bottom).

(D) GST-RB (379-928) represses human U6 snRNA gene transcription by RNA
polymerase III. Approximately 2 uL of HeLa cell nuclear extract was incubated with 100,
250, and 500 ng of GST-RB (379-928) (lanes 2-4) or GST protein (lanes 5-7). Lane 1
shows the level of transcription with the untreated extract. Correctly initiated transcripts
from the U6 promoter (labeled U6 5') were detected by RNase T1 protection essentially
as described (31).

52

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53

elements. As shown in Figure 2-1D, increasing amounts of GST-RB (379-928)
significantly reduced RNA polymerase III transcription that is correctly initiated from the
human U6 promoter (lanes 2-4). Again, comparable levels of the GST control protein had
no significant effect in these assays (lanes 5-7). Therefore, RB effectively represses in

vitro transcription by RNA polymerase III.

The human U6 snRNA and adenovirus VAI promoters have distinct factor
requirements for transcriptional repression by RB.

In order to repress gene transcription by RNA polymerase III, RB must target specific
factors required for transcription of these genes. To identify these factors,
chromatographic fractions previously demonstrated to reconstitute human U6 and Ad
VAI transcription were tested for their ability to restore transcription in extracts that were
treated with GST-RB (379-928). Potentially, these chromatographic fractions also
contain the factor(s) that are targeted by RB and these may act as a dominant inhibitor of
RB function. Both Ad VAI and human U6 transcription can be reconstituted by using a
combination of fractions obtained from the purification of HeLa cell extracts over a
phosphocellulose P-ll column. These fractions include the P1 l-B fraction (containing
RNA polymerase III as well as 0.38M TFIIIB; ref. 32) and the P1 l-C fi'action (containing
RNA polymerase III, TFIIIC and SNAPc). For human U6 transcription, the Pll-C
fraction can be replaced with a chromatographic fraction further enriched for SNAPc
(Mono-Q) and additional recombinant TBP (48). When recombinant TBP and
chromatographic fractions containing SNAPc were initially tested for their ability to

directly restore human U6 transcription in RB treated extracts, none were able to counter

54

RB repression (data not shown), perhaps because these reactions contain an excess of RB
(379-928). Therefore, whether chromatographic fractions could restore transcription in
RB-repressed extracts that have had the GST-RB (379-928) removed by affinity
purification was tested. First, the amounts of GST-RB (379-928) required for specific
repression under these conditions were established. To perform these experiments, GST-
RB (379-928) or GST was pre-incubated with HeLa cell nuclear extracts. Subsequently,
GST-RB (379—928) was removed by affinity purification with glutathione agarose beads
and extracts were then tested for ability to support U6 transcription. As shown in Figure
2-2A, diminished U6 transcription was observed with extracts affinity depleted with
increasing amounts of GST-RB (379-928) (lanes 3-5) compared to extracts treated with
similar amounts of GST protein (lanes 6-8). Therefore, GST-RB (379-928) can
specifically repress human U6 transcription under these conditions and transcription

levels remain low afier removal of GST-RB (3 79-928) by affinity depletion.

To determine the identity of factors targeted by GST-RB (379-928), the ability of
chromatographic fractions to reconstitute human U6 gene transcription in GST-RB (379-
928) affinity depleted extracts was then assessed. In the experiment shown in Figure 2-
2B, the GST-RB (379-928) depletion conditions are similar to that shown in lane 5 of

Figure 2-2A; however, less extract was tested for transcription thus the starting levels of

55

Figure 2-2. Different factors reconstitute adenovirus VAI and human U6 snRNA
gene transcription in GST-RB (379-928) treated extracts.

(A) GST-RB (379-928) affinity depletion specifically inhibits U6 transcription. HeLa cell
nuclear extracts (8 uL) were incubated with Dignam buffer D (mock lane 2) or 1, 2, or 3
ug purified GST-RB (379-928) (lanes 3-5) or GST (lanes 6-8) for 30 minutes at 30°C.
The recombinant proteins and associated factors were then removed by affinity
purification with glutathione agarose. One half of each treated extract was then tested for
the ability to support human U6 snRNA transcription. Lane 1 shows the transcription
supported by 4 ILL of the untreated extract.

(B) SNAPc but not TFIIIB acts cooperatively with TBP to reconstitute human U6 snRNA
gene transcription. HeLa cell extracts were incubated with GST-RB (379-928) (lanes 2-
9). After treating with glutathione agarose beads, 5 1.1L of each treated extract was tested
for the ability to support U6 transcription in the absence (lane 2) or presence of
chromatographic fractions containing SNAPc (2.7, 8, and 8 uL, lanes 4-6) or TFIIIB (2.7,
8, and 8 uL; lanes 7-9). Reactions shown in lanes 3, 6, and 9 were also complemented
with 10 ng recombinant TBP (Promega). Lane 1 shows transcription supported by 2 uL
of the untreated extract.

(C) TFIIIB but not SNAPc reconstitutes adenovirus VAI gene transcription. HeLa cell
nuclear extracts were incubated with Dignam buffer D (lane 2), GST-RB (379-928)
(lanes 3-7), or GST (lane 8) for 30 min at 30°C. GST-RB (379-928) and GST were
removed by incubating treated extracts with glutathione agarose beads for 4 hr at 4°C. 8
uL of depleted extracts were then tested for ability to support VAI gene transcription in
the absence (lane 3 and 8) or presence of chromatographic fractions containing SNAPc
(2.7 and 8 [.1]; lanes 4 and 5, respectively) or TFIIIB (2.7 and 8 [11; lanes 6 and 7,
respectively). Lane 1 shows transcription supported by 2 11L of the untreated extract.

(D) SNAPc levels are reduced in RB-treated extracts. HeLa cell nuclear extract was
treated with 6 ug of GST-RB (379-928) or GST as described above. 15 uL of depleted
extracts and proteins associated with the beads after extensive washing were separated by
12.5% SDS-PAGE and tested by western blot analysis using rabbit anti-SNAP43 antisera
(top panel). The membrane was then stripped and reprobed using mouse anti-TBP
antibodies (bottom panel). Lanes 1-5 contain 12, 6, 3, 1.5, and 0.75 1.1L of HeLa cell
nuclear extract, respectively. The GST-RB (379-928)- and GST-treated extracts are
shown in lanes 6 and 7, respectively. Lanes 8 and 9 contain proteins associated with the
GST-RB (379-928) and GST agarose beads, respectively.

56

 

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58

transcription is reduced. Under these conditions, U6 transcription is abolished by GST-
RB (379-928) afiinity depletion (lane 2) compared to the untreated extract (lane 1).
Addition of recombinant TBP alone restored significant activity to depleted extracts (lane
3). Therefore, TBP or a TBP-containing complex required for U6 snRNA gene
transcription is functionally limiting in these RB-treated extracts. Neither fractions
containing SNAPc (lanes 4, 5) nor TFIIIB (lanes 7, 8) restored transcription when added
alone even though these fiactions contain significant levels of TBP. However, when
chromatographic fractions containing SNAPc are complemented with recombinant TBP,
enhanced activity is now observed (lane 6). This level is approximately 3-fold greater
than that observed for TBP alone (lane 3) and significantly greater than SNAPc alone
(lane 5). The cooperative effect observed for TBP with SNAPc is specific because this is
not observed with T BP complemented with chromatographic fractions containing TFIIIB
(lane 9). Therefore, SNAPc alone can not restore transcription, but together SNAPc and
recombinant TBP act cooperatively to restore U6 snRNA transcriptional activity to

fractions treated with GST-RB (379-928).

The above results suggest that RB may target TBP or alternatively target SNAPc to
control TBP activity whereas TFIIIB may not be involved in RB repression of these
genes. However, TFIIIB was previously described as a target for RB repression of RNA
polymerase III activity (7, 29). To determine whether RNA Polymerase III-transcribed
genes with intragenic promoter elements have similar factor requirements for relief from
RB repression, the ability of chromatographic fractions to restore Ad VAI transcription

was also tested. As shown in Figure 2-2C, the GST-RB (379-928) treated extract is

59

compromised for Ad VAI transcriptional activity (lane 3) as compared to either mock
treated (lane 2) or untreated HeLa cell nuclear extracts (lane 1). Transcriptional activity
was not restored by addition of chromatographic fractions containing SNAPc (lanes 4, 5).
This result was expected because SNAPc is not required for transcription of these genes.
However, Ad VAI transcriptional activity is effectively reconstituted by addition of
increasing amounts of chromatographic fractions containing TFIIIB (lanes 6, 7).
Transcription in these samples is comparable to levels obtained with either the mock
treated (lane 2) or GST-treated (lane 8) extracts. Therefore, fractions containing TFIIIB
effectively reconstitute Ad VAI transcription and this is specific because restoration is
not observed with SNAPc-containing fractions. This data is consistent with that
previously described (7, 29) and supports the hypothesis that TFIIIB is one target for

gene regulation by RB.

One explanation for the reduced U6 transcription following affinity depletion with GST-
RB (379-928) is that TBP or higher order complexes containing TBP and SNAPc are
removed. Thus, affinity depletion of nuclear extracts using GST-RB (379-928) should
also result in a measurable reduction in the levels of these factors. Therefore, a western
blot analysis was performed using anti-SNAP43 antibodies (C848; Henry et a1. 1995) and
anti-TBP antibodies (SL2; ref. 33) to determine whether treatment of nuclear extracts
with GST-RB (379-928) alters the levels of SNAPc or TBP. As shown in Figure 2D (top
panel), depleting extracts with GST-RB (379-928) results in a significant decrease in
SNAP43 levels (lane 6) compared to amounts present in extracts depleted with the GST

control protein (lane 7). Comparison of SNAP43 in the GST-RB (379-928) treated

60

sample and in decreasing amounts of HeLa cell nuclear extract (lanes 1-5) indicate that at
least 50% of endogenous SNAP43 was removed by affinity depletion with GST-RB
(379-928). This same membrane was then re-probed using antibodies directed against
TBP and the results are shown in Figure 2-2D (lower panel). In this case, no significant
difference in TBP levels was observed for the GST-RB (379-928) and GST treated
samples suggesting that TBP is not effectively depleted in these assays. This may mean
that RB does not target TBP. Alternatively, since TBP is present in numerous TBP-
containing complexes and RB may target only some of these complexes, the removal of a
minor proportion of the total TBP may not be detectable in these assays. Indeed, TBP
was associated with the GST-RB (379-928) agarose beads (lane 8) but not with the GST-
agarose beads (lane 9) suggesting that a minor proportion of the total TBP can associate
specifically with GST-RB (379-928). Our results are also consistent with the notion that

GST-RB (379-928) is targeting SNAPc in a stable fashion.

Endogenous RB associates with SNAPc

The above experiments suggest that high levels of GST-RB (379-928) can target SNAPc
to potentially control human U6 gene transcription. However, it is important to determine
whether an association between endogenous RB and SNAPc is possible. To determine
whether endogenous RB and SNAPc can interact, co-immunoprecipitation experiments
were performed by incubating HeLa cell nuclear extracts with either anti-HA or anti-RB
antibodies. Proteins bound by these antibodies were eluted by boiling in Laemmli buffer

and separated by SDS-PAGE for analysis by anti-SNAP43 western blotting. As shown in

61

Figure 2-3. Endogenous RB is associated with SNAPc.

(A) Endogenous SNAP43 is co-immunoprecipitated with RB. Approximately 300 ILL
HeLa cell nuclear extract was incubated with mouse anti-RB (G3-245; Pharmingen) (lane
5) or anti-HA (12CA5) (lane 4) antibodies overnight. Protein-antibody complexes were
removed by affinity purification using Protein-G agarose beads (Gibco-BRL). The beads
were washed extensively and bound proteins were resolved by 12.5% SDS-PAGE.
SNAP43 association was detected by western blot analysis using antibodies specific to
SNAP43 (CS48; ref. 20). Lanes 1-3 show the amount of SNAP43 present in 10, 3, and l

uL nuclear extract.

(B) Endogenous RB is co-immunoprecipitated with SNAPc. HeLa cell nuclear extracts
were incubated with antibodies directed against the SNAP43 subunit of SNAPc (lanes 6
and 7) or rabbit pre-immune antibodies (lanes 4 and 5) covalently coupled to protein A
agarose beads (Boehringer Mannheim). After protein binding and extensive washing,
each reaction was divided in half. Bound proteins were competitively eluted in buffer
containing either a specific peptide (CSH375: ref. 20) (lanes 4 and 6) or an irrelevant
peptide (CSH374) (lanes 5 and 7). Eluted proteins were precipitated with TCA, size
fractionated by 12.5% SDS-PAGE and the levels of RB were detected by western blot
analysis. Lanes 1-3 show the levels of RB detected in 10, 3, and l uL of HeLa cell
nuclear extract after precipitation with TCA. The experiment in part B was performed by
R. William Henry.

62

 

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63

Figure 2-3A, significant levels of SNAP43 were detected in the samples
immunoprecipitated with the anti-RB (lane 5) but not with the anti-HA (lane 4)
antibodies. To confirm these results, the reciprocal experiment was performed testing the
co-immunoprecipitation of RB through immunoprecipitation of the SNAP43 subunit of
SNAPc (Figure 2-3B). In these experiments the immunoprecipitation methodology was
modified to reduce the high non-specific background that was observed due to cross-
reaction of the secondary antibody with the heavy chain from the anti-SNAP43
antibodies. HeLa cell nuclear extracts were incubated with agarose beads covalently
crosslinked with either rabbit anti-SNAP43 (CS48: ref. 20) or preimmune antibodies.
Each sample was then divided in half. Bound proteins were competitively eluted either
with the specific peptide used to generate the anti-SNAP43 antibodies (1mg/ml, peptide
CSH375: ref. 20) or a non-specific peptide. These elution conditions were chosen to
minimize the disruption of protein-protein interactions and reduce contamination of the
eluted proteins with the heavy chain fi'om the SNAP43 antibodies. Eluted proteins were
then concentrated by precipitation with trichloroacetic acid, size fractionated by 12.5 %
SDS-PAGE, and analyzed by western blot analysis using antibodies directed against RB.
As shown in Figure 3, a significant amount of endogenous RB is co-immunoprecipitated
using anti-SNAP43 antibodies and eluted with the specific peptide (lane 6). The co-
immunoprecipitation of RB with SNAPc is specific because it is not non-specifically
eluted from the anti-SNAP43 antibodies (lane 7) and it also is not observed under any
immunoprecipitation conditions using pre-immune antibodies (lanes 4, 5). Therefore, a

sub-population of endogenous RB associates with endogenous SNAPc.

64

If the association between RB and SNAPc is stable, then it is possible that a significant
amount of endogenous RB will co-fractionate with SNAPc during extensive
chromatographic purification of SNAPc. Therefore, to further test the association
between endogenous RB and SNAPc, SNAPc was purified from HeLa cell extracts and
the co-purification of RB was monitored by western blot analysis. The purification
scheme typically used to fractionate SNAPc is shown in Figure 2-4A. This scheme
applies to the purification of SNAPc both from HeLa cell nuclear and S-100 extracts.
Briefly, HeLa cell extracts were selectively precipitated by ammonium sulfate prior to
fractionation using a phosphocellulose P-ll column. Proteins bound to this column were
step eluted in buffers containing increasing concentrations of KCl to generate the P1 l-A,
B, C, and D fractions. The majority of SNAPc is present in the P1 l-C fraction. This was
then directly passed over a Cibacron blue affigel column (CB) and bound proteins were
eluted with a linear gradient from 500 mM KCl (0% ethylene glycol) to 2.5 M KCl (25 %
ethylene glycol). Fractions generated from this column were dialyzed against Q100
buffer and then tested for DNA binding activity as previously described (48).
Electromobility shift analysis (EMSA) of the CB fractions revealed that the majority of
PSE binding activity is present in CB fractions 29-59 (Figure 2-4B). This peak
corresponds to fractions eluted at KCl concentrations between 1.8 to 2.5 M KCl and is
indicative of SNAPc activity. These fractions were then tested for the presence of RB by
western blot analysis and the results are shown in Figure 2-4C. A significant level of RB

is present in the CB fractions with a peak observed in fractions 29-39 indicating that RB

65

Figure 2-4. Endogenous RB co-fractionates with SNAPc during chromatographic
purification.

(A) Schematic representation of the chromatographic purification used to purify SNAPc.

(B) Characterization of chromatographic fractions for PSE binding activity.
Approximately 10 UL aliquots of the P1 l-C (load; ca. 0.5 mg/ml protein) and fractions
obtained from the cibacron blue (CB) step of purification were tested for DNA binding
activity by electromobility shift assay using radioactive probes containing a high affinity
mouse U6 PSE and TATA box. The positions of the unbound probe (free probe) and
SNAPc bound to DNA (SNAPc) are labeled. The peak of SNAPc is contained in
fractions 27-59 and corresponds to fractions eluted in buffer containing between 1.8-2.5
M KCl.

(C) Anti-RB western blot analysis of ‘ SNAPc -containing fractions shown in B.
Approximately 10 11L aliquots of each fraction were tested for the presence of RB using
mouse anti-RB monoclonal antibodies that recognize an epitope between amino acids
300-380 of human RB (Pharrningen, antibody G3-245). Significant levels of RB are
detected in CB fractions 29-39.

(D) Anti-RB western blot analysis of highly purified SNAPc fiactions. The peak of
SNAPc activity from the cibacron blue column (CB 29-59) was pooled and further
purified by anion exchange column chromatography using a mono-Q HR 5/5 column
(Pharmacia). The peak of SNAPc elutes from this column as a single peak in buffer
containing 250 mM KCl. Increasing amounts of the Mono-Q peak fractions (lanes 1-3:
approximately 0.8, 2.4, and 7.5 ug total protein, respectively) and nuclear extract starting
material (lanes 4-6: approximately 10, 30, and 100 ug total protein, respectively) were
analyzed by 12.5% SDS-PAGE and western blotting using antibodies directed against
RB.

These experiments were performed by R. William Henry.

66

A NJCIOI' Extract

18-3296 amrrlroriun sulfate
phosphocialluloao P-11

A B E 6

(MM) (0-35 M) (05“ (1.0M)

clbacron blue

2.5!
40'!- E6

 

r

1.8 .3
0.5' i2 I

12% E6 MONO-Q
OISUI 4:0 I
01' 0.25 I

025 I

I
Mono-Q pair
SNAPc activity

B
CB Fraction #

911131517192123252729313335373941434547495153555759

C‘Midc 4 — SNAPc

‘5
a

”B
a
3:3.

Free
Probe

 

67

lilla-l.

3634

Load

CB Fraction #

rim

3%
11° g 9111315171921232527 29 313335373941434547495153555759

 

 

a! .....- “
Nuclear

I MonoQ Extract

__-‘ ...-u‘

sa—I‘

---—-J—RB
l 2 3 4 5 6

68

co-fi'actionates with SNAPc. However, RB is present only in a subset of the fractions
containing significant levels of SNAPc activity. For example, CB fractions 39, 41, and 43
contain similar levels of DNA binding activity and yet contain distinctly different levels

of RB. This suggests that RB is associated with a sub-population of SNAPc.

To firrther characterize the association of RB with SNAPc, the peak of DNA binding
activity from the Cibacron blue column was purified by anion exchange chromatography
using a Mono-Q column (Pharmacia). The vast majority of SNAPc is eluted from this
column isocratically in buffer containing 250 mM KCl. By this stage of purification
SNAPc has been purified approximately 2000-fold (data not shown). To test for the
presence of RB in these fractions, increasing amounts of the Mono-Q peak (3, 10, and 30
11L: ca. 0.25 mg/mL protein) were analyzed for the presence of RB and the results are
shown in Figure 2-4D. Again, significant levels of RB are present in these fractions
containing highly purified SNAPc (lanes 1-3). More RB is detected in these Mono-Q
fractions than that in HeLa cell nuclear extracts (1, 3, and 10 uL; ca. 10 mg/mL protein)
shown in lanes 4-6. These data indicate that RB is approximately 1-3 fold more
concentrated in the Mono-Q fractions than in HeLa cell nuclear extracts.
Correspondingly, this represents an estimated 30-100 fold purification of RB during the
purification of SNAPc. Therefore, a sub-population of endogenous RB is associated with

a sub-population of endogenous SNAPc.

69

RB can interact with SNAPc

RB associates with SNAPc as detected both by co-immunoprecipitation from nuclear
extracts and co-fractionation during SNAPc purification. However, it is not known
whether this association is mediated by additional factors or RB directly targets SNAPc.
Therefore, to determine whether direct interactions between RB and SNAPc are possible,
GST-pulldown assays were performed with GST-RB (379-928) and individual subunits

of SNAPc. Each SNAPc subunit was expressed separately in rabbit reticulocyte lysates

and proteins were labeled with 35S-methionine. These were then mixed with GST-RB
(3 79-928) that was pre-bound to glutathione agarose beads. Proteins were also tested for
interactions with GST protein or beads alone as negative controls. The results of these
GST pulldown assays are shown in Figure 5A. Significant interactions were observed
between GST-RB (379-928) and two SNAPc subunits: SNAP43 and SNAPSO. These
interactions are specific as no interaction was detected between these proteins and either
the GST or beads-alone control samples. In contrast, no significant interactions were
observed between GST-RB (379-928) and any other SNAPc subunits. As an additional

control, the reciprocal experiment was performed as shown in Figure 5B. Full-length RB

(1-928) was expressed in vitro and labeled with 35S-methionine. This was tested for
interactions with GST-SNAP43 and GST-SNAPSO. Again, full-length RB interacted with
both SNAP43 and SNAPSO, and this is specific because little cross-reaction with the

GST-alone or beads-alone samples was observed. Therefore, the association of RB with

70

Figure 2-5. RB interacts with two components of SNAPc.

(A) GST-pulldown experiment performed to test interactions between individual SNAPc
subunits and GST-RB (379-928). Each SNAPc subunit was expressed in vitro using

rabbit reticulocyte lysates and proteins were labeled with 35S-methionine. Lane 1

contains 10% of the 35S-labeled proteins used as inputs. These were tested for
interactions with GST-RB (379-928) (lane 2), GST (lane 3), or glutathione agarose beads
alone (lane 4). Proteins were size fractionated by 12.5% SDS-PAGE and visualized by
autoradiography. The identity of each SNAPc subunit is indicated.

(B) Full length RB was expressed in vitro and labeled with 35S-methionine. Lane 1

contains 10% of 35S-labeled RB used as input. This was tested for interaction with GST-
SNAP43 (lane 2), GST-SNAPSO (lane 3), GST (lane 4), and beads alone (lane 5) as

above.

71

—SNAP190

o3 ._
#8

83.2.8
man—.8

.8... *2 a.

- SNAP50

.

- SNAP45

-SNAP43
-SNAP19

!
4

1 2

SNAPc previously observed may involve direct protein-protein interactions between RB

and SNAPc.

Discussion

RB is an important tumor suppressor protein that acts to regulate cell grth in part by
controlling progression through the cell cycle. Typically, RB is thought to act by
regulating expression of genes that are important for executing specific cell cycle
functions. The discovery that RB represses transcription by RNA polymerases I (4) and
III (63) reveals that RB also acts to regulate expression of highly transcribed genes

encoding non-translated RNAs.

The ability of RB to regulate gene expression is determined by targeting RB to specific
gene promoters. Control of RNA polymerase II transcription typically involves recruiting
RB to gene promoters by direct protein-protein interactions with the transcriptional
regulatory protein E2F (11). Interactions between RB and other regulatory proteins are
also important for RB function. Nonetheless, RB does not directly regulate transcription
of most genes by RNA polymerase II. In contrast, RB appears to generally repress RNA
polymerase III activity (63). This suggests that RB interacts with the general
transcriptional machinery required for RNA polymerase III transcription to effect
repression. Indeed, the general transcription factor TFIIIB, composed of TBP and BRF, is
important for expression of SS rRNA and tRNA genes and also appears important for
regulation of these genes by RB. In our experiments, addition of chromatographic

fractions containing TFIIIB restored adenovirus VAI transcription to extracts that were

73

affinity depleted using GST-RB (379-928). This result suggests that TFIIIB is limiting
for VAI transcription in extracts affinity depleted using GST-RB (379-928) and is
consistent with the previously suggested hypothesis that RB targets TFIIIB for repression

of these genes (7, 29).

The human U6 gene family has distinctly different promoter elements contained entirely
in the 5' region flanking these genes. In vitro, RB also effectively represses transcription
of these genes and in our assays repression of U6 gene transcription is observed at
moderately lower concentrations of RB than that required for repression of adenovirus
VAI gene transcription (Figure 2-1 and data not shown). Many possible explanations
might explain the differential regulation of these two genes by RB, but one explanation
that we favor is that RB is specifically interacting with different factors specialized for
transcription of these genes and these interactions are important for repression.
Transcription of human U6 snRNA genes by RNA polymerase 111 requires the general
transcription factors SNAPc, BRF2, and TBP (37, 48), whereas TFIIIB, consisting of
BRF and TBP, appears not to be required (39). Fractions containing TFIIIB were unable
to restore U6 snRNA gene transcription in extracts that were affinity depleted using GST-
RB (379-928). Therefore, TFIIIB appears to be important for RB repression of

adenovirus VAI but not human U6 snRNA gene transcription.

In order to test whether the general transcription factor SNAPc has a role in mediating
RB regulation, chromatographic fractions containing SNAPc were added to GST-RB

(379-928) treated extracts. In these experiments, SNAPc fractions did not restore U6

74

snRNA gene transcription, which would suggest that SNAPc is not involved in RB
repression. One possibility is that RB also present in these fractions is repressing SNAPc
activity. We consider this possibility unlikely because SNAPc fractions obtained by
biochemical fractionation efficiently restore U6 transcription in extracts immuno-
depleted of endogenous SNAPc (data not shown) suggesting that the levels of RB present
are not inhibitory for SNAPc function. A second possibility is that other factors not
contained in the SNAPc fractions are targeted and removed from extracts by affinity
depletion using GST-RB (379-928). Indeed, recombinant TBP alone restored
transcription from the U6 promoter when added to GST-RB (379-928) affinity depleted
extracts. This result suggests that TBP or higher order complexes containing TBP are

important for RB regulation of human U6 snRNA genes.

Although recombinant TBP alone restored U6 transcription, the levels of transcription
observed were increased by addition of fractions containing SNAPc, but not TFIIIB, to
reactions containing recombinant TBP. This result suggests that SNAPc is also involved
in the pathway for RB regulation of human U6 genes. Consistent with these observations,
the levels of endogenous SNAPc were reduced in nuclear extracts affinity depleted with
an excess of GST-RB (379-928). One function of SNAPc is to recruit TBP to the TATA
box contained in human U6 snRNA gene promoters (40). However, high levels of
recombinant TBP can overcome the requirement for SNAPc for U6 transcription (data
not shown). The ability of TBP to function alone in these assays, therefore, may be
because high levels of TBP can bypass the requirement for SNAPc. Thus, RB may

control SNAPc to affect TBP activity at these promoters. If RB targets SNAPc to repress

75

human U6 transcription then it was expected that there should be a measurable
association between endogenous RB and SNAPc. Indeed, a fraction of the total
endogenous RB associates with SNAPc. First, a modest level of RB was observed to co-
immunoprecipitate with SNAPc. Furthermore, co-purification of RB with SNAPc was
observed during the biochemical fractionation of SNAPc. In support of these results, RB
can interact with the SNAP43 and SNAPSO subunits of SNAPc, which suggests that
direct interactions between RB and SNAPc may be important for regulating U6 gene
expression. These results, however, do not rule out the possibility that additional factors
may also contribute to the function of RB at these promoters. It remains to be determined
whether the recently described alternatively spliced forms of human BRF (37) also

participate in RB repression of human U6 gene transcription.

For RNA polymerase II transcription, two models have been proposed to explain the
mechanism for gene repression by RB. In one model, RB binds E2F at the promoter to
abrogate the potential of E2F to activate transcription. E2F recruits the general
transcription factor TFIID to the promoter in a TFIIA-dependent manner and this is
sensitive to the presence of RB. After TFIID-TFIIA complex formation, gene expression
becomes resistant to repression by RB (46). In a second model, RB represses
transcription of some cell cycle responsive genes that contain E2F binding sites via
recruitment of a histone deactylase (HDAC) (2, 35, 36). Thus, transcriptional repression
by RB may involve modification of chromatin structure. HDACs remove acetyl groups
from histones and consequently re-configure the chromatin structure to a non-permissive

state for transcription. The recruitment of HDACs by RB may be direct (13, 36) or

76

require a tethering protein (28). However, not all promoters are sensitive to recruitment
of HDACs and thus it appears that these two models for RB repression are promoter

selective (35).

How does RB regulate U6 snRNA gene transcription by RNA polymerase III?
Potentially, histone deacetylation is an attractive model to explain regulation of U6
snRNA gene transcription in vivo. In chromatin reconstitution experiments, the human
U6 snRNA gene contains a positioned nucleosome located between the distal sequence
element (DSE) and the PSE (53). Transcription of the U6 wild-type gene is enhanced
after chromatin assembly, and thus, modification of histones potentially could repress
gene activity. However, in our in vitro system we observe repression of RNA polymerase
III transcription using naked DNA templates. Thus, it appears that chromatin
modification is not essential for repression of RNA polymerase III in vitro. In an
alternative model, RB acts to inhibit pre-initiation complex assembly at human U6
promoters. For example, RB could disrupt interaction between the transcriptional
activator Oct-1 and SNAPc (Figure 4-6). Direct contacts between the Oct-1 POU domain
and the SNAP190 subunit of SNAPc facilitate SNAPc binding to the PSE (14, 41).
Interactions between RB and SNAPc may prevent interactions between Oct-l and
SNAPc and therefore prevent recruitment of SNAPc to human U6 gene promoters. This
model is reminiscent of the role of RB for repressing RNA polymerase II transcription by

modulating E2F-mediated pre-initiation complex assembly with TFIID and TFIIA (46).

77

Figure 2-6. Model for RB repression of human U6 snRNA gene transcription. There
are several mechanisms by which RB may repress U6 snRNA gene expression by RNA
polymerase III. RB could interact with SNAPc and prevent DNA binding by SNAPc.
Alternatively, RB could disrupt protein-protein communications that are important for U6
transcription. Targeting interactions between SNAPc and either the transcriptional
activator protein Oct-l or TBP would be predicted to repress human U6 snRNA gene
transcription. Finally, RB may also interact with both SNAPc and TBP/BRF2/B"
simultaneously to block further pre-initiation complex assembly.

78

l. 116::
ll R11
SNAP;
l for it
lplt‘u
A
RIIB"

 

79

RB may also interfere with pre-initiation complex assembly by preventing DNA binding
by SNAPc or TBP. By binding to SNAPc, RB may directly prevent this core promoter
complex from binding to the PSE in the promoter of human U6 snRNA genes. Another
interesting possibility is that RB disrupts communication between SNAPc and TBP. The
binding of SNAPc to the PSE and recombinant TBP to the TATA box is cooperative and
this is important for transcription of the U6 snRNA gene (40). Therefore, RB could
disrupt TBP recruitment by interfering with the function of SNAPc for TBP recruitment.
Interestingly, SNAPc interacts well with TBP and this may involve the SNAP43 subunit
(20), which also binds RB in our assays. Thus, potential interactions between SNAP43

and RB may modulate simultaneous interaction between SNAP43 and TBP.

RB plays an important role in coordinating RNA polymerase III activity and our data
indicates that RB does this by targeting TFIIIB and SNAPc fTBP. Clearly, regulating
TBP is important and RB appears to target core-promoter complexes that are important
for TBP function. TFIIIB and SNAPc play crucial early roles in pre-initiation complex
assembly at RNA polymerase III-gene promoters and therefore, these are attractive
targets for regulating RNA polymerase III activity. By understanding the mechanisms by
which expression of non-translated RNAs is controlled, we can define the contribution of
these RNAs to the regulation of normal cell growth. Importantly, the availability of
essential non-translated RNAs could act to limit cell growth and RB repression of genes

encoding these RNAs would likely have to be overcome prior to tumor progression.

80

Acknowledgments

We are indebted to Dr. Nouria Hernandez for her generous support, helpful advice, and
contribution of numerous reagents. We also gratefully thank Beicong Ma for constructing
the GST-RB (379-928) expression plasmid and Drs. David Arnosti, Zachary Burton, and
Craig Hinkley for critical reading of the manuscript. This work was supported by grants
from the American Cancer Society (RPG-00-263-01-GMC) and the Michigan State
University Intramural Research Grant Program. Generous support was also provided by
the Michigan State University College of Human Medicine and College of Natural

Science.

81

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88

Chapter 3

Retinoblastoma protein and RNA polymerase III concurrently occupy a

repressed human U6 promoter

Abstract

The Retinoblastoma protein (RB) regulates the cell cycle and differentiation by
repressing RNA polymerase II transcription of key target genes. Postulated repression
mechanisms suggest that RB directly blocks pre-initiation complex assembly or recruits
additional co-factors such as histone deacetylases or ATP-dependent chromatin
remodeling machines whose activities impair RNA polymerase II access to promoters.
RB also represses transcription of non-translated RNAs that are transcribed by RNA
polymerases I and III, potentially to control cell growth. To analyze the mechanism by
which RB represses RNA polymerase III transcription we examined human U6 snRNA
gene transcription by RNA polymerase III. Herein, we show that RB co-occupies the U6
promoter with RNA polymerase III in vivo and RB repression in vitro does not preclude
RNA polymerase III recruitment. These results suggest a novel mechanism wherein RB
represses transcription at steps subsequent to RNA polymerase recruitment to gene

promoters.

89

Introduction

The Retinoblastoma protein (RB) provides important functions that contribute to cell
cycle control, cellular differentiation, apoptosis, and general grth control. Mutations in
the gene encoding RB are found in a wide variety of human cancers (14), highlighting the
central importance of this protein as a tumor suppressor. Typically, RB functions to
repress RNA polymerase II transcription of genes that act at control points in these key
cellular processes (6, 7, 8). The classical understanding of RB function is exemplified by
cell cycle control by RB (reviewed in 30). For example, RB can target cell cycle control
genes that are required to enter into S phase and whose promoters contain binding sites
for the E2F family of transcriptional activator proteins (4, 11, 20, 31). By binding to E2F
at gene promoters, RB can directly block the transactivation firnction of E2F (21).
Alternatively, RB can recruit co-regulatory proteins, such as histone deacetylases (l, 16,
17) or ATP-dependent chromatin remodeling machines (25, 28, 37) that alter chromatin

structure surrounding these genes to repress transcription.

In addition to regulating transcription of key protein-encoding genes by RNA polymerase
11, RB can also repress transcription of non-translated RNAs that are transcribed by RNA
polymerases I (3) and III (35). Interestingly, transcription of diverse RNA polymerase
III-transcribed genes is elevated in cell lines whose RB function is disrupted, suggesting
that RB can play an important role in regulating RNA polymerase III transcription
generally (35). In contrast, global RNA polymerase II transcription in these cells is

unaffected. It was proposed that RB may regulate transcription of key biosynthetic genes

90

by RNA polymerase I and III to control grth (32). This suggestion is consistent with
observations that RNA polymerase I and III transcription in eukaryotic cells is tightly
regulated in response to cell growth rate and differentiation status, as well as during the
cell cycle (23, 33, 34). Cells lacking RB would be expected to have increased
proliferation potential as a result of increased available biosynthetic machinery and this

increased potential may contribute to unregulated growth during tumor formation.

Even though RB can globally regulate RNA polymerase III transcription, genes
transcribed by this polymerase exhibit diverse promoter architectures and factor
requirements for transcription. This factor diversity presents a puzzle as to how RB can
recognize and regulate general RNA polymerase III transcription. The RNA polymerase
III-specific factor TFIIIB, which is an essential factor required for the transcription of
most RNA polymerase III-transcribed genes that contain intragenic promoter elements, is
one proposed target for RB (15, 26, 35). Human TFIIIB is composed of the TATA box
binding protein (TBP) and at least two additional TBP associated factors called Brfl (l 8,
29) and thpl (22). Together these factors form a platform at the promoter that is
recognized by RNA polymerase III. Within TFIIIB, the Brfl component is one target for
RB (15, 26). Indeed, RB can disrupt interactions between TFIIIB and TFIIIC (26) and
may prevent pre-initiation complex assembly at some RNA polymerase III-transcribed

genes.

In contrast to most RNA polymerase III-transcribed genes, human U6 snRNA gene

transcription does not require Brfl (18) and yet is regulated by RB (13). Thus, additional

91

factors must be important for RB regulation of these genes. Clues as to the identity of the
potential targets for RB regulation of these genes are derived from the architecture of the
U6 promoter. The U6 core promoter region contains a proximal sequence element (PSE)
and a TATA box, which are centered around -57 and -27 bp upstream from the start site,
respectively. The PSE recruits a multi-protein complex called the small nuclear RNA
activating protein complex (SNAPc) (10) that is also known as the proximal sequence
element transcription factor (PTF) (19, 36). Endogenous RB associates with SNAPc both
during co-immunoprecipitation and chromatographic purification of SNAPc, and RB can
interact with the SNAP43 and SNAPSO subunits of SNAPc (13). Thus, SNAPc may
contribute to the ability of RB to specifically target human U6 snRNA genes. Although
SNAPc is important both for human U6 transcription and RB repression, additional data
suggested that TBP or TBP-containing complexes likely contribute to RB regulation of

these genes (13).

It is interesting that human U6 snRNA genes are transcribed by RNA polymerase 111, but
contain extragenic promoter sequences like typical protein-encoding genes transcribed by
RNA polymerase 11. Thus, these genes appear to be a hybrid between genes transcribed
by RNA polymerase II and III. Consequently, an investigation of human U6 snRNA
gene transcription was initiated to examine the mechanism for RB repression of these
unusual genes. In contrast with previous hypotheses that suggest RB disrupts pre-
initiation complex assembly and RNA polymerase recruitment, the data presented herein
suggests that RNA polymerase 111 can occupy a human U6 promoter simultaneously with

RB and under conditions where in vitro U6 transcription is repressed. Together, these

92

observations suggest a novel mechanism for RB repression where RB likely interferes
with critical steps in transcription that occur subsequently to RNA polymerase

recruitment.

Materials and Methods:

Tissue culture: Human mammary epithelieal cells (184B5) were a gift from Susan
Conrad. Cells were maintained in Dulbecco’s Minimum Essential Media (DMEM -
Gibco) plus 10% fetal bovine serum (Gibco), 200 mM glutamine, and penicillin-

streptomycin in 37°C incubator with 5% C02.

Antibodies: Anti-SNAP43 (C848) and anti-TBP (SL2) antibodies have been described
previously (10). Anti-thpl (C8913) and OI-BRFl/BRFZ (C81043) antibodies were gifts
from Nouria Hernandez (22). The anti-Galectin-3 antibody (Mac-2) was a gift from Patty
V053 and John Wang (Michigan State University). Mouse anti-RB antibodies (G3-24S)
used for western analysis were purchased from Pharmingen. The goat anti-RB antibodies
(C-15) and pre-immune control IgGs were purchased from Santa Cruz Biotechnologies.
The RNA polymerase II antibodies (Covance Research Products — 8WG16) were gifts

from Fransisco Herrera and Steve Triezenberg (Michigan State University)

Chromatin immunoprecipitation: Chromatin immunoprecipitations were performed
similarly to that described previously (Braunstein et al., 1993). Human 184BS cells were
grown to 75% confluency and then crosslinked with formaldehyde for 30 minutes. After

cell lysis and sonication, chromatin immunoprecipitations were performed

93

(approximately 1 x 107 cells per immunoprecipitation) in Dilution buffer containing 400
mM NaCl using 1 ug of each antibody overnight at 4°C. Cross-links were reversed at
65°C overnight and recovered chromatin was suspended in 50 111 TE buffer. PCR analysis
was performed using 5 1.1L of immunoprecipitated chromatin or input chromatin. The
primers used for each gene are:

U6 forward — 5’-GTACAAAATACGTGACGTAGAAAG- 3’,

U6 reverse — 5’-GGTGTT‘TCGTCCTTTCCAC- 3’,

U1 forward — 5’-CACGAAGGAGTTCCCGTG-3’,

U1 reverse — S’-CCCTGCCAGGTAAGTATG-3’,

U2 forward — 5’-AGGGCGTCAATAGCGCTGTGG-3’,

U2 reverse — 5’-TGCGCTCGCCTTCGCGCCCGCCG-3’,

GAPDH forward — 5’-AGGTCATCCCTGAGCTGAAC-3’, and

GAPDH reverse — 5’-GCAATGCCAGCCCCAGCGTC-3’.

PCR products were separated by 2% agarose electrophoresis in TBE buffer and

visualized using Kodak imaging software.

Recombinant protein expression and purification:

Mini-SNAPc: Recombinant SNAP43, SNAP50, and SNAP190 (1-505) were expressed
individually in E. coli BL21 DE3 codon+ cells (Stratagene) using the vectors pGST-
SNAP43, pGST-SNAPSO, and pGST—SNAP190 (1-505), respectively (12). The GST-
tagged proteins were purified by binding to glutathione agarose beads and cut with
thrombin in HEMGT-150 buffer (20 mM HEPES pH 7.9, 5 mM EDTA, 10 mM MgCl2,

10% glycerol (v/v), 0.1 % Tween-20 (v/v), and 150 mM KCl). Mini-SNAPc was

94

assembled from approximately 100 ug of each protein at room temperature for 2 hours.
Assembled mini-SNAPc was purified by chromatography using a Mono-Q (HR 5/5)
column (Pharmacia) and eluted proteins were assayed for SNAPc activity by EMSA. The
purified mini-SNAPc was estimated to contain approximately 5 ng/uL of each SNAPc
protein.

T HP: TBP was expressed as a GST-fusion protein in E. coli BL21 DE3 at 37 °C in 28-
M9 media as previously described (12). GST-TBP was bound to glutathione agarose
beads prior to cleavage with thrombin as described above. TBP was concentrated using a
centricon YM-lO spin column (Millipore) and dialyzed against Dignam buffer D
(Dignam et al., 1983).

Brfl and thp1: Recombinant BRF 2 and thp1 were expressed as previously described
(22) using the pSBET-hBRFU and pSBET-hB’ expression plasmids, respectively.
Recombinant thpl was affinity purified using Ni-NTA beads (Qiagen) as described
(22). Subsequently, thpl was dialyzed against Dignam buffer D containing 80 mM KCl
prior to concentration by centrifugation using a centricon YM-30 spin column
(Millipore).

GST-RB (3 79-928) and GST: Recombinant GST-RB (379-928) and GST were expressed

and purified as described previously (13).

In vitro transcription: In vitro transcription assays were performed essentially as
described previously (9). 2 u] of HeLa cell nuclear extract was used for U6 and Ad VAI

reactions, 4 ul HeLa nuclear extract was used for each AdML reaction, and 9 ul whole

cell extract was used for each U1 transcription reaction. The templates used for each

95

reaction are pU6/Hae/RA.2, M13-VAI, Ml3-AdML, and pUl for the U6, VAI, AdML,
and U1 transcription reactions, respectively. Transcription reactions were supplemented

with 200 and 800 ng of GST-RB (379-928) or GST as designated in the figure legend.

Co-immunoprecipitation and GST-Pull down experiments: Approximately 300 LIL of
HeLa cell nuclear extract (ca. 8 mg/mL) was incubated with 2 ug of antibodies directed

against de1 (CS913; (22), BRF l/BRF2 (C81043; (22)), or 1 ug rabbit IgG overnight at
4°C as indicated in figure legend. Reactions were diluted to 1 ml in HEMGT-ISO buffer
and stable complexes were affinity purified by incubation with Protein-G Fast Flow
sepharose beads (Upstate Biotechnology) for 4 hours at 4°C. Beads were washed 3 times
in HEMGT-lSO and boiled for 3 minutes in Laernmli Buffer. Bound proteins were
separated by 12.5% SDS-PAGE, transferred to nitrocellulose and associated proteins
were determined by western blot analysis using antibodies directed against RB (G3-245;
Pharmingen). The same membrane was stripped and probed sequentially with antibodies
directed against TBP (SL-2) and then Galectin 3 (Mac-2). GST pull down experiments

were performed as described previously ( l 3).

Electrophoretic Mobility Shift Assays:

The EMSA experiments shown in figure 2 were performed in 10 mM HEPES, pH
7.9,30mM Tris-HCl, pH 8.4, 60 mM KCl, 7.5 mM MgC12, 20% glycerol, 6mM B-
mercaptoethanol, 20 mM DTT and 50 mM NaF. Each binding reaction was supplemented
with 0.2 pg pUCll9 DNA and 0.6 pg poly deC-deC (Amersham-Pharrnacia).

Approximately 15 ng SNAPc, 200 ng TBP, 100 ng Brf2, and 20 ng del were used as

96

indicated. Reactions were incubated on ice for 20 minutes prior to addition of
radiolabeled probe. DNA binding reactions were canied out at 30°C for 30 minutes.
Approximately 20 ng del was then added to appropriate DNA binding reactions for 40
minutes at room temperature. Next, approximately 3 ug GST-RB (379-928) or GST was
added to appropriate reactions for 20 minutes at room temperature. Resulting DNA-
proteins complexes were separated on a 4% polyacrylamide gel in 0.5x TBE running
buffer at 150V. Complexes were visualized by autoradiography and phosphoimager

analysis.

Sequential Chromatin Immunoprecipitations:

Soluble chromatin fraction was prepared fiom 184BS cells as above. Primary IPs were
performed scaled up 5 fold (~ 5 x 107 cells per 1P) using 5 ug or-RB, or-SNAP43, or-
RNAPII, and IgG. The immunoprecipitations were performed at room temperature for 1
hour and then incubated with protein G agarose for an additional hour at room
temperature. After extensive washing, precipitated protein-DNA complexes were eluted
in dilution buffer containing 15 mM DTT at room temperature for 30 minutes. One sixth
of the recovered material was used for a second ChIP and material was processed exactly

the same as chromatin immunoprecipitations described in figure 3-1.

Cross-linked Immunoprecipitation of Repressed Promoters:

In vitro transcription reactions were performed as described above with the following
exceptions: transcription was carried out for 15 minutes rather than 1 hour and 0.25 ug

pUCl 19 was added to each reaction to serve as a negative control DNA. Each of the 20

97

III transcription reactions was set up in triplicate due to volume constraints and all three
reactions were pooled at the end of the 15 minute transcription time. One third of this
reaction was processed as described above by T1 RNase protection (Figure 4A). The
remaining two-thirds was diluted to 1 ml with ChIP Dilution buffer and was cross-linked
in 1% formaldehyde for 15 minutes at room temperature and quenched in 0.125 M
glycine. Ten 111 of each cross-linked reaction was used as starting material to perform
sequential immunoprecipitations as described in the sequential chromatin
immunoprecipitation section. RSP22 and USP21 primers were used in PCR reactions for

analysis of the negative control pUC 1 l9 plasmid.

Results

Endogenous RB occupies a human U6 snRNA promoter

A key step in the direct regulation of gene expression by any transcription factor is the
ability of that factor to associate with promoter regions of target genes. In order for RB
to efficiently regulate U6 expression, RB may be directed to U6 promoters either by
direct binding to specific DNA control elements or through recruitment by other trans-
acting factors. Additional data suggest that RB can directly interact with SNAPc on
promoter DNA in vitro rather than direct binding to DNA sequences in the U6 promoters
region as determined by EMSA (Chapter 4). However, it is also important to determine
whether RB association at human U6 promoters can be observed in vivo. To determine
whether RB occupies human snRNA promoters in the cell, chromatin
immunoprecipitation experiments (ChIP) were performed. Normal human mammary

epithelial cells (184B5) were treated with formaldehyde to covalently cross-link proteins

98

to DNA and other neighboring proteins. Subsequently, lysates were prepared and
sonicated to fragment genomic DNA into segments approximately 500-800 bp in size
such that there should not be more than one promoter per segment. Lysates were then
subjected to immunoprecipitation using antibodies directed against SNAP43 or RB, and
the resultant protein-DNA complexes were affinity purified using protein-G agarose
beads. Ideally, proteins precipitated in this manner will carry along DNA segments to
which they are covalently cross-linked. The recovered DNA segments were analyzed by
PCR using primers specific to human U1, U2, and U6 snRNA promoters as well as
primers specific to GAPDH exon 2 as a negative control. A ten-fold serial dilution of
input chromatin was also analyzed for each gene of interest to serve as a standard curve,
allowing estimation of relative levels of promoter DNA precipitated and to illustrate that
PCR conditions for each set of primers was in the dynamic range. As shown in Figure 3-
1A, U6 promoter DNA was immunoprecipitated with anti-RB (lane 5) and anti-SNAP43
antibodies (lane 7) but not with pre-immune serum (lane 6). The level of U6 promoter
enrichment is significant as compared to the levels of enrichment of the negative control
GAPDH exon 2 DNA. This observation indicates that RB is present at this human U6
gene promoter in these cells. In contrast, the level of RB associated with the U1 and U2
snRNA gene promoters is not significantly higher than that observed for the GAPDH
negative control, indicating that RB does not associate with the promoters of the RNA
polymerase II-transcribed U1 and U2 snRNA genes. Thus, RB can associate in vivo with
the promoters of RNA polymerase III-transcribed snRNA genes but not with those

transcribed by RNA polymerase II.

99

Figure 3-1: RB selectively occupies U6 snRNA promoters in vivo and specifically
represses U6 snRNA transcription in vitro.

(A) Chromatin immunoprecipitation experiments were performed from human 184B5
cells using antibodies specific to RB (lane 5), SNAP43 (lane7) and IgG control (lane 6).
Precipitated DNAs were analyzed by PCR for enrichment of U6 snRNA, U1 snRNA, U2
snRNA promoters and GAPDH exon 2 as a negative control. Lanes 1-4 show a 10 fold
serial dilution from 10% to 0.01% of input chromatin.

(B) Schematic representation of selected RNA polymerase II and RNA polymerase III
promoters used for in vitro transcription assays.

(C) In vitro transcription assays for the RNA polymerase II and RNA polymerase III-
transcribed genes were performed using HeLa cell nuclear extract. Lanes 2 and 3 were
treated with 200 and 800 ng of GST-RB (379-928), respectively. Lanes 4 and 5 were
treated with 200 and 800 ng of GST, respectively, as a negative control.

100

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The specific association of RB with a human U6 promoter but not with the U1 or U2
snRNA promoters raised the possibility that RB occupancy of snRNA promoters
correlates with the ability of RB to repress gene expression. To determine whether RB
represses expression of RNA polymerase II-transcribed snRNA genes, in vitro repression
assays were performed. A schematic representation of the promoters tested is shown in
Figure 3-1B. Two classes of RNA polymerase II-specific promoters were assayed for
susceptibility to RB repression: the adenovirus major late gene (AdML) promoter and a
human U1 snRNA promoter. Two classes of RNA polymerase III-specific promoters
were also examined: the adenovirus VAI (Ad VAI) gene and a human U6 snRNA
promoter. For these in vitro repression experiments, HeLa cell extracts were pre-
incubated with increasing amounts of GST-RB containing amino acids 379-928, hereafter
referred to as GST-RB, or GST proteins. As shown in Figure 3-1C, addition of increasing
amounts of GST-RB decreased RNA polymerase III transcription from the AD VAI and
U6 snRNA promoters, consistent with our previous observations (13). Repression of both
human U6 and Ad VAI by GST-RB is specific to RB since the addition of equivalent
amounts of GST protein did not reduce the relative amount of gene expression observed
(lanes 4 and S). In contrast, addition of GST-RB did not repress expression from either
the AdML or U1 snRNA promoters. Together, these results suggest that RB effectively
represses expression of these two RNA polymerase III-transcribed genes but not the RNA
polymerase II-transcribed genes. These results are consistent with the observation that

RB is present at a human U6 promoter but not at the U1 and U2 promoters in vivo.

102

RB promoter recruitment depends upon SNAPc and an snRNA-specific TFIIIB
complex

Our previous data suggests that RB may target SNAPc to regulate human U6 snRNA
gene transcription (13). However, SNAPc is present at both RNA polymerase II- and III-
transcribed snRNA genes (U1 and U6, respectively), whereas RB is present at only the
U6 promoter, and RB does not repress human U1 transcription in vitro. Therefore, we
tested whether other factors present at U6 snRNA promoters may lend specificity to RB
recruitment. Unlike U1 snRNA promoters, the U6 core promoter contains a TATA box
that recruits TBP and additional components of a TFIIIB complex including de1 and
Ber (2, 22, 27, 12). To determine whether RB can associate with TFIIIB, co-
immunoprecipitation experiments were performed. HeLa cell nuclear extracts were
incubated with antibodies specific to del or with antibodies that recognize both Brfl
and Brf2 (22). As a negative control, co-immunoprecipitations were also performed using
IgG. Irnmunoprecipitated protein complexes were analyzed by Western blot using
antibodies specific to RB. As shown in Figure 3-2A (top panel), a significant amount of
RB precipitated with de1 (lane 3) as well as with Brfl/Brf2 (lane 4). The association
between RB and both de1 and the Brf proteins appears specific because RB was not
immunoprecipitated using IgG (lane 2). This membrane was then re-probed with anti-
TBP antibodies (middle panel) to determine whether TBP associates with these proteins.
Indeed, significant amounts of TBP co-immunoprecipitated with del and with Brf-
1/Brf2. Finally, the membrane was stripped and re-probed with an antibody specific to
Galectin-3 as a negative control (bottom panel). Galectin-3 functions in mRNA splicing

(5) and is not expected to associate with TFIIIB. In contrast to the observed association of

103

Figure 3-2: RB interacts with components of TFIIIB complexes

(A) Co-immunoprecipitation assays were performed from HeLa cell extract using IgG
(lane 2), adel (lane 3), or orBrfl/2 (lane 4) antibodies. Western blot analysis were
performed to detect RB, TBP and Galectin-3 association.

(B) GST pull-down assays were performed using recombinant GST-RB (379-928) (lane
2), GST (lane 3), or glutathione agarose beads (lane 4). Interactions were tested with the

indicated in vitro translated, 358-methionine labeled proteins. Associated proteins were
size fractionated by SDS-PAGE and visualized by autoradiography.

104

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105

the TFIIIB proteins with both RB and TBP, Galectin-3 was not precipitated under any
conditions. Therefore, these results suggest that endogenous RB can associate with

components of TFIIIB complexes.

To determine whether RB can interact directly with individual components of TFIIIB
complexes, GST-pulldown experiments were performed. As shown in Figure 3-28,
significant amounts of del , Brf-l, and TBP interacted with GST-RB. These interactions
appear specific because no interaction with GST or the beads alone were observed. In
contrast, neither Brf-2 nor Oct-1 interact with GST-RB. Therefore, RB can interact with
all three proteins (de1, Brf-1, and TBP) comprising the TFIIIB complex involved in
tRNA gene expression but only two out of three proteins (de1 , Ber, and TBP) that are

recruited to the TATA element of human U6 snRNA genes.

One possibility for how RB represses RNA polymerase III transcription is that RB
interferes with the DNA binding of the RNA polymerase III transcription machinery (26).
We and others (15, 26) have shown that RB can interact with components of TFIIIB, and
therefore, we used electrophoretic mobility shift assays (EMSA) to determine whether
RB affects the recruitment of TFIIIB to the U6 promoter (Figure 3—3A). Neither GST-RB
nor GST were capable of binding alone to the DNA probe containing a high affinity PSE
and wild type TATA box (compare lanes 2 and 3 to lane 1). However, addition of GST-
RB to reactions containing recombinant mini- SNAPc plus TFIIIB (TBP, Brfl, and
de1) resulted in the formation of a slower migrating complex (labeled RB*, lane 5) that

was not observed in the presence of comparable amounts of GST (lane 6) or in reactions

106

Figure 3-3: TFIIIB acts as a selectivity factor to recruit RB specifically to RNA
polymerase III transcribed snRNA promoters.

(A) Electrophoretic mobility shift assays were performed using a 32P labeled DNA probe
containing a high affinity PSE and wild type TATA. Recombinant mini-SNAPc,
recombinant TFIIIB, and GST-RB (379-928) were added to DNA binding reactions as
indicated. DNA-protein complexes were resolved by polyacrylamide electrophoresis and
visualized by autoradiography.

(B) Phospho-imager analysis quantitation for RB recruitment. The lower mobility
complex formed by the addition of RB was quantitated for net intensity value. Each value
was normalized to the probe alone shift such that the figure reflects fold increase in RB
recruitment.

107

RB Recruitment

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containing the general factors alone (lane 4). This suggests that RB recruitment to the
promoter is dependent on the presence of TFIIIB and SNAPc. To determine the relative
contribution of TFIIIB and SNAPc for RB recruitment in these assays, each of these
factors was tested individually with GST-RB and quantitation of this recruitment is
shown in figure 3-3B. Weak recruitment of GST-RB was observed in the presence of
SNAPc alone (lane 8) or TFIIIB alone (lane 11). More specifically, GST-RB recruitment
by SNAPc plus TFIIIB was 26-fold better than SNAPc alone and 8-fold better than
TFIIIB alone. In these experiments, both GST-RB and GST diminished TFIIIB binding
(lane 12), suggesting that TFIIIB association with this promoter is unstable. It is possible
that SNAPc stabilizes TFIIIB on DNA while TFIIIB provides stronger RB contacts.
Altogether, these results indicate that efficient RB recruitment depends upon multiple
interactions between RB and the general transcription machinery. More importantly, the
presence of RB does not prevent binding of the general transcription machinery to the

promoter.

To determine the contribution of each of the individual factors found at U6 snRNA
promoters to RB recruitment, EMSA experiments were performed in which a single
component was left out of the DNA binding reaction. These single-elimination reactions
were then tested for the ability to recruit RB to a DNA probe containing a wild type PSE
and TATA elements similar to those found at U6 promoters. A representative result from
these EMSA experiments is shown in Figure 3-4A. The amount of RB recruitment was

measured using phospho-imager analysis and is illustrated in the graph shown in Figure

109

Figure 3—4: Contribution of individual components of TFIIIB to RB recruitment to a
U6 snRNA promoter:

(A) EMSA experiments were performed to determine which components of TFIIIB and
SNAPc are important for RB recruitment to a U6 snRNA promoter. DNA binding
reactions 'similar to those performed in the previous figure were assembled minus one
individual component as shown above. The amount of recruitment for each combination
was then determined by phospho-imager analysis.

(B) Graphical representation of the amount of RB recruitment observed for each
combination of SNAPc and TFIIIB components. The lower mobility complex formed by
the addition of RB was quantitated for net intensity value. Each value was normalized to
the amount of RB recruited when all proteins are present. This value was set to 100%
such that the recruitment of RB is expressed as a percentage.

110

- SNAPc SNAPc SNAPc SNAPc -

 

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3-4B. The amount of RB recruitment when all components of SNAPc and TFIIIB are
present (Figure 3-4A lanes 4-6) was considered 100%. In DNA binding reactions
missing de1 (Figure 3-4A lanes 7-9), the amount of RB recruitment was decreased to
40% (2.5 fold decrease) of the amount of RB recruitment observed when all of the factors
(SNAPc, TBP, del, and Brf2) are present (Figure 3-4A lanes 4-6). Interestingly, the
absence of Brf2 (Figure 3-4A lanes 10-12) results in a drastic drop in RB recruitment.
Without Brf2, the amount of RB recruitment observed is 9% (11 fold decrease) of that
seen when all factors are present. This result is surprising since there was no detectible
association between RB and Brf2 in the GST-pull down experiment shown above.
Leaving TBP out of the DNA binding reactions (Figure 3-4A lanes 13-15) results in 15%
(7 fold decrease) RB recruitment as compared to the amount of RB recruitment seen with
all SNAPc and TFIIIB proteins. Lanes 16-18 show the effects of leaving SNAPc out of
the DNA binding reactions. Longer exposure of these experiments show that TFIIIB can
bind to the DNA probe by itself although much less efficiently than when SNAPc is also
present. The amount of RB recruited to the U6 promoter in vitro in the absence of
SNAPc is approximately 15% (7 fold decrease) of the amount of RB recruitment
observed when all components of SNAPc and TFIIIB are present. Overall these
experiments indicate that while TFIIIB is important for RB recruitment to the U6
promoter, it is not sufficient to do so alone. Therefore, both SNAPc and TFIIIB play

important cooperative roles in the recruitment of RB to human U6 snRNA promoters.

112

RB and RNA polymerase III co-occupy a human U6 promoter in vivo and in vitro
The data above suggest that rather than preventing the binding of TFIIIB to the snRNA
gene promoter, RB is capable of co-occupying the same U6 promoter with TFIIIB and
SNAPc. To establish whether these factors co-occupy the same promoter and whether RB
precludes binding of RNA polymerase III to the U6 snRNA gene promoter in vivo,
sequential chromatin immunoprecipitations were performed (Figure 3-5). Soluble
chromatin fractions isolated from cells were subjected to a primary a—RB
immunoprecipitation (top left panel) and the recovered precipitated material was
Subsequently re-immunoprecipitated with antibodies specific to the transcription
jI'lrachinery involved in snRNA gene expression, antibodies specific to RB, or control IgG.
As expected, secondary immunoprecipitations performed using or-RB antibodies (lane 7)
Significantly enriched U6 promoter DNA compared to reactions performed with IgG
( lane 1) or RNA polymerase 11 (lane 5). Secondary immunoprecipitations performed
using (II-SNAP43 (lane 3) and or-TBP (lane 4) antibodies also enriched U6 promoter
DNA, consistent with results in the above EMSA (see Figure 2C). Interestingly, U6
promoter DNA was also enriched in the or-RNA polymerase III immunoprecipitation.
None of the secondary precipitations significantly enriched for U2 snRNA promoter
]DNA or GADPH exon 2 DNA, suggesting that the observed association between RB and

I{NA polymerase III with this human U6 promoter is specific.
To confirm these observations, similar sequential ChIP assays were performed using or-
S NAP43 antibodies in the first round of immunoprecipitation (top right panel) to enrich

for both a U6 snRNA promoter, which directs RNA polymerase III transcription, and U2

113

Figure 3-5: RB co-occupies the same endogenous U6 snRNA promoter as SNAPc,
TFIIIB and RNA polymerase III.

Sequential chromatin immunoprecipitations were performed from human 184BS cells to
determine RB co-occupancy with other factors. Precipitated material was recovered after
the second immunoprecipitation and analyzed by PCR for enrichment of U6 and U2
promoter DNA or GAPDH exon 2 as a negative control. Lane 1 shows 10% input
chromatin.

114

 

 

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snRNA promoters, which direct RNA polymerase 11 transcription. As expected, or-RNA
polymerase III immunoprecipitation enriched for U6 but not U2 snRNA promoters

whereas or-RNA polymerase II immunoprecipitation enriched for U2 but not U6 snRNA

promoters. Furthermore, secondary immunoprecipitations with either (Jr-SNAP43 or or-
TBP antibodies enriched both U6 and U2 snRNA promoters, consistent with the
suggestion that these factors function for transcription of human snRNA genes by both
RNA polymerases II and 111 (Henry et al., 1998; Henry et al., 1995). Importantly,
secondary immunoprecipitations with or-RB antibodies enriched for U6 but not U2
snRNA promoter sequences. This result confirms the original observation that RB and
SNAPc can reside simultaneously at the same U6 gene. In contrast, none of the secondary
immunoprecipitations enriched U6 DNA when primary immunoprecipitations were
performed using either or-RNA polymerase II or IgG antibodies (bottom left and bottom
light panel, respectively). RNA polymerase II was found simultaneously with SNAP43
and TBP, but not with RB, on U2 promoters. Taken together, these results suggest that
RB does not prevent binding of SNAPc, TFIIIB, or RNA polymerase III to the U6
snRNA gene promoter. Rather, RB can co-occupy the same U6 snRNA promoter as
SNAPc (or-SNAP43 1P), TFIIIB (or-TBP IP), and RNA polymerase III (or-RNAPIII IP).

This result is unexpected because previous models suggested that RB functions to prevent

RNA polymerase access to the promoter.
To directly test whether RB repression of U6 transcription occurs simultaneously with the
association of the RNA polymerase III transcription machinery, in vitro U6 transcription

reactions were performed with HeLa cell nuclear extracts that were either left untreated

116

Figure 3-6: RB and RNA polymerase III co-occupy the same repressed U6 snRNA
promoter in vitro.

(A) In vitro transcription reactions were performed using HeLa cell extracts. Lane 1
shows untreated extract and lane 4 shows no addition of nuclear extract. 500 ng GST-
RB(379-928) or GST was added to lanes 2 and 3 respectively.

(B) Sequential crosslinked immunoprecipitations were performed from the transcription
reactions from part A using or-RNA polymerase III antibodies for the primary
immunoprecipitation and IgG (lane 2), or-RNA polymerase III (lane 3), and or-RB (lane
4) antibodies for the secondary immunoprecipitation. PCR analysis was performed on the
recovered DNA using primers specific to the U6/Hae/RA.2 reporter or pUC119 as a
negative control.

117

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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118

or treated with GST-RB or GST. Portions of these reactions were analyzed by RNase
protection assays for full-length transcripts driven from the U6 promoter while the
remainder was subjected to formaldehyde cross-linking followed by or-RNA polymerase
III immunoprecipitation. Secondary immunoprecipitations were then performed using
I gG, or-RNA polymerase III, or or-RB antibodies. The precipitates were then analyzed for
the presence of the U6 reporter construct (pU6/Hae/RA.2) or a negative control plasmid
(pUC119) that was included in the original transcription reactions. Figure 3-6A shows the
level of U6 gene transcription from samples containing HeLa cell nuclear extract (lanel),
extract plus GST-RB (lane 2), or extract plus GST (lane 3). Transcription from samples
lacking any nuclear extract is shown in lane 4. As expected, GST-RB effectively
:repressed U6 transcription as compared to the effect of GST. The result of the sequential
immunoprecipitations from these transcription reactions is shown in Figure 3-6B. RNA
polymerase III was detected at the U6 promoter-containing plasmid in transcription
reactions performed with untreated nuclear extract (top panel) but not in reactions to
Vvhich no extract was added Gmttom panel). Significantly, RNA polymerase III was also
detected at this promoter DNA in transcription reactions that were completely repressed
by GST-RB (second panel). In these GST-RB repressed transcription assays, RB also
associated with the U6 promoter but not with the irrelevant plasmid. The pattern of factor
association with the U6 promoter plasmid in the GST-treated samples (third panel) was
Similar to that observed with samples containing the untreated nuclear extract. Together,
tlrese observations indicate that under repressed conditions, RB does not displace RNA

polymerase 111, but rather RB and RNA polymerase 111 can co-occupy the same repressed

promoter.

119

Discussion

To better understand how RB acts to prevent tumor progression it is important to
understand how RB regulates gene expression. Previous studies illustrate that RB can
repress transcription by RNA polymerase I (3), II (31), and III (35) suggesting a role for
RB in the regulation of a diverse set of genes. This versatility allows RB to participate in
many processes critical to normal cell function including cell cycle progression,
apoptosis, DNA replication, and differentiation. RB’s contribution to each of these

processes may play a role in the suppression of tumor formation.

Additionally, RB may regulate cell growth by the regulation of RNA polymerase III gene
expression. The non-translated genes transcribed by RNA polymerase III provide
metabolic building blocks that increase the biosynthetic capacity of the cell. An increase
in a cell’s biosynthetic capacity elevates the cell’s growth potential, which unchecked,
could lead to tumorigenesis. Bypass of RB regulation of non-translated RNAs could

represent a significant obstacle to overcome during the progression of tumor formation.

RB can repress transcription by RNA polymerase 111 both in vivo (35) and in vitro (15,
35) including transcription of U6 snRNA genes (13). The data herein illustrate that RB
can associate with the U6 snRNA promoter, a step that may be important for subsequent
repression of transcription. The chromatin immunoprecipitation assays clearly show the
RB protein occupying U6 snRNA promoter in vivo suggesting that RB does in fact target

this gene for transcriptional regulation. In contrast, RB is not present at the U1 and U2

120

RNA polymerase II transcribed snRNA genes. Interestingly, RB occupancy of snRNA
promoters in vivo correlates with the ability to repress transcription as demonstrated by in
vitro transcription experiments. Whereas RB can repress U6 snRNA transcription by
RNA polymerase 111, it is unable to repress transcription by RNA polymerase II initiated
from a U1 snRNA promoter. Indeed, the data presented herein, illustrate that RB

Preferentially targets and represses RNA polymerase III transcribed snRNA genes.

One potential mechanism for the recruitment of RB to target promoters is through
interaction with general factors required for transcription. In the case of the U6 snRNA
gene both SNAPc and TFIIIB could be potential recruitment factors for RB. Previous
studies demonstrated that RB can associate with SNAPc (13). However, if SNAPc alone
were capable of mediating RB repression of U6 snRNA genes, then it should also direct
RB repression activities to other genes at which SNAPc is present such as the U1 or U2
snRN A genes. Because RB was not detected at these genes, it is possible that SNAPc is
not the sole determining factor for selective RB targeting RNA polymerase III transcribed
snRNA genes. One obvious difference between snRNA gene transcribed by RNA
polymerase II and III is the presence of a TATA element in core promoter region of
RNAP 111 type snRNA genes. One possibility is that proteins or protein complexes
recruited to this distinguishing promoter element are candidate selectivity factors

directing RB solely to RNAP III transcribed snRNA genes rather than all snRNA genes.

121

Indeed, TFIIIB is one candidate selectivity factor that binds to the TATA elements of U6
snRNA promoters. The results in Figure 3-2 demonstrate that TFIIIB associates with
endogenous RB by co-immunoprecipitation and that individual components of TFIIIB
(del and TBP) interact with RB in GST-pulldown experiments. Thus, an alternative
TFIIIB complex consisting of TBP, Brf2, and deI that associates with U6 snRNA
promoters via the TATA element may be important for ensuring efficient RB recruitment
to human U6 snRNA genes. In EMSA experiments, TFIIIB, like SNAPc, can recruit RB
to U6 promoter DNA. RB recruitment is significantly enhanced in the presence of
SNAPc, suggesting cooperative interactions between TFIIIB and SNAPc direct RB to the
U6 snRNA promoter. Additionally, all components of SNAPc and TFIIIB are necessary

for efficient recruitment of RB.

The results of the EMSA experiments indicate that RB can interact with SNAPc and
TFIIIB on a U6 snRNA DNA probe rather than simply displacing these factors to repress
transcription. This result is also supported by the promoter occupancy studies of the U6
snRNA gene using sequential chromatin immunoprecipitation analysis. Sequential
chromatin immunoprecipitation analysis revealed that RB, SNAPc and TFIIIB are
present at the same U6 snRNA promoter in vivo again indicating that association of these
basal factors is not interupted by RB. The unique surprising result from these sequential
ChIP experiments is that RB and RNA polymerase III occupy the same U6 snRNA
promoter. Further in vitro analysis also indicates the RB and RNA polymerase 111 can

occupy the same repressed U6 snRNA promoter.

122

The results presented herein are inconsistent with the previous model in which RB
represses transcription by blocking the binding of RNA polymerase III to cognate
promoters. Sutcliffe and co-workers (26) proposed a mechanism of RNA polymerase III
transcriptional repression in which RB sequesters required factors. RB was proposed to
bind to TFIIIB interrupting important interactions with TFIIIC. Since interaction with
TFIIIC is critical for recruitment to tRNA genes, RB effectively sequesters TFIIIB away
from the promoter thereby not allowing pre-initiation complex formation to occur.
Similarly, RB was proposed to repress some RNA polymerase II transcribed genes by
targeting the TFIID-TFIIA complex formation step to prevent PIC formation for RNA
polymerase II (21). Since RB and RNA polymerase III occupy the same promoter under
repressed conditions, RB is clearly not using a commonly accepted mechanism,
disrupting pre-initiation complex formation, to repress transcription at the U6 snRNA

promoter.

Again, the results described herein disagree with accepted mechanisms of RB repression.
Instead, we propose that, at least for human U6 transcription, RB impedes critical steps in
RNA polymerase III transcription that occur subsequently to polymerase recruitment to
the promoter. For example, RB may create a physical barrier in the chromatin at the start
site of transcription that would block the progression of the polymerase. Potentially RB
could recruit histone modification activities that would modify local chromatin structure
preventing transcription by RNA polymerase 111. However, several studies have
indicated that HDAC mediated RB repression is promoter specific and may not occur at

all RB regulated promoters (l6). HDAC mediated repression of U6 genes may be

123

unlikely as data from other groups suggest that RB can repress expression of U6 gene in

an HDAC independent fashion (26).

Another method by with RB could use to create a physical barrier in chromatin to prevent
U6 gene expression is through the recruitment of ATP-dependent chromatin remodeling
complexes such as SWI-SNF. Previous studies have demonstrated interaction between
RB and SWI-SNF complexes and that RB required SWI-SNF to repress transcription
initiated from some promoters (28, 37). In vivo, the U6 promoter contains a positioned
nucleosome between the DSE and PSE that is vital to proper gene expression (38).
Recruitement of chromatin remodeling factors by RB to a U6 promoter could potentially

mis-align this nucleosome and thus effectively abolish U6 gene expression.

Additionally, RB may hinder steps that allow for promoter escape and subsequent
transcription of the U6 snRNA gene. Furthermore, RB could inhibit steps that convert an
initiating polymerase to an elongating polymerase such as phosphorylation of the
enzyme. Perhaps RB interferes with elongation resulting in a stalled complex in the
middle of the U6 snRNA gene. Whichever case may apply, it represents a novel

mechanism for RB regulation of gene expression.

Acknowledgments
We gratefully thank S. Conrad, P. Voss, J. Wang, F. Herrera, S. Triezenberg, and N.
Hernandez for supplying reagents as well as C. Hinkley, D. Arnosti, S. Triezenberg, and

G. Chen for critical reading of the manuscript. We also thank J. Mann for technical

124

assistance. This work was supported by grants from the National Institutes of Health and

American Cancer Society to RWH.

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Weintraub, S. J., Prater, C. A., and Dean, D. C. (1992). Retinoblastoma protein
switches the E2F site from positive to negative element. Nature 358, 259-61.

White, R. J. (1997). Regulation of RNA polymerases I and III by the retinoblastoma
protein: a mechanism for grth control? Trends Biochem Sci 22, 77-80.

White, R. J., Gottlieb, T. M., Downes, C. S., and Jackson, S. P. (1995). Cell cycle
regulation of RNA polymerase III transcription. Mol Cell Biol 15, 6653-62.

White, R. J., Gottlieb, T. M., Downes, C. S., and Jackson, S. P. (1995). Mitotic

regulation of a TATA-binding-protein-containing complex. Mol Cell Biol 15, 1983-
92.

127

35.

36.

37.

38.

White, R. J., Trouche, D., Martin, K., Jackson, S. P., and Kouzarides, T. (1996).
Repression of RNA polymerase III transcription by the retinoblastoma protein.
Nature 382, 88-90.

Yoon, J. B., Murphy, 8., Bai, L., Wang, Z., and Roeder, R. G. (1995). Proximal
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Zhang, H. 8., Gavin, M., Dahiya, A., Postigo, A. A., Ma, D., Luo, R. X.,
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Zhao, X., Pendergrast, P. 8., and Hernandez, N. (2001). A positioned nucleosome

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128

Chapter 4

The intact Large A/B pocket domain and C-terminus of RB is required

for efficient repression of RNA polymerase III transcription.

Abstract

The Retinoblastoma tumor suppressor protein plays a central role in many cellular
processes including cell cycle regulation, apoptosis, differentiation, and growth control.
This protein contributes to growth control through its regulation of RNA polymerase III
transcription. Here we dissect the region of RB required for repression of transcription
and interaction with basal RNA polymerase III transcription factors. The region of RB
between amino acids 379-870 is sufficient to repress both U6 snRNA and Ad VAI
transcription since the ability to regulate transcription is lost in a RB mutant protein
containing only amino acids 379-772. Interestingly this phenomenon correlates with the
ability to interact with SNAP43, a component of the SNAP complex required for snRNA

expression.

Introduction

RB is an important regulator of eukaryotic cellular growth. Its ability to perform this
function can partially be attributed to regulation of RNA polymerase III activity (8, 18,
19). RB contains 928 amino acids and can be divided into at least three regions: the N-
terminal region from amino acids 1-378, the NB region from 393-772, and the C region

from 768-869 (16, 17). Most functions ascribed to RB including tumor suppressor

129

activity and interactions with regulatory target proteins require either the NB and/or C
regions. When re-introduced into RB -/- mice, the region of RB between amino acids
379-928 was sufficient to rescue the phenotype and allow mice to develop normally (21).
This same region of RB also appears to be the minimal region necessary for effective
tumor suppression (21). Interestingly, the regions required for tumor suppression and
transcription repression are different. Previous data has shown that the region from 379-
792 was sufficient to repress transcription from RNA polymerase II transcribed reporter
constructs (3). Additionally, the region of RB from amino acids 379-772 was shown to
be sufficient to recruit histone deacetylase activity to repress RNA polymerase II

transcription (11).

RB interacts with several components of RNA polymerase III basal machinery. Both
SNAP43 and SNAPSO of the s_rI_RNA activating protein pomplex (SNAPc) interact
strongly with RB (7). RB also interacts with TATA binding protein, TBP, (9 and HAH
unpublished data) and B double prime, de1, which are components of all TFIIIB
complexes. The TFIIB related factor 1, Brfl, a component of TFIIIB complexes involved
in tRNA transcription, also weakly interacts with RB (9 and HAH unpublished data). RB
does not appear to interact with RNA polymerase III itself (15 and HAH unpublished
results). Additionally, RB has been shown to interact with TFIIIC (4, 15), a basal factor
required for tRNA and SS rRNA expression, although this complex will not be studied
filrther in this paper. Further characterization of RB interaction with basal factors and
further insight into the mechanisms RB uses to regulate gene expression is essential in

understanding the function of this critical tumor suppressor protein function.

130

Materials and Methods

Tissue culture: Human mammary epithelieal cells (18485) were a gift from Susan
Conrad. Cells were maintained in Dulbecco’s Minimum Essential Media (DMEM -
Gibco) plus 10% Fetal Bovine serum (Gibco), 200 mM Glutarnine, and penicillin-

streptomycin in 37°C incubator with 5% C02.

Recombinant protein expression and purification:

Mini-SNAPc: Plasmids encoding GST-SNAP43 Ex-XB, pET-GST-SNAPSO, and pET-
GST-SNAP190(1-505) were transformed into E. coli BL21 DE3 codon + (Stratagene)
and grown at 37°C in ZBM9 media until OD at 600 nm is 0.5. Temperature was lowered
to 16°C and protein expression was induced with 1 mM IPTG for 20 hours. Pelleted
bacteria were resuspended in HEMGT-150 (20 mM Hepes pH 7.9; 0.5 mM EDTA; 10
mM MgC12; 10% glycerol; 0.1% Tween-20) containing protease inhibitors (0.1 mM
PMSF, 1 mM sodium bis-sulfate, 1 mM benzamidine, 1 11M pepstatin A) and 1 mM DTT
and sonicated using a Branson sonifier 4 times for 30 pulses output=6 duty 60%. Debris
was pelleted at 13,000 RPM at 4°C for 30 minutes. Soluble protein was bound to
glutathione sepharose (Pharmacia) overnight at 4°C and washed 4 times in HEMGT-150
plus protease inhibitors and twice in HEMGT-150 without protease inhibitors. Proteins
were cleaved from the beads using 20 units thrombin (Sigma) on ice for 1.5 hours
vortexing every 15 minutes. SNAPSO and SNAP190(1-505) were collected as fractions
in HEMGT-150. Appropriated fractions were pooled and dialyzed against Dignam buffer
D80 (5). SNAP43 was collected in step wise salt gradient (150 to 1000 mM KCL in

HEMGT buffer) and appropriate fractions pooled. Mini-SNAPc was assembled from

131

approximately 100 ug of each protein at room temperature for 2 hours. The assembly
reaction was diluted such that the final salt concentration was less than 80 mM KCl and
the final glycerol concentration was less than 5%. The diluted assembly reaction was
allowed to equilibrilate on ice for 1 hour. Assembled mini-SNAPc was then loaded onto
a 1 ml Mono-Q column. Bound proteins were eluted from the column in 0.5 ml fractions
in a 0 to 500 mM salt gradient in Dignam buffer D80 (5% glycerol). Fractions were
adjusted to 20% glycerol and assayed for SNAPc activity by SDS-PAGE and EMSA.
The peak of SNAPc activity corresponded to fractions 20-24 (~380-420 mM KCl).
Recombinant RB proteins were expressed and purified as before (7) with the exception
that RB mutants were grown at a temperature of 30°C to an CD. of 0.6 and induced at

16°C for 20 hours.

Chromatin immunoprecipitation: Chromatin immunoprecipitations were performed
similarly to that described previously (2). Human 184BS cells were grown to 75%
confluency and then crosslinked with formaldehyde for 30 minutes. After cell lysis and
sonication, chromatin immunoprecipitations were performed (approximately 1 x 107 cells
per immunoprecipitation) in Dilution buffer containing 400 mM NaCl using 1 ug of each
antibody overnight at 4°C. Cross-links were reversed at 65°C overnight and recovered
chromatin was suspended in 50 111 TE buffer. PCR analysis was performed using 5 11L of
immunoprecipitated chromatin or input chromatin. The primers used for each gene are:
U6 forward — 5’-GTACAAAATACGTGACGTAGAAAG-3’,

U6 reverse — 5’-GGTGTTTCGTCCTTTCCAC-3’,

U1 forward - 5’—CACGAAGGAGTTCCCGTG-3’,

132

U1 reverse — 5’-CCCTGCCAGGTAAGTATG-3 ’,

U2 forward — 5’-AGGGCGTCAATAGCGCTGTGG-3 ’,

U2 reverse - 5’-TGCGCTCGCCTTCGCGCCCGCCG-3 ’,

GAPDH forward - 5’-AGGTCATCCCTGAGCTGAAC-3’,

GAPDH reverse — 5’-GCAATGCCAGCCCCAGCGTC-3’,

tRNA-lys forward — 5’-GGTTTCCCTCAAGGAGGGGG-3 ’,

tRNA-lys reverse — 5 ’-GCCCGGATAGCTCAGTCGGTAG-3 ’

PCR products were separated by 2% TBE-agarose electrophoresis and visualized using

Kodak imaging software.

In vitro transcription: In vitro transcription assays were performed essentially as
described previously (6). 2 ul of HeLa cell nuclear extract (~15 mg/ml) was used for the
transcription reactions using 0.25 11g pU6/Hae/RA.2 or M13-VAI reporter plasmids.
Transcription reactions were supplemented with 250 and 1000 ng of GST-RB (3 79-928),
GST-RB (379-870), GST-RB (379-772), GST-RB (379-577) or GST as designated in the

figure legend.

Cross-linked immunoprecipitation of repressed promoters: In vitro transcription
reactions were performed as described above with the following exceptions. Three-
quarters of this reaction was processed as described above by T1 RNase protection
(Figure 4A). The remaining fourth was diluted to 150 u] with ChIP Dilution buffer (as
described in chapter 3) and was cross-linked in 1% formaldehyde for 15 minutes at room

temperature and quenched in 0.125 M glycine. Ten ul of each cross-linked reaction was

133

used as starting material to perform immunoprecipitations as described in the
immunoprecipitation section in chapter 3 using 10 ml of glutathione sepharose beads
(Pharmacia). Recovered DNA was analyzed using the following primers:

U6 forward — 5’-GTACAAAATACGTGACGTAGAAAG-3’,

U6 reverse — 5’-GGTGTTTCGTCCTTTCCAC-3 ’,

VAI forward — 5’-TCCGTGGTCTGGTGG-3’,

VAI reverse — 5’-CGGGGTTCGAACCCGG-3’.

Electrophoretic Mobility Shift Assays:

DNA binding reactions were performed in 60 mM KCl, 20 mM HEPES pH 7.9, 5 mM
MgC12, 0.2 mM EDTA, 10% glycerol, 0.5 ug poly(dI-dC), and 0.5ug pUC119 plasmid.
Approximately 25 ng of SNAPc were incubated with increasing amount of GST-RB
(379-928) or GST at room temperature for 20 minutes. 5000 CPM DNA probe (prepared
as described previously (6)) containing with a high affinity PSE or mutant PSE was
added to each reaction and incubated for 30 minutes at room temperature. The resulting
complexes were separated by 5% Tris-Glycine polyacrylamide containing 5% glycerol
and visualized by autoradiography. For the EMSA performed in figure 2, 1 ug goat or-
RB (Santa Cruz) antibodies or goat IgG was added to the binding reactions at the times

indicated.

GST-Pulldown: GST-pulldowns were performed as described previously (7).

134

Results

Characterization of the RNA polymerase III repression domain in RB

In order to determine the minimal region of RB required to regulate RNA polymerase III
transcription, C-terrninal truncation mutagenesis was performed on RB separating the
protein into its pocket regions (Figure 4-1A) based on the structural domains observed in
the RB crystal structure (10). These mutantions were created by Ms. Liping Gu in the
Henry lab. Truncated RB proteins were expressed as GST fusion proteins in E. coli and
affinity purified using gluthione-sepharose resin. Eluted proteins were desalted and
concentrated using spin columns. Each RB protein was purified to near homogeneity
(>90%) as shown by SDS-PAGE analysis and subsequent coomassie blue staining

(Figure 4-1B).

In vitro repression assays were used to test the ability of the RB truncation protein
mutants to repress RNA polymerase III transcription. The two representative RNA
polymerase III promoters examined, U6 snRNA and Adenovirus VAI, are diagramed in
Figure 4-1C. For these experiments, HeLa nuclear extracts were pre-incubated with GST-
RB, the RB truncation mutants, or GST as a negative control. Transcription was initiated
by the addition of template and nucleotides. Correctly initiated transcripts were visualized
by autoradiography. As shown in Figure 4-1D, addition of increasing amounts of GST-
RB (379-928) (lanes 2-3) significantly decreases the amount of transcription correctly

initiated from the U6 snRNA or VAI promoters as compared to untreated extract (lane 1)

135

Figure 4-1: Amino acids 379-870 of RB contain the minimal region necessary to
repress RNA polymerase III transcription.

(A) Schematic representation of the RB truncation mutants used in these experiments.

(B) Analysis of GST-RB truncation mutants and GST proteins used in transcription
reactions. GST-RB (379-928) (lane 2), GST-RB (379—870) (lane 3), GST-RB (379-772)
(lane 4), GST-RB (379-577) (lane 5) and GST (lane 6) were expressed in E. coli and
purified by affinity chromatography using glutathione agarose beads and competitive
elution with glutathione. After dialysis against Dignam buffer D proteins were separated
by SDS-PAGE and visualized by staining with Coomassie blue. Lane 1 contains a protein
size standard.

(C) Schematic representation of the adenovirus VAI and human U6 snRNA promoters.

(D) GST-RB (379-928) and GST-RB (379-870) repress adenovirus U6 snRNA and VAI
transcription by RNA polymerase III. Approximately 2 uL of HeLa cell nuclear extract
(approx. 7.5 ug/uL) was incubated with 250 and 1000 ng of GST-RB (379-928) (lanes 2-
3), GST-RB (379-870) (lanes 4-5), GST-RB (379-772) (lanes 6-7), GST-RB (379-577)
(lanes 8-9) or GST protein (lanes 10-11) at 30°C for 30 minutes. Lane 1 shows the level
of transcription with the untreated extract. Correctly initiated transcripts from the U6
promoter (labeled U6 5') were detected by RNase T1 protection essentially as described
in Chapters 2 and 3 (20). In vitro transcription of the Ad VAI gene (bottom) was then

initiated by addition of template, cold rNTPs, [a32P]CTP, and transcription buffer.

Sample handling was monitored by a non-specific RNA handling control transcript
(bottom panels).

136

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and extract treated with a molar equivalent of GST (lane 10). Removal of the extreme C-
terminus (GST-RB (379-870)) results in lowered, but still significant repression (lanes 4-
5) of U6 snRNA and VAI gene expression. However, deletion of the C-pocket (GST-RB
(379-772)) results in abolition of RB’s repressor ability (lanes 6-7) even though A/B
pocket integrity should still be maintained in this mutant. In fact, genes transcribed by
RNA polymerase II have been shown to be regulated by RB truncation mutants
containing only amino acids 379-772 (3, 11). Similarly, the A pocket alone (GST-RB
(379-577)) was unable to repress U6 or VAI gene expression. Together, these results
suggest that the NB pocket as well as the C region of RB is necessary for repression of
RNA polymerase III repression. Interestingly, this corresponds to the region of RB that is

required for grth suppression.

RB occupancy of RNA polymerase III promoters in vitro and in vivo

To ascertain whether the ability to repress transcription correlates with the ability to
occupy a target RNA polymerase III transcribed promoter, cross-linked immuno-
precipitations were performed from the transcription reactions used in the previous
figure. In vitro U6 transcription reactions were performed with HeLa cell nuclear extracts
that were either left untreated or treated with GST-RB mutants or GST. Portions of these
reactions were analyzed by RNase protection assays for full-length transcripts driven
from the U6 promoter (see above) while the remainder was subjected to formaldehyde
cross-linking followed by affinity purification, using glutathione sepharose to isolate the
RB mutant proteins. The precipitates were then analyzed for the presence of the U6

reporter construct (pU6/Hae/RA.2) or the Ad VAI reporter construct (M13-Ad VAI)

138

Figure 4-2: RB occupancy of RNA polymerase III transcribed promoters in vitro
and in viva

(A) GST-pull down experiments were performed from the crosslinked U6 5’ transcription
reactions in figure 4-1D. Associated plasmid DNA was detected by PCR using primers
specific to U6 promoter.

(B) GST-pull down experiments were performed from the crosslinked Ad VAI
transcription reactions in figure 4-1D. Associated plasmid DNA was detected by PCR
using primers specific to M1 3-Ad VAI promoter.

(C) Chromatin immunoprecipitation experiment to determine RB occupancy of class 2
and class 3 promoters in viva. Chromatin immunoprecipitation experiments were
performed similar to those described in Chapter 3. A ten fold serial dilution of input
chromatin from 10% to 0.01% is shown in lanes 1-4. Immunoprecipitation reactions
were set up using pre-immune sera (lane 5), or-SNAP43 (lane 6), a-RB (lane 7), or or-
TBP (lane 8). Recovered DNA was analyzed using primers specific to U2 and U6 snRNA
promoters, tRNA (lys), and GAPDH.

Part C of this figure was performed by Gauri Jawdekar.

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using primers specific to each of these plasmids. As shown in Figure 4-2A, GST-RB
(3 79— 928) exhibits significant amounts of U6 promoter occupancy compared to untreated,
GST treated or reactions containing no nuclear extract. This result correlates to its ability
to completely repress transcription. Additionally, GST-RB (3 79-870) also associates with
the U6 reporter construct, although to a lesser extent, suggesting that the lowered ability
to repress transcription may be due to a lowered affinity for the U6 promoter. Again this
result suggests an important role for the extreme C-terminus of RB in the regulation of
U6 snRNA gene expression. Interestingly, GST-RB (379-772) can also occupy the U6
reporter construct promoter to some extent, even though it does not repress transcription,
indicating that recruitment to the promoter may not be enough for repression and that
there may be additional co-factors required to repress transcription. These potential co-
repressors may require the intact C pocket region of RB to be recruited to the U6
promo ter. The RB mutant containing amino acids 379-577 did not appear to support
Significant promoter occupancy similar to its inability to repress transcription.
Surprisingly, none of the RB mutants occupied the Ad VAI reporter constructs even
though they were capable of repressing transcription to varying extents (Figure 4-2B).
This t‘esult implies that RB may have fundamentally different mechanisms for regulating

the e"EIDIession of class 2 versus class 3 RNA polymerase III transcribed genes.

T0 fl-ll‘ther define the mechanisms used for repression of class 2 and class 3, the in vivo
RB 0Cczupancy of a tRNA(lys) and a U6 snRNA promoter were analyzed by chromatin
imuno131*ecipitation. Normal human mammary epithelial cells (184B5) were treated with

formal(idehyde to covalently cross-link proteins to DNA and other neighboring proteins.

141

Subsequently, lysates were prepared and sonicated to fragment genomic DNA into
segments approximately 500-800 bp in size such that there should not be more than one
promoter per segment. Lysates were then subjected to immunoprecipitation using
antibodies directed against SNAP43, RB or TBP, and the resultant protein-DNA
complexes were affinity purified using protein-G agarose beads. The recovered DNA
segments were analyzed by PCR using primers specific to human tRNA, U2 and U6
snRNA promoters as well as primers specific to GAPDH exon 2 as a negative control. A
ten fold serial dilution of input chromatin was also analyzed for each gene of interest to
serve as a standard curve, allowing estimation of relative levels of promoter DNA
precipitated and to illustrate that PCR conditions for each set of primers was in the
dynamic range. As shown in Figure 4-2C, immunoprecipitations performed with on-
SNAP43 antibodies were significantly enriched for U6 snRNA and U2 snRNA promoter
DNA but not tRNA(lys) or GAPDH as compared to lgG precipitations as expected (23).
Similarly, precipitations performed with a—TBP antibodies were enriched for U6 snRNA,
U2 snRNA, and tRNA(lys) promoter DNA as has been shown previously (23). As
observed previously, immunoprecipitations performed using Ot-RB antibodies were
enriched for U6 snRNA promoter DNA but not U2 or GAPDH promoter DNA.
Interestingly, the oc-RB immunprecipitations were not enriched for tRNA promoter DNA,
correlating to the results seen in the in vitro assays shown above. Together, these results
suggest that RB may regulate RNA polymerase III gene expression by two different

mechanisms.

142

Characterization of the region of RB interacts with SNAPc bound to the PSE

In order for RB to efficiently regulate U6 expression, RB may be directed to U6
promoters either by direct binding to specific DNA control elements or through
recruitment by other trans-acting factors. Interaction between RB and SNAPc has been
shown previously, and therefore, it is possible that RB could be recruited to human U6
snRNA promoters through interaction with SNAPc. To determine whether RB can
interact with SNAPc bound to DNA, electrophoretic mobility shift assays (EMSA) were
performed. It should be noted here that these assays were performed using a much
different gel system as compared to chapter 3. This tris-glycine gel system is optimized
for the best possible SNAPc-RB interaction, all other gel conditions did not support
SNAPc and RB interaction. The reason that this type of gel system was not used in
chapter 3 is that the TFIIIB complexes will not form. For these experiments, a mini-
SNAPc (12) was assembled fiom recombinant proteins (SNAP43, SNAPSO, and
SNAP190 (1-505)) and was incubated with a radio-labeled probe containing either a high
affinity or mutant PSE in the presence and absence of GST-RB (379-928). Since RB
interacts efficiently with both SNAP43 and SNAPSO (7), a complex containing these
factors could suffice to recruit RB to the U6 promoter. As shown in Figure 4-3, DNA
binding reactions performed with increasing amounts of GST-RB (379-928) result in a
protein-DNA complex with slower mobility than that observed for reactions containing
only mini-SNAPc (compare lanes 4-6 to lane 3), suggesting that GST-RB (379-928) is
forming a complex with mini-SNAPc on DNA. In contrast, addition of a GST control
protein had no effect on DNA binding by mini-SNAPc (lanes 8 -lO). In all cases, DNA

binding is dependent upon the PSE because no binding of any type is observed for

143

Figure 4-3: SN APc can recruit RB to promoter DNA.

Left panel: Electrophoretic mobility shift assays were performed with purified
recombinant mini-SNAPc alone (lane 3) or with mini-SNAPc plus 300 ng, 1000 ng and
3000 ng GST-RB (379-928) (lanes 4 -7) or GST (lanes 8-1 I). Radio-labeled DNA probe
containing either wild type or mutant PSE was then incubated with each reaction. The
resulting protein-DNA complexes were resolved by 5% acrylamide gel electrophoresis
and were visualized by autoradiography. Addition of RB to reactions containing SNAPc
results in formation of a lower mobility complex (lanes 4-6) that is PSE specific (lane 7)
whereas GST does not affect SNAPc/DNA binding. Addition of GST-RB also results in
increased SNAPc binding activity whereas GST alone does not. As shown in lanes 12
and 13, RB alone does not bind to DNA. GST also does not bind DNA (lanes 14 and 15).
Right Panel: A cross titration of GST-RB (379-928) and GST was performed to illustrate
that the increased DNA binding activity observed in the left panel is a specific effect of
GST-RB and not a non-specific protein effect.

144

  

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reactions containing a mutant PSE probe (lanes 7 and 11). Neither GST-RB (379-928)
nor GST bind to DNA containing either a wild type or mutant PSE (lanes 12-15).
Therefore, RB can associate with SNAPc bound to the promoter of a U6 gene in vitro.
Interestingly, RB also appears to enhance mini-SNAPc binding to the PSE. In order to
determine whether the observed effects of GST-RB (379—928) are perhaps due non-
specific protein effects, EMSA analysis was performed maintaining the total protein level
constant while varying the proportion of GST-RB (379-928) and GST in each reaction.
When the total amount of protein is kept constant, an increasing proportion of GST-RB
(379-928) correlates with an increased amount of mini-SNAPc bound to the PSE (Figure
4-3; lanes 2-5), suggesting that RB can enhance DNA binding by SNAPc or that RB has
associated with the complex and that the supershified complex is not resolvable in this

system.

To further demonstrate that the effects of RB on DNA binding by mini-SNAPc are
indeed due to RB, EMSA analysis was performed with mini-SNAPc plus GST-RB (379-
928) in the presence of an antibody specific to the extreme C-terminus of RB. As shown
in Figure 4-4, addition of the anti-RB antibody disrupted the slower mobility complex,
consisting of SNAPc and RB, and also abolished the enhanced DNA binding by SNAPc
(compare lane 2 to lane 1). An order of addition experiment was also performed to
determine whether the timing of antibody addition relative to DNA binding by SNAPc
and RB was important. Pre-incubating GST-RB (379-928) with the anti-RB antibodies
(lane 4) or adding the anti-RB antibodies, SNAPc, and RB at the same time (lane 6)

results in the loss of the lower mobility complex and also in reduced DNA binding by

146

Figure 4-4: Addition of (X-RB antibodies precludes interaction with SNAPc.

The effects of anti-RB antibodies on DNA binding by GST-RB (379-928) and SNAPc
when added at various incubation time points were studied in electrophoretic mobility
shift assays. DNA binding reactions were set up by the addition of RB followed by
SNAPc and lastly, DNA. Equivalent amounts of anti-RB antibodies or non-specific IgG
antibodies were added with RB (A), with SNAPc (B), after SNAPc (C), with DNA (D),
or afier DNA (E).

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148

mini-SNAPc. However, the formation of the lower mobility complex was unaffected
when the anti-RB antibodies were added after GST-RB (379-928) and mini-SNAPc

interacted with each other. For example, addition of the anti-RB antibodies prior to DNA
binding by GST-RB (379-928) plus mini-SNAPc (lane 8) or after DNA binding (lane 10)
had no effect, which suggests that the epitope on RB recognized by this antibody is
masked by pre-incubating RB with mini-SNAPc. Non-specific rabbit IgG antibodies had
no effect on DNA binding by mini-SNAPc and GST-RB (379-928) (lanes 3, 5, 7, 9, and
11) indicating that the effect of the anti-RB antibodies is specific. These observations
suggest RB is in fact part of the lower mobility complex and RB does not displace

SNAPc from DNA.

Since RB can interact with SNAPc bound to the PSE in EMSA experiments, the region of
RB required for this interaction was determined. Using DNA binding assays similar to
those described above, RB mutant proteins were tested for the ability to associate with
SNAPc bound to DNA. Increasing amounts of GST-RB truncation proteins were
incubated with SNAPc prior to DNA binding and were resolved using polyacrylamide gel
electrophoresis. Resulting protein-DNA complexes were visualized by autoradiography.
For this experiment, lowered amounts of RB proteins were used that would result in
enhanced binding of SNAPc but not the formation of the upper complex. As shown in
Figure 4-5, the amount of binding observed from SNAPc alone is illustrated in lane 2.
Addition of increasing amounts of GST-RB (379-928) (lanes 3-5) results in a significant
increase in SNAPc binding as compared to GST (lanes 15-17), recapitulating the results

seen above and indicating RB association with SNAPc bound to DNA. Similarly, GST-

149

Figure 4-5: Characterization of the region of RB necessary for interaction with
SNAPc bound to U6 promoter DNA.

EMSA analysis was done to determine which region of RB can associate with SNAPc on
DNA. Purified recombinant mini-SNAPc alone (lane 2) or mini-SNAPc plus 30 ng,
100mg, or 300 ng GST-RB (379-928) (lanes 3-5), GST-RB (379-870) (lanes 6-8), GST-
RB (379-772) (lanes 9-1 1), GST-RB (379-577) (lanes 12-14) or GST (lanes 15-17) were
incubated for 20 minutes at room temperature. Radio-labeled DNA probe containing a
wild type PSE was then incubated with each reaction. The resulting protein-DNA
complexes were resolved by 5% acrylamide gel electrophoresis and were visualized by
autoradiography.

This experiment was performed by Gauri J awdekar.

150

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GST-RB GST-RB GST-RB GST-RB

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151

RB (379-870) (lanes 6-8) also increases binding indicating that this region of RB can still
associate with the U6 snRNA promoter in vitro, which is consistent with repression and
promoter occupancy data. However, GST-RB (379-772) (lanes 9-11) seems to associate
with SNAPc bound to DNA with a slightly lower affinity correlating to its lack of ability
to repress transcription. GST-RB (379-577) (lanele-l4) does not seem to have as
significant effect on SNAPc binding as the other truncation mutants, again consistent

with transcription data showing a lack of repression.

RB interaction with components of RNA polymerase III basal machinery

To better understand how RB is acting to regulate expression of genes transcribed by
RNA polymerase 111, it is important to determine which regions of RB are necessary for
interaction with components of core promoter complexes expected to be present at RNA
polymerase III promoters. Interaction with basal machinery (i.e. SNAPc and TFIIIB)
represents a mechanism by which RB is recruited to target promoters to repress
transcription. To determine which regions of RB are requisite for interaction with these
basal factors, GST pull down experiments were performed. RB mutant proteins or GST

were mixed with components of RNA polymerase III basal machinery that were

individually expressed in rabbit reticulocyte lysate and labeled with 35S-methionine.

Expression of these 35S-labeled proteins is shown in Figure 4-6 (lane 1). Stably

interacting proteins were isolated using glutathione sepharose beads, washed extensively

and stable protein complexes were eluted into Laemmli buffer. Recovered proteins were

152

Figure 4-6: Characterization of the region in RB that can interact with RNA
polymerase III basal machinery.

GST-pull down analysis to determine the region of RB that can interact with each
component of RNA polymerase III basal machinery. SNAP43, SNAPSO, TBP, Brfl,

Brf2, and Oct-l were expressed in vitro and labeled with 35S-methionine. 10% input for
each in vitro expressed protein is shown in lane 1. Each protein was incubated with
GST-RB truncation proteins or GST and stable protein complexes were purified using
glutathione sepharose. The beads were extensively washed and bound proteins were
separated by SDS-PAGE. Associated proteins were visualized by autoradiography.

This experiment was performed by Gauri Jawdekar.

153

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154

separated by SDS-polyacrylamide gel electrophoresis and detected by autoradiography.
As shown in figure 4-6, SNAP43 interacts strongly with the full length GST-RB (379-
928) (lane 2) but not with GST (lane 6) or beads alone (lane 7) demonstrating the
specificity of interaction of SNAP43 with GST-RB (379-928). SNAP43 also interacts
with GST-RB (379-870) (lane 3) suggesting that the extreme C-terminus of RB is not
critical for SNAP43 interaction. However, when the region of RB containing the C
pocket of RB is removed, interaction is abolished, suggesting that the C pocket of RB is
required for SNAP43 interaction. Further truncation of the RB protein resulting in the A
pocket alone (lane 5) also lacks the ability to interact with SNAP43. A similar pattern of
RB interaction is also observed for TBP in that the removal of the C pocket results in
lowered capacity for interaction. Together these results indicate that the NB pocket
domain as well as the C pocket is crucial for RB’s interaction with both SNAP43 and

TBP.

Unlike SNAP43, the C pocket of RB does not appear to be required for SNAPSO
interaction. SNAPSO interacts strongly with GST-RB (379-928) and GST-RB (379-870)
but not GST or beads alone as was the case with SNAP43. Interestingly, SNAPSO can
still interact with GST-RB (379-772) in which the C-pocket has been removed. GST-RB
(379-57 7) did not interact with SNAPSO indicating that the intact A/B pocket is required
for interaction. These data imply that the NB pocket of RB is required and sufficient for
interaction with SNAPSO. Similarly, Brfl and de1 also interact with GST-RB (379-

928), GST-RB (379-870), and GST-RB (379-772) although to a lesser extent than

155

SNAPSO. Oddly, GST-RB (379-870) interacts more strongly than GST-RB (379-928). As
expected, none of the RB mutants tested interact with the transcriptional activator Oct-1
or with Brf2, a component of variant TFIIIB complex both of which are important for
expression of snRNA genes transcribed by RNA polymerase 111. Overall, these assays.
indicate that interaction with SNAP43 and TBP require the NB pocket and C pocket of

RB whereas SNAPSO, Brfl , and de1 require only the NB pocket

The region of SNAP43 contained within amino acids 234-368 is sufficient for RB
interaction

Since there is a strong correlation between the region of RB that interacts with SNAP43
and the region that is able to repress U6 snRNA transcription, the RB interaction domain
within SNAP43 was further characterized using SNAP43 truncation mutant proteins.
Each truncation mutant as shown in Figure 4-7 was tested for RB interaction in GST-pull
down assays similar to those described above. As shown in Figure 4-7, SNAP43 (1-368)
interacts strongly with RB whereas SNAP43 (1-93), SNAP43 (1-168), and SNAP43 (l-
233) do not. This suggests that the region of SNAP43 between 1-233 is not sufficient for
interaction with RB. Therefore, N-terminal truncation mutants of SNAP43 were tested
for interaction with RB. Truncation mutant proteins containing amino acids SNAP43 (94-
368), SNAP43 (169-368), and SNAP43 (234-368) interact strongly with RB. However, a
truncation proteins containing amino acids 169-300 no longer interacts with RB,
suggesting that amino acids 301 to 368 are important for interaction. Accordingly, a
SNAP43 truncation containing amino acids 301 to 368 was tested and was shown not to

interact with RB. This result suggests that amino acids 301 to 368 are required for

156

Figure 4-7: SNAP43 (234-368) is the minimal region required for interaction with

RB
GST-pull down analysis to determine the minimal region of SNAP43 required to interact

with RB. Individual SNAP43 mutants (cloned by Liping Gu) were expressed in vitro and
labeled with 35S-methionine. These proteins were incubated with GST-RB (379-928)
(lane 2), GST (lane 3), or beads alone (lane 4). Bound proteins were washed extensively
and separated by 17% SDS-PAGE. Proteins were visualized by autoradiography. 10%
input is shown in lane 1.

157

         
 

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158

interaction with RB but are not alone sufficient to support RB interaction. As shown in
Figure 4-7, the smallest region of SNAP43 that interacts with RB in this assay is SNAP43
(234-268). Interestingly, RB does not interact with SNAP43 (116-199), which has been
shown to interact with both SNAPSO and TBP. Altogether, the results of this assay
suggest that the region of SNAP43 containing amino acids 234-368 is sufficient for

interaction with RB.

Discussion

As discussed earlier, RB plays a central role in many cellular processes. Studies from
other labs have determined that distinct regions of RB are necessary for participation in
different cellular processes. The A and B pockets of RB are sufficient to regulate cell
cycle progression (13, 14), interact with E2F or HDACs (3, ll, 22), and repress
transcription by RNA polymerase II (1, 3, 14). Conversely, growth control and tumor
suppression have been assigned to the A, B, and C pockets (17). In fact, tumor
suppression can be rescued by re-introduction of RB 379-928 into Rb-/- mouse
embryonic fibroblasts and can reverse embryonic lethality and other development defects

in Rb-/- mice (21).

RB’s role in grth regulation may significantly depend on it’s ability to regulate
transcription by RNA polymerase III. It was therefore interesting to determine whether
the region of RB that repressed RNA polymerase III activity correlates to the region
sufficient to regulate growth control. As shown by the mutational analysis of RB, the

minimal region of RB necessary for repression of RNA polymerase III transcription is

159

amino acids 379-870. This region of RB was sufficient for repression of both U6 snRNA
and Ad VAI transcription if not quite as potent as the truncation protein 379-928.
Potentially, the extreme C-terminus of RB has important contributions to RNA
polymerase IIII repression but is not absolutely required. The RB truncation mutant
containing amino acids 379-772 is not sufficient to support repression of RNA
polymerase III even though it can repress RNA polymerase II transcription (3, 11)
suggesting an required role for the C-pocket. The results of this study suggest a link
between regulation of RNA polymerase III transcription, growth control, and tumor

suppression based on the regions of RB required for each of these processes.

In order to repress U6 snRNA transcription, RB is recruited to the promoter via a
complex network of interactions. RB can interact with multiple components of complexes
needed for pre-initiation complex formation at the U6 snRNA gene. Previously, SNAPc
has been shown to be an important determinant in the regulation of U6 snRNA genes by
RB. RB association with SNAPc has been described by a variety of methods including
co-immunoprecipitation, biochemical co-purification, and GST-pull down analysis in
which RB was shown to preferentially interact with the SNAP43 and SNAPSO subunits
(7). Treatment of nuclear extracts with RB to deplete for RB interacting factors exhibit a
marked depletion of SNAPc as well as comprised ability to continue to support correctly
initiated U6 gene expression. When supplemented with a combination of SNAPc and
TBP, transcription of U6 snRNA genes is restored, suggesting that while SNAPc is
important to the ability of RB to regulate U6 genes, it is not sufficient to do so alone.

Data presented in this report demonstrates that RB can in fact be recruited to a U6

160

promoter in vitro by interaction with SNAPc albeit not very efficiently. An alternative
form of TFIIIB that assembles at the TATA box of U6 snRNA promoters also plays a
role in the recruitment of RB (Chapter 3). Endogenous RB co-immunoprecipitates with
TFIIIB complexes. Additionally, TBP and del in the TFIIIB complex specific for U6
snRNA transcription also interact significantly. Together, interactions with SNAPc and

TFIIIB act cooperatively to recruit RB to the U6 snRNA promoter.

Structure function analysis of RB interaction with these basal factors RB illustrates that
all three pocket domain are important for protein-protein interactions. The A/B pocket +
C pocket regions are sufficient for interaction with SNAP43 and TFIIIB components and
the C pocket is critical for this interaction. Conversely, the A and B pockets were
sufficient to interact with SNAPSO and removal of the B region abolishes the interaction.
Interestingly, the loss of ability to repress U6 snRNA transcription (removal of the C
pocket) correlates with the loss of ability to interact with SNAP43 and a lowered affinity
for the U6 promoter. Similarly, the region of RB containing the A, B, and C pockets was
sufficient to significantly associate with SNAPc bound to a U6 snRNA promoter in

EMSA experiments.

The mechanisms RB uses to repress RNA polymerase III are still unclear. One proposed
model for RB repression of tRNA genes suggests that RB target the TFIIIB complex
involved in tRNA expression (20). Through direct interactions with the Brf-l subunit,
RB inhibits important interactions with TFIIIC. It has been proposed that RB disrupts

pre-initiation complex formation including polymerase recruitment at tRNA gene

161

promoters (Figure 4-8) (9). In this model, RB is not expected to be present at a tRNA
promoter as it has sequestered TFIIIB away from the target gene (15). The data presented
in this study are consistent with this mechanism, as RB does not appear to associate with

tRNA promoters in vivo or in vitro under conditions of repression.

However, RB appears to employ a different mechanism for the repression of class 3
genes (U6 snRNA). As shown by both in vivo and in vitro promoter occupancy
experiments, RB occupies the U6 snRNA promoter (a class 3 gene) in contrast to a lack
of promoter occupancy at a tRNA promoter (class 2) (Figure 4-8). One explanation for
RB association at the U6 snRNA promoter and not Ad VAI or tRNA promoter lies in the
strength of interaction with the basal factors. Overall, RB interacts more strongly with
SNAPc components than with TFIIIB component and this strength of interaction could
contribute to a more stable complex formation at the U6 snRNA promoter. Additionally,
SNAPc and TFIIIB bind very tightly to the promoter elements in the U6 snRNA gene
perhaps making them more resistant to RB displacement and encouraging a stable

complex including RB to form on the DNA.

162

Figure 4-8: RB repression of class 2 and class 3 RNA polymerase III transcribed
genes.

RB potentially uses to different mechanisms to repress RNA polymerase III transcription
of different classes of promoters. For repression of tRNA genes, RB may specifically
target the TFIIIB complex, sequestering it away from the tRNA promoter interrupting
pre-initiation complex formation. In this model RB would not be expected to associate
with the target promoter. Conversely, RB does associate with the U6 snRNA promoter
through recruitment by SNAPc and TFIIIB. The molecular step inhibited by RB is still
not yet known but may be subsequent to pre-initiation complex assembly.

163

 

 

 

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References:

10.

ll.

Adnane, J., Z. Shao, and P. D. Robbins. 1995. The retinoblastoma susceptibility
gene product represses transcription when directly bound to the promoter. J Biol
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Braunstein, M., A. B. Rose, S. G. Holmes, C. D. Allis, and J. R. Broach. 1993.
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Chow, K. N., and D. C. Dean. 1996. Domains A and B in the Rb pocket interact
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Chu, W. M., Z. Wang, R. G. Roeder, and C. W. Schmid. 1997. RNA
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Dignam, J. D., P. L. Martin, B. S. Shastry, and R. G. Roeder. 1983.
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Henry, R. W., E. Ford, R. Mital, V. Mittal, and N. Hernandez. 1998. Crossing
the line between RNA polymerases: transcription of human snRNA genes by
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Hirsch, H. A., L. Gu, and R. W. Henry. 2000. The retinoblastoma tumor
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Larminie, C. G., H. M. Alzuherri, C. A. Cairns, A. McLees, and R. J. White.
1998. Transcription by RNA polymerases I and III: a potential link between cell
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Larminie, C. G., C. A. Cairns, R. Mital, K. Martin, T. Kouzarides, S. P.
Jackson, and R. J. White. 1997. Mechanistic analysis of RNA polymerase III
regulation by the retinoblastoma protein. EMBO J 16:2061-71.

Lee, J. 0., A. A. Russo, and N. P. Pavletich. 1998. Structure of the
retinoblastoma tumour-suppressor pocket domain bound to a peptide from HPV
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Magnaghi-Jaulin, L., R. Groisman, I. Naguibneva, P. Robin, S. Lorain, J. P.
Le Villain, F. Troalen, D. Trouche, and A. Harel-Bellan. 1998. Retinoblastoma
protein represses transcription by recruiting a histone deacetylase. Nature
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Mittal, V., B. Ma, and N. Hernandez. 1999. SNAP(c): a core promoter factor
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domain. Genes Dev 13: 1807-21 .

Sellers, W. R., B. G. Novitch, S. Miyake, A. Heith, G. A. Otterson, F. J. Kaye,
A. B. Lassar, and W. G. Kaelin, Jr. 1998. Stable binding to E2F is not required
for the retinoblastoma protein to activate transcription, promote differentiation,
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Sellers, W. R., J. W. Rodgers, and W. G. Kaelin, Jr. 1995. A potent
transrepression domain in the retinoblastoma protein induces a cell cycle arrest
when bound to E2F sites. Proc Natl Acad Sci U S A 92:11544-8.

Sutcliffe, J. E., T. R. Brown, S. J. Allison, P. H. Scott, and R. J. White. 2000.
Retinoblastoma Protein Disrupts Interactions Required for RNA Polymerase III
Transcription. Mol Cell Biol 20:9192-9202.

Welch, P. J., and J. Y. Wang. 1993. A C-terminal protein-binding domain in the
retinoblastoma protein regulates nuclear c-Abl tyrosine kinase in the cell cycle.
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Welch, P. J ., and J. Y. Wang. 1995. Disruption of retinoblastoma protein
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White, R. J. 1997. Regulation of RNA polymerases I and III by the
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White, R. J. 1998. Transcription factor IIIB: An important determinant of
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White, R. J., D. Trouche, K. Martin, S. P. Jackson, and T. Kouzarides. 1996.
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Yang, H., B. 0. Williams, P. W. Hinds, T. S. Shih, T. Jacks, R. T. Bronson,
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166

Chapter 5

Summary

The Retinoblastoma tumor suppressor protein plays a central role in many regulatory
processes in the cell including cell cycle progression, apoptosis, DNA replication and
repair, differentiation, and growth control. The key to RB’s ability to function in all of
these cellular processes is the ability to act as a transcription factor to regulate the
transcription of genes involved each process. Previous studies illustrate that RB can
repress transcription by RNA polymerase I (3), II (31), and III (35) suggesting a role for
RB in the regulation of a diverse set of genes. This versatility allows RB to participate in
many processes critical to normal cell function. RB’s contribution to each of these

processes may play a role in the suppression of tumor formation.

RB acts to regulate cell growth in part by controlling the production of biosynthetic
building blocks. The discovery that RB represses transcription by RNA polymerases I (3)
and III (18) reveals that RB also acts to regulate expression of highly transcribed genes
encoding non-translated RNAs. By understanding the mechanisms by which expression
of non-translated RNAs is controlled, we can define the contribution of these RNAs to
the regulation of normal cell growth. Importantly, the availability of essential non-
translated RNAs could act to limit cell growth and RB repression of genes encoding these

RNAs would likely have to be overcome prior to tumor progression.

167

Unlike regulation of RNA polymerase II, in which only small subset of genes is
regulated, RB appears to regulate global RNA polymerase III activity. Nuclear run off
assays performed from cells that were deficient for RB activity either through mutation
(osteosarcoma cells) or from targeted gene disruption (Rb-/- mouse embryonic
fibroblasts) show a net elevation in RNA polymerase III activity as compared to matched
sister cell lines with active RB (9, 18). While the RNA polymerase III activity increases
in RB deficient cells, wholesale RNA polymerase II activity is largely unaffected.
Similarly, RNA polymerase III activity is repressed when RB is re-introduced into these

cells via transient overexpression (9, 18).

Transcription by RNA polymerase III is also regulated in a cell cycle dependent manner
(15, 17). Transcription of these genes is low in G0 and G1, increases and peaks in S
phase, decreases by G2 and is severely diminished in M phase. The lack of transcription
by RNA polymerase III corresponds to phases of the cell cycle during which RB is
expected to be active, whereas expression RNA polymerase III transcribed genes is high
during cell cycle phases when RB is phosphorylated and inactive. Furthermore, over-
expression of cyclin D/cdk4 and cyclin E/cdk2 activates transcription by RNA
polymerase III in transient transfection assays (15). Together these two results suggest
that inactivation of RB is important for the expression of RNA polymerase III transcribed

genes.

The mechanisms RB uses to repress RNA polymerase III are still unclear. One proposed

model for RB repression of tRNA genes suggests that RB target the TFIIIB complex

168

involved in tRNA expression (18). Through direct interactions with the Brf-l subunit,
RB inhibits important interactions with TFIIIC. It has been proposed that RB disrupts
pre—initiation complex formation including polymerase recruitment at tRNA gene
promoters (Figure 4-8) (9). Similarly, RB was proposed to repress some RNA
polymerase II transcribed genes by targeting the TFIID-TFIIA complex formation step to
prevent pre-initiation complex formation for RNA polymerase II (I 1). In this model, RB
is not expected to be present at a tRNA promoter as it has sequestered TFIIIB away from
the target gene (16). However, not all RNA polymerase III transcribed gene have a Brf-l
requirement (10, 14). For example, the class 3 genes such as U6 snRNA do not require
Brf-1 or the TFIIIB complex containing Brf-1 for efficient expression. Nonetheless,
these genes are still repressed by RB suggesting that RB has multiple mechanisms for
repressing RNA polymerase III transcribed genes. 1 therefore wanted to explore the
different mechanisms used by RB to repress RNA polymerase III transcription paying

special attention to the class 3 genes.

At the beginning of my thesis research, I needed to establish a functional assay to study
the effects of RB on transcription by RNA polymerase III. I therefore developed in vitro
repression assays using two representative RNA polymerase III transcribed genes. I chose
the Ad VAI gene as a representative of class 2 genes and the U6 snRNA gene as a
representative of class 3 genes. Both of the genes tested were repressed in the presence
of recombinant RB but not by the control GST protein. Overall, these experiments
recapitulated the original observation that RB repressed transcription by RNA

polymerase III.

169

In order for a repressor or activator transcription factor to directly affect the transcription
of a target gene, it may associate with the promoter of that target gene. Therefore, it was
important to determine whether RB could associate with U6 promoter in vivo. To this
end, chromatin immunoprecipitation assays, a powerful tool to study in vivo promoter
occupancy, were developed using normal human mammary epithelial cells. The
chromatin immunoprecipitation assays clearly show the RB protein occupying U6
snRNA promoter in viva suggesting that RB does in fact target this gene for
transcriptional regulation. In contrast, RB is not present at the U1 and U2 RNA
polymerase II transcribed snRNA genes. Interestingly, RB occupancy of snRNA
promoters in vivo correlates with the ability to repress transcription as demonstrated by in
vitro transcription experiments. Whereas RB can repress U6 snRNA transcription by
RNA polymerase III, it is unable to repress transcription by RNA polymerase II initiated
from a U1 snRNA promoter. Indeed, the data presented herein, illustrate that RB

preferentially targets and represses RNA polymerase III transcribed snRNA genes.

To efficiently regulate U6 expression, RB may be directed to U6 promoters either by
direct binding to specific DNA control elements or through recruitment by other trans-
acting factors. Since RB does not seem to have innate DNA binding capability, it is most
likely recruited to target promoters through protein-protein interactsions. Potentially, RB
could be recruited to a U6 snRNA promoter by the protein complexes that bind to U6

snRNA promoter elements such as the proximal sequence element and TATA box.

170

The snRNA activating protein complex (SNAPc) is one potential candidate protein
complex for recruiting RB to a U6 snRNA promoter. This protein complex consisting of
at least five subunits binds to the PSE to nucleate pre-initiation complex formation and is
important for subsequent gene expression (4-8, 12, 13, 19). SNAPc is an important
determinant in the regulation of U6 snRNA genes by RB. Treatment of nuclear extracts
with RB to deplete for RB interacting factors exhibit a marked depletion of SNAPc as
well as comprised ability to continue to support correctly initiated U6 gene expression.
When supplemented with a combination of SNAPc and TBP, transcription of U6 snRNA
genes is restored, suggesting that while SNAPc is important to the ability of RB to
regulate U6 genes, it is not sufficient to do so alone. Potentially, TBP or TBP containing
complexes are also important for RB regulation of U6 snRNA genes. Importantly, the
TBP containing TFIIIB complex utilized in tRNA transcription was not able to restore
U6 snRNA transcription in these GST-RB treated extracts. RB association with SNAPc
has been described by a variety of methods including co-immunoprecipitation,
biochemical co-purification, and GST-pull down analysis in which RB was shown to
preferentially interact with the SNAP43 and SNAPSO subunits. Data presented in this
report demonstrates that RB can in fact be recruited to a U6 promoter in vitro by
interaction with SNAPc albeit not very efficiently. Together, these data suggest that
SNAPc is important for RB repression of U6 snRNA transcription but it is not the sole

factor targeted during RB repression.

When searching for clues as to what other factor(s) play a role recruiting RB to U6

snRNA to subsequently repress transcription, the results of chromatin

171

immunoprecipitation and in vitro transcription experiments presented strong evidence for
an additional candidate target for RB repression. Interestingly, RB was only present at
RNA polymerase III transcribed snRNA genes in vivo and capable of transcriptional
repression of only RNA polymerase III transcribed snRNA genes in vitro. It would stand
to reason that if SNAPc alone were capable of mediating RB repression of U6 snRNA
genes, then it should also direct RB repression activities to other genes at which SNAPc
is present. This is clearly not the case, suggesting that SNAPc is not the sole determining
factor for RB selectively targeting the RNA polymerase III transcribed snRNA genes.
The obvious difference between snRNA genes transcribed by RNA polymerase II and III
is the presence of a TATA element in core promoter region of RNA polymerase III
transcribed snRNA genes. It follows that proteins or proteins complexes that are
recruited to this distinguishing promoter element are candidate selectivity factors

directing RB solely to RNAP III transcribed snRNA genes rather than all snRNA genes.

An alternative TFIIIB complex consisting of TBP, Brf2, and del associates with U6
snRNA promotes (but not U1) in vivo through association with the TATA element and is
thought to recruit RNA polymerase III for ensuing gene expression (1, 2, 14). As a
candidate target for mediating RB repression of U6 genes, I demonstrated by
coimmunoprecipitation, that TFIIIB associates with endogenous RB and that individual
components of TFIIIB (del and TBP) interact with RB in GST-pulldown experiments.
TFIIIB, like SNAPc, can recruit RB to a U6 promoter but most likely does not act
unaided to mediate RB association. In vitro RB promoter recruitment is significantly

enhanced in the presence of SNAPc, suggesting cooperative interactions between TFIIIB

172

and SNAPc to direct RB to the U6 snRNA promoter. Surprisingly, Brf2, which does not
interact directly with RB, appears to play a vital role in RB interaction at U6 promoters in
vitro. Potentially, Brf2 could function within the TFIIIB complex to present the proper
facade for RB association. Alternatively, Brf2 may help to stabilize de1 binding at the
TATA element increasing the chances of a stable del-RB interaction. RB does
however interact with the two subunits (del and TBP) that are common to both types of
TFIIIB complexes suggesting a potential mechanism for targeting RB to promoters of

RNA polymerase III transcribed genes.

Importantly, EMSA experiments show that RB does not repress transcription of U6
snRNA genes simply by displacing required general factors. In fact, RB appears to be
able to bind to both SNAPc and TFIIIB bound to a U6 snRNA promoter. This idea of
RB co-occupying a U6 snRNA promoter with required general factors is further
recapitulated in the results of sequential chromatin immunoprecipitation experiment.
These experiments clearly show RB, SNAPc, and TFIIIB occupying the same U6 snRNA

promoter in vivo.

One potential mechanism RB could use to repress transcription is to block RNA
polymerase 111 access to the U6 snRNA promoter. For example RB could be recruited to
the promoter through interaction with both SNAPc and TFIIIB and further block the
interactions necessary for RNA polymerase III association. Surprisingly, sequential
chromatin immunoprecipitation experiments indicate that this may not be the case. These

assays show that RB and RNA polymerase 111 can co-occupy the same U6 snRNA

173

promoter in viva. This experiment, however, does not tell us whether these co-occupied
promoter are repressed. Therefore, I returned to an in vitro repression system to
determine whether RB and RNA polymerase III co-occupy a repressed U6 snRNA
promoter. These experiments plainly indicate that RB and RNA polymerase 111 can co-

occupy the same repressed U6 snRNA promoter.

The results presented herein are inconsistent with the previous model in which RB
represses transcription by blocking the binding of RNA polymerase III to cognate
promoters. Sutcliffe and co-workers (26) proposed a mechanism of RNA polymerase III
transcriptional repression in which RB sequesters required factors. RB was proposed to
bind to TFIIIB interrupting important interactions with TFIIIC. Since interaction with
TFIIIC is critical for recruitment to tRNA genes, RB effectively sequesters TFIIIB away
from the promoter thereby not allowing pre-initiation complex formation to occur. Since
RB and RNA polymerase III occupy the same promoter under repressed conditions, RB
is clearly not using a commonly accepted mechanism, disrupting pre-initiation complex
formation, to repress transcription at the U6 snRNA promoter. Instead, we propose that,
at least for human U6 transcription, RB impedes critical steps in RNA polymerase III

transcription that occur subsequently to polymerase recruitment to the promoter.

How then does RB regulate transcription of U6 snRNA genes? We suggest that RB is

recruited to U6 core promoter regions cooperatively by both SNAPc and TFIIIB where it

174

can subsequently repress transcription through many potential mechanisms. Potentially
RB could recruit histone modification activities that would modify local chromatin
structure preventing transcription by RNA polymerase 111. However, in an in vitro system
we observe repression of RNA polymerase III transcription using naked DNA templates.
Thus, it appears that chromatin modification is not essential for repression of RNA
polymerase III in vitro. Additionally, HDAC mediated repression of U6 genes may be
unlikely as data from other groups suggest that RB can still repress expression of U6

gene even in the presence of HDAC inhibitors ( l 6).

Another method by with RB could prevent U6 gene expression is through the recruitment
of ATP-dependent chromatin remodeling complexes such as SWI-SNF. In viva, the U6
promoter contains a positioned nucleosome between the DSE and PSE that is vital to
proper gene expression. Association of RB in combination with chromatin remodeling
factors a U6 promoter could potentially mis-align this nucleosome and thus effectively

abolish U6 gene expression.

Additionally, RB may hinder steps that allow for promoter escape and subsequent
transcription of the U6 snRNA gene. Furthermore, RB could inhibit steps that convert an
initiating polymerase to an elongating polymerase such as phosphorylation of the
enzyme. Perhaps RB interferes with elongation resulting in a stalled complex in the
middle of the U6 snRNA gene. Whichever case may apply, it represents a novel

mechanism for RB regulation of gene expression.

175

Why regulate snRNA gene expression at all? RB regulation of snRNA gene expression
serves as a brake system to help shut down growth capacity of the cell. Small non-
translated RNAs such as snRNAs or tRNA are building blocks that contribute to the
biosynthetic capacity of the cell and thereby increase growth potential. The cell’s ability
to overcome the “brake system” imposed by RB would directly influence the cell’s

growth potential possibly encouraging progression into a tumor state.

176

References

10.

ll.

Cabart, P., and S. Murphy. 2002. Assembly of human small nuclear RNA gene-
specific transcription factor IIIB complex de novo on and off promoter. J Biol
Chem 277:26831-8.

Cabart, P., and S. Murphy. 2001. BRFU, a TFIIB-like factor, is directly
recruited to the TATA-box of polymerase 111 small nuclear RNA gene promoters
through its interaction with TATA-binding protein. J Biol Chem 276:43056-64.

Cavanaugh, A. H., W. M. Hempel, L. J. Taylor, V. Rogalsky, G. Todorov,
and L. I. Rothblum. 1995. Activity of RNA polymerase I transcription factor
UBF blocked by Rb gene product. Nature 374:177-80.

Ford, E., M. Strubin, and N. Hernandez. 1998. The Oct-1 POU domain
activates snRNA gene transcription by contacting a region in the SNAPc largest
subunit that bears sequence similarities to the Oct-1 coactivator OBF- 1. Genes
Dev 12:3528-40.

Henry, R. W., E. Ford, R. Mital, V. Mittal, and N. Hernandez. 1998. Crossing
the line between RNA polymerases: transcription of human snRNA genes by
RNA polymerases II and III. Cold Spring Harb Symp Quant Biol 63:111-20.

Henry, R. W., B. Ma, C. L. Sadowski, R. Kobayashi, and N. Hernandez.
1996. Cloning and characterization of SNAPSO, a subunit of the snRNA-
activating protein complex SNAPc. EMBO J 15:7129-36.

Henry, R. W., V. Mittal, B. Ma, R. Kobayashi, and N. Hernandez. 1998.
SNAP l 9 mediates the assembly of a functional core promoter complex (SNAPc)
shared by RNA polymerases II and III. Genes Dev 12:2664-72.

Henry, R. W., C. L. Sadowski, R. Kobayashi, and N. Hernandez. 1995. A
TBP-TAF complex required for transcription of human snRNA genes by RNA
polymerase II and III. Nature 374:653-6.

Larminie, C. G., C. A. Cairns, R. Mital, K. Martin, T. Kouzarides, S. P.
Jackson, and R. J. White. 1997. Mechanistic analysis of RNA polymerase III
regulation by the retinoblastoma protein. EMBO J 16:2061-71.

Mital, R., R. Kobayashi, and N. Hernandez. 1996. RNA polymerase III
transcription from the human U6 and adenovirus type 2 VAI promoters has
different requirements for human BRF, a subunit of human TFIIIB. Mol Cell Biol
16:7031-42.

Ross, J. F., X. Liu, and B. D. Dynlacht. 1999. Mechanism of transcriptional
repression of E2F by the retinoblastoma tumor suppressor protein. Mol Cell
3: 195-205.

177

12.

13.

I4.

15.

l6.

17.

18.

19.

Sadowski, C. L., R. W. Henry, R. Kobayashi, and N. Hernandez. 1996. The
SNAP45 subunit of the small nuclear RNA (snRNA) activating protein complex
is required for RNA polymerase II and III snRNA gene transcription and interacts
with the TATA box binding protein. Proc Natl Acad Sci U S A 93:4289-93.

Sadowski, C. L., R. W. Henry, S. M. Lobo, and N. Hernandez. 1993.
Targeting TBP to a non-TATA box cis-regulatory element: a TBP-containing
complex activates transcription from snRNA promoters through the PSE. Genes
Dev 7 :1535-48.

Schramm, L., P. S. Pendergrast, Y. Sun, and N. Hernandez. 2000. Different
human TFIIIB activities direct RNA polymerase III transcription from TATA-
containing and TATA-less promoters. Genes Dev 14:2650-63.

Scott, P. H., C. A. Cairns, J. E. Sutcliffe, H. M. Alzuherri, A. McLees, A. G.
Winter, and R. J. White. 2001. Regulation of RNA polymerase III transcription
during cell cycle entry. J Biol Chem 276:1005-14.

Sutcliffe, J. E., T. R. Brown, S. J. Allison, P. H. Scott, and R. J. White. 2000.
Retinoblastoma Protein Disrupts Interactions Required for RNA Polymerase III
Transcription. Mol Cell Biol 20:9192-9202.

White, R. J., T. M. Gottlieb, C. S. Downes, and S. P. Jackson. 1995. Cell cycle
regulation of RNA polymerase III transcription. Mol Cell Biol 15:6653-62.

White, R. J., D. Trouche, K. Martin, S. P. Jackson, and T. Kouzarides. I996.
Repression of RNA polymerase III transcription by the retinoblastoma protein.
Nature 382:88-90.

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-l. Mol Cell Biol 18:368-
77.

178

Appendix A

Recruitment of Basal Factors to the U6 snRNA promoter

Figure A-2 was included in the following manuscript:

Hinkley, C. S., Hirsch, H. A., Gu, L., Lamere, B., and Henry, R. W., (2003). The
Small Nuclear RNA-activating Protein 190 Myb DNA Binding Domain Stimulates
TATA Box-binding Protein-TATA Box Recognition. J. Biol. Chem. 278: 18649-
18657.

179

SNAPc assembly at human U6 snRNA promoter in vitro

To study the effects of RB on SNAPc binding to the U6 snRNA promoter, an assay for
studying SNAPc binding to the proximal sequence element was required. To this end,
the components of mini-SNAPc: SNAP190 (1-505), SNAPSO, and SNAP43 were
expressed as GST-fusion proteins in E. coli (3, 5) and purified by affinity
chromatography using glutathione sepharose. The proteins were cleaved from the affinity
matrix using thrombin and assayed for purity using SDS-PAGE analysis. Both SNAP43
and SNAP190 (1-505) purified to >90% homogeneity whereas SNAPSO contained at
least three major contaminating proteins. These purified proteins were assembled into a
mini-SNAPc complex and fiirther purified by anion exchange chromatography to remove
contaminating proteins and unincorporated subunits. The resulting fractions were
analyzed for SNAPc activity by EMSA and analyzed for purity by SDS-PAGE analysis.
Figure A-IA illustrates the chromatogram associated with SNAPc purification over a
mono-Q column. Protein concentration was monitored by UV absorbance at both 280 and
220 nm wavelengths. Figure A-IB shows the SDS-PAGE analysis of the fractions
illustrating the removal of contaminating bands in fractions 20 to 22 as compared to the
starting material. As shown in Figure A-lC, SNAPc activity, as monitored by DNA

binding, peaks in fractions 20 to 22.

180

 

q?

Figure A-l: SNAPc assembly and binding to U6 snRNA promoter DNA in vitro:
(A) Chromatograph of protein fractions obtained during column chromatographic

purification of SNAPc. (B) SDS-PAGE of SNAPc starting material and eluted column 1

fractions. (C) EMSA analysis for SNAPc activity.

181

 

 

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183

SNAPc stimulates TFIIIB assembly at human U6 promoters

To study the effects of RB on both SNAPc and TFIIIB complexes at the U6 promoter, an
assay for measuring the assembly of both of the complexes needed to be established. It
has already been shown that SNAPc and TBP act cooperatively to bind to U6 promoter
DNA (3-5). Additionally, other studies have shown that TBP+Brf2 and TBP+Brf2+del
can form complexes on 7SK DNA probes that are similar to U6 promoters (l, 2).
However, these papers do not take SNAPc into consideration when looking at TFIIIB
complex association. Therefore both Brf2 and del association with the U6 snRNA

promoters in the presence of SNAPc and TBP were analyzed.

Brf2:

SNAPc binding to the PSE is a crucial early event during the assembly of a functional
pre-initiation complex at human U6 promoters. As described, one fimction of SNAPc is
to stimulate TBP recruitment to the TATA box. However, additional events are required
including the assembly of additional TFIIIB components at these promoters. As a marker
for TFIIIB assembly, we followed the binding of Brf2 to human U6 promoter probes in
electrophoretic mobility shifis assays. As shown in Figure A-2, neither TBP alone (lanes
2, 10, 18, and 26) nor Brf2 alone (lanes 3, 11, 19, and 27) bound efficiently to any of the
U6 promoter probes. In contrast, mini-SNAPc alone bound efficiently to wild-type PSE
probes (lanes 4 and 20) but not to mutant PSE probes (lanes 12 and 28) as expected.
When recombinant TBP was added to reactions containing mini-SNAPc, modest

formation of a slower migrating complex was observed (lane 5: labeled mini-SNAPc +

184

Figure A-2: Mini-SNAPc can recruit Brf2 to a U6 snRNA promoter.

Electrophoretic mobility shift assays were performed with 1 ug rTBP (lanes 2, 10, 18, and
26), 120 ng Brf2 (lanes 3, ll, 19, and 27), 10 ng mini-SNAPc (lanes 4, 12, 20, and 28),
rTBP and mini-SNAPC (lanes 5, 13, 21, and 29), Brf2 and mini-SNAPc (lanes 6, 14, 22,
and 30), or Brf2, mini-SNAPc, and increasing amounts of rTBP (0.1, 0.3 and 1.0 pg:
lanes 7-9, 15—17, 23-25, and 31-33, respectively). The DNA probes used contain a high-
affinity mouse U6 snRNA PSE and a human U6 snRNA TATA box (lanes 1-9), a mutant
PSE and wild-type TATA box (lanes 10-17), a wild-type PSE and mutant TATA box
(lanes 18-25), or a mutant PSE and mutant TATA box (lanes 26-33). Lane 1 contains
probe alone. The presence (+) or absence (—) of rTBP, Brf2, and mini-SNAPc is indicated
above each lane. The positions of protein/DNA complexes are indicated.

185

 

 

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rTBP) and surprisingly, complex formation was not affected by TATA box mutation
(lane 21). This complex was inferred to contain both mini-SNAPc and TBP because it
was only observed in the presence of both mini-SNAPc and TBP. SNAPc stimulation of
TBP binding was also less than that observed in experiments presented in Figures 1 and 3
presumably because these experiments were performed using different electrophoresis
conditions. Interestingly, a slower migrating complex was now observed when Brf2 was
added to reactions containing mini-SNAPc (lane 6: labeled mini-SNAPc + Brf2). This
complex contained Brf2 because antibodies directed against the histidine tag on Brf2
were able to supershift this complex (data not shown). Mutation of the PSE severely
debilitated SNAPc/Brf2 complex formation (lane 14) whereas TATA box mutations did
not affect this complex (lane 22). Thus, mini-SNAPc can recruit Brf2 to a U6 promoter in
a PSE-dependent manner. Next, the ability of SNAPc/Brf2 to recruit TBP was tested. As
increasing amounts of TBP were added to reactions containing mini-SNAPc and Brf2
(lanes 7-9), formation of a new complex was observed (labeled mini-SNAPc + Brf2 +
rTBP). The presence of Brf2 in this new complex was confirmed using antibody
supershift assays (data not shown). In these reactions, the levels of the SNAPc/Brf2
complex was reduced concomitantly with added TBP whereas SNAPc binding was
unchanged, suggesting that TBP preferentially recognized the SNAPc/Brf2 complex to
form the SNAPc/Brf2/TBP complex. Mutation of the PSE severely impaired, but did not
completely abrogate, the formation of this new complex (lanes 15-17). This observation
suggests that TBP and Brf2 together can help stabilize SNAPc binding to a weak PSE. In
these reactions, a faster migrating complex is also observed, which co-migrates with the

SNAPc/Brf2 complex. However, this complex likely corresponds to a complex

187

containing Brf2 and TBP because the SNAPc/Brf2 complex does not form on probes
containing a mutated PSE (lane 14). Indeed, in subsequent experiments the SNAPc/Brf2
and TBP/Brf2 complexes were observed to co-migrate (data not shown). As expected,
when mutations were introduced into both the PSE and TATA box, DNA binding by all
factors was abolished (lanes 26-33). Therefore, SNAPc can recruit Brf2 in a PSE-specific
manner and together, these factors further stimulate TBP recruitment to a human U6

promoter.

de1:

Although TFIIIB complex formation at U6 snRNA promoters can be partially monitored
by incorporation of Brf2, deI must also associate with the U6 promoter to complete the
complex. Therefore, recombinant del was expressed in E. coli, purified, and
introduced into the DNA binding reactions described above to create a complex
containing SNAPc and TFIIIB bound to the U6 snRNA promoter in vitro. As shown in
figure A-3, TBP, Brf2, and deI do not bind to DNA alone (lanes 2-4). The complex
formed by the addition of SNAPc to U6 probe is shown in lane 5. Addition of TBP to
SNAPc is shown in lane 6 resulting in increased binding of SNAPc. Addition of Brf2 to
these proteins and DNA (lane 7) results in a lower mobility complex consistent to those
seen in the figure above. Addition of de1 (lane 8) to these reactions results in the
formation of a lower mobility complex indicated in the figure as SNAPc + TFIIIB. This

complex is inferred to contain SNAP and TFIIIB.

188

Figure A-3: SNAPc and TFIIIB assembly at a U6 snRNA promoter in vitro.

The assembly of SNAPc and TFIIIB complexes was analyzed using EMSA assays as
described in figure A-2. Lane 1 contains probe alone illustrating the position of free
probe. TBP (300 ng), Brf2 (200 ng), and del (40 ng) alone do not bind to the probe
(lanes 2-4 respectively). The amount of binding supported by SNAPc (25 ng) is shown in
lane 5 and is labeled as SNAPc. Addition of TBP to SNAPc containing reactions is
shown in lane 6. SNAPc, TBP, and Brf2 (lane 7) create a lower mobility complex similar
to that observed in the previous figure. The complex formed by both SNAPc and TFIIIB
is labeled as such in lane 8.

189

 

 

 

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Materials and Methods:

Recombinant protein expression and purification:

Mini-SNAPc: GST-SNAP43 Ex-XB, pET-GST-SNAPSO, and pET-GST-SNAP190(1-
505) were transformed into E. Coli BL21 DE3 codon + (Stratagene) and grown at 37°C
in ZBM9 media until OD600= 0.5. Temperature was lowered to 16°C and protein
expression was induced with 1 mM IPTG for 20 hours. Pelleted bacteria were
resuspended in HEMGT-150 and sonicated using a Branson sonifier 4 times for 30 pulses
output=6 duty 60%. Debris was pelleted at 13,000 RPM at 4°C for 30 minutes. Soluble
protein was bound to glutahione sepharose (Pharmacia) overnight at 4°C and washed 4
times in HEMGT-150 plus protease inhibitors and twice in HEMGT-150 without
protease inhibitors. Proteins were cleaved from the beads using 20 units Thrombin
(Sigma) on ice for 1.5 hours vortexing every 15 minutes. SNAPSO and SNAP190(l-505)
were collected as fractions in HEMGT-150. Appropriated fractions were pooled and
dialyzed against Dignam Buffer D80. SNAP43 was collected in step-wise salt gradient
(150 to 1000 mM KCL in HEMGT buffer) and appropriate fractions pooled. Mini-
SNAPc was assembled from approximately 100 ug of each protein at room temperature
for 2 hours. The assembly reaction was diluted such that the final salt concentration was
less than 80 mM KCl and the final glycerol concentration was less than 5%. The diluted
assembly reaction was allowed to equilibrilate on ice for 1 hour. Assembled mini-
SNAPc was then loaded onto a 1 m1 Mono-Q column. Bound proteins were eluted from
the column in 0.5 m1 fiactions in a O to 500 mM salt gradient in Dignam Buffer D80 (5%

glycerol). Fractions were adjusted to 20% glycerol and assayed for SNAPc activity by

191

SDS-PAGE and EMSA. The peak of SNAPc activity corresponded to fractions 20-24

(~380-420 mM KCl).

TBP: TBP was expressed as a GST-fusion protein in E. Cali BL21 DE3 at 37 °C in ZB-
M9 media and induced with 1 mM IPTG for 3 hours. Pelleted bacteria were resuspended
in HEMGT-150 and sonicated using a Branson sonifier 4 times for 30 pulses output=6
duty 60%. Debris was pelleted at 13,000 RPM at 4°C for 30 minutes. Soluble protein
was bound to glutahione sepharose (Pharmacia) overnight at 4°C and washed 4 times in
HEMGT-150 plus protease inhibitors and twice in HEMGT-150 without protease
inhibitors. TBP was cleaved from the beads using 20 units Thrombin (Sigma) on ice for
1.5 hours vortexing every 15 minutes. Appropriate fractions were pooled, concentrated
to a working concentration of 400 ng/ul using centricon YM-IO spin columns

(Millipore), and dialyzed against Dignam Buffer-D80.

Brfl: pSBET-hBRF U plasmid was a gift from N. Hernandez. BRF U was expressed and

purified as described previously (7).

del: pSBET-hB” plasmid was a gift from N. Hemadez. hB” was transformed into E.
Coli BL21 DE3 codon + (Stratagene) and grown at 37°C until OD600= 0.5. Temperature
was lowered to 16°C and protein expression was induced with 1 mM IPTG for 20 hours.
hB” was affinity purified using Ni-NTA beads (Qiagen) as described previously (7). hB”
was dialyzed against Dignam Buffer D-80 and concentrated using centricon YM-30 spin

columns (Millipore).

192

Electrophoretic mobility shift assays:

SNAPc: Approximately 25 ng of SNAPc was used in EMSA using DNA probes
containing a wild-type or mutant mouse U6 PSE with a wild-type or mutant human U6
TATA box as described previously (4, 6). All DNA binding reactions were performed in
20 ul total volume in a buffer containing 60mM KC], 20 mM HEPES pH 7.9, 5 mM
MgC12, 0.2 mM EDTA, 10% glycerol, 0.5 ug poly(dI-dC), and 0.5ug pUC119 plasmid.
Reactions were incubated 20 minutes at room temperature after which 5000 cpm of probe
was added and reactions were incubated an additional 20 minutes. Samples were
fractionated on a 5% nondenaturing polyacrylamide gel (39 : 1) in TGE running buffer

(50 mM Tris base, 380 mM glycine, 2 mM EDTA).

SNAPc and TFIIIB: Reactions also containing Brf2 were performed in a buffer
containing 50 mM NaF, 10 mM HEPES pH 7.9, 20 mM Tris pH 8.4, 60 mM KCI, 7.5
mM MgC12, 10% glycerol, 6 mM B-mercaptoethanol, 12 mM DTT, 0.2 ug poly(dG-dC),
and 0.2 ug pUC119 plasmid. Reactions were incubated 20 minutes at on ice after which
5000 cpm of probe was added and reactions were incubated an additional 30 minutes at
30°C. The samples were fractionated on a 4% nondenaturing polyacrylamide gel (39:1)
containing 1 X TBE (90 mM Tris, 90 mM boric acid, 2 mM EDTA) and 2.5% glycerol in

0.5 X TBE running buffer.

193

References

1.

Cabart, P., and S. Murphy. 2002. Assembly of human small nuclear RNA gene-
specific transcription factor IIIB complex de novo on and off promoter. J Biol
Chem 277:26831-8.

Cabart, P., and S. Murphy. 2001. BRFU, a TFIIB-like factor, is directly
recruited to the TATA-box of polymerase 111 small nuclear RNA gene promoters
through its interaction with TATA-binding protein. J Biol Chem 276:43056-64.
Hinkley, C. S., H. A. Hirsch, L. Cu, B. LaMere, and R. W. Henry. 2003. The
Small Nuclear RNA-activating Protein 190 Myb DNA Binding Domain
Stimulates TATA Box-binding Protein-TATA Box Recognition. J Biol Chem
278:18649-57.

Mittal, V., and N. Hernandez. 1997. Role for the amino-terminal region of
human TBP in U6 snRNA transcription. Science 275:1136-40.

Mittal, V., B. Ma, and N. Hernandez. 1999. SNAP(c): a core promoter factor
with a built-in DNA-binding damper that is deactivated by the Oct-l POU
domain. Genes Dev 13: 1807-21.

Sadowski, C. L., R. W. Henry, S. M. Lobo, and N. Hernandez. 1993.
Targeting TBP to a non-TATA box cis-regulatory element: a TBP-containing
complex activates transcription fiom snRNA promoters through the PSE. Genes
Dev 7:1535-48.

Schramm, L., P. S. Pendergrast, Y. Sun, and N. Hernandez. 2000. Different
human TFIIIB activities direct RNA polymerase III transcription from TATA-
containing and TATA-less promoters. Genes Dev 14:2650-63.

194

Appendix B:

Potential co-factors that regulate snRNA gene expression

Actin association with potentially active U6 snRNA promoters

Previous studies have shown that actin proteins may be in complexes with chromatin
remodeling machines. Potentially actin and actin related proteins may be important for
regulation of gene expression (1, 6). Data produced in our laboratory by Ms. Liping Gu
suggests that actin may be in a complex with SNAPc and may therefore contribute to the

regulation of snRNA genes.

In order to determine whether actin is present at the U6 snRNA promoter, perhaps
through recruitment by SNAPc, sequential chromatin immunoprecipitations were
performed (Figure B-l) similar to those described in chapter 3. Sequential ChIP assays
were performed using a—SNAP43 (Figure B-lA), a—RNAPIII (Figure B-lB), or or-RB
(Figure B-lC) antibodies in the first round of immunoprecipitation. As expected,
secondary immunoprecipitations using either or-SNAP43 (lane 3), a—TBP (lane 4), 0:-
RNA polymerase III (lane 5) or Ot-RB (lane 6) antibodies enriched the U6 snRNA
promoter DNA but not GAPDH DNA, consistent with the data shown in chapter 3.
Interestingly, when secondary precipitations were performed using or-actin antibodies

(lane 2) were enriched for U6 snRNA promoter DNA only when primary precipitations

195

Figure B-l: Actin and SNAPc or RNA polymerase III co-occupy the same U6
snRNA promoter in viva.

Sequential chromatin immunoprecipitations were performed from human 18485 cells to
determine actin co-occupancy with other factors. Precipitated material was recovered

after the second immunoprecipitation and analyzed by PCR for enrichment of U6 DNA
or GAPDH exon 2 as a negative control. Lane I shows 10% input chromatin.

196

 

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were performed with a-SNAP43 or or-RNAPIII. Secondary immunoprecipitations using
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not enriched for U6 snRNA promoter DNA. If it is assumed that the U6 genes
precipitated with a-RB antibodies in the first round are in the repressed state, it may be
the case that actin is associating with only the actively transcribed U6 snRNA gene

perhaps suggesting that actin plays a role in the activation of U6 snRNA genes.

HMG-1 association with snRNA promoters:

High mobility group protein (HMG 1&2) bind to DNA without sequence specificity
similar to histone proteins and appear to have a role in the assembly of nucleoprotein
complexes in viva (8, 10). It is thought that HMG proteins may act like H1 linker
histones during higher order chromatin assembly. These proteins are sometimes referred
to as architectural transcription factors due to their ability to help stabilize higher order
structures containing chromatin and other transcription factors at the promotes of specific
genes. For example, HMG proteins have been shown to interact with the transcriptional
activator Oct-l and help is bind stably to DNA (1 1). Interestingly, the region of snRNA
genes between the Oct-l binding sites in the DSE and region containing the PSE have
been shown to be resistant to micro-coccal nuclease digestion indicating the presence of a
higher order chromatin structure (9). Two possible explanations for this protection of
promoter DNA is the presence of a nucleosomes or a higher order complex composed of

HMG proteins and DNA.

198

Figure B-2: HMG-l occupies snRNA promoters in viva.
Chromatin immunoprecipations were performed from HeLa cells using antibodies

specific to HMG-l (lane 3) and SNAP43 (lane 4). The precipitated DNA was then
analyzed by PCR for the presence of U1 and U6 snRNA promoter DNA or the U1

upstream negative control. 1% input is shown in lane 1.

199

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In order to determine whether HMG proteins are present at snRNA promoters, chromatin
immunoprecipitations were performed from HeLa similar to the experiments described
previously. As shown in Figure B-2, chromatin immunoprecipitations performed with Ot-
SNAP43 antibodies were enriched for U6 and U1 snRNA promoter DNA but not the U1
upstream control DNA as compared to precipitations performed using control IgG.
Additionally, chromatin immunoprecipitations performed using (X-HMG antibodies were
significantly enriched for both U6 and U1 snRNA promoter DNA indicating that HMG-l

proteins are present at snRNA promoters in viva.

p70: a potential co-repressor for RB?

During the biochemical purification of SNAPc, a protein of 70 kDa was identified that
co-purified with SNAPc. This protein, designated p70, bears some sequence similarity to
yeast de1. p70 has been shown to interact with SNAP43 and TBP by GST pull down
analysis (Brandon Lamere, unpublished data) and can be directly crosslinked to U6
promoter DNA during UV-photocrosslinking experiments using biochemically purified
SNAPc (R. William Henry, unpublished data). Thus far, the function of p70 remains
unknown. It is not required for snRNA transcription (3) and immuno-depeletion of this

factor does not affect U1 or U6 transcription (R. William Henry unpublished data).

To better define a role for p70 in snRNA transcription, chromatin immunoprecipitation
experiments were performed fi'om HeLa cells to see if p70 associates with snRNA
promoters (Figure B-3A). As expected, chromatin immunoprecipitations using 01-

SNAP43 antibodies (lane 4) were enriched for both U6

201

Figure B-3: p70 associates with the U6 snRNA promoter in vivo and interacts with
RB.

(A) Chromatin immunoprecipitation to determine the in viva occupancy of p70 at snRNA
promoters. Chromatin immunoprecipations were performed from HeLa cells using
antibodies specific to p70 (lane 2) and SNAP43 (lane 4). The precipitated DNA was then
analyzed by PCR for the presence of U1 and U6 snRNA promoter DNA or the U1
upstream negative control. 10% input is shown in lane 1.

(B) p70 associates with endogenous RB. Co-immunoprecipitation was performed from
HeLa cell nuclear extract using antibodies specific to p70. RB association was
determined by Western Blot analysis using Ot-Galectin 3 antibodies as a negative control.
10% input is shown in lane 1. i

(C) p70 directly interacts with GST-RB (379-928). ) GST pull-down assays were
performed using recombinant GST-RB (379-928) (lane 2), GST (lane 3), or glutathione
agarose beads (lane 4). Interactions were tested with the indicated in vitro translated,
3SS-methionine labeled p70 protein. Associated proteins were size fractionated by SDS-
PAGE and visualized by autoradiography.

 

 

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203

and U1 snRNA promoter DNA as compared to control IgG precipitations (lane 3).
Additionally these IPs were not enriched for the U1 upstream control DNA supporting
the specificity of the precipitations. When the chromatin immunoprecipitations were
performed using antibodies specific for p70 (lane 2), only the U6 snRNA promoter was
precipitated in the reaction. This result suggests that p70 is specifically recruited to the
RNA polymerase III transcribed snRNA genes and that p70 could potentially play a role

in U6 snRNA gene expression.

Interestingly, p70 was present at the same snRNA promoter to which RB is specifically
recruited, suggesting that p70 may have a connection to RB regulation of U6 snRNA
genes. Accordingly, association between p70 and RB was investigated. As shown in
Figure B-3B, immunoprecipition of HeLa cell extract using p70 (lane 3) demonstrates
RB association as analyzed by Western Blot. This association appears to be specific, as
Galectin-3 is not precipitated with the p70 antibody. Together these results provide

evidence for association of endogenous p70 with endogenous RB.

To determine whether p70 directly interacts with RB, GST-pull down experiments were
performed similar to those described in previous chapters. For these experiments, p70
was expressed in vitro and labeled with 35S-methionine. The expression of this protein
is shown in lane 1 of Figure B-3C. These in vitro expressed proteins were then mixed
with GST-RB (379-928) or GST. Stable protein complexes were isolated with glutathione
sepharose and size fractionated by 12.5% SDS-PAGE. Proteins were then visualized by

autoradiography. As shown in lane 2, p70 interacts very strongly with GST-RB (379-

204

928). This interaction is specific as p70 did not interact with GST (lane 3) or beads alone
(lane 4). The results of this experiment indicate that p70 can directly interact with RB.
Overall the experiments suggest that p70 may have a role in U6 snRNA transcription

potentially in connection with RB.

Materials and Methods:

Antibodies: Anti-SNAP43 (CS48) and anti-TBP (SL2) antibodies have been described
previously (5, 7). Anti-p70 (CS160) antibodies were gifts from Nouria Hernandez. The
anti-Galectin-3 antibody (Mac-2) was a gift from Patty V053 and John Wang (Michigan
State University). Mouse anti-RB antibodies (G3-245) used for western analysis were
purchased from Pharrningen. The goat anti-RB antibodies (C-IS), HMG-l, and pre-
immune control IgGs were purchased from Santa Cruz Biotechnologies. Actin antibodies

were purchased from Sigma.

Chromatin immunoprecipitation: Chromatin immunoprecipitations were performed
similarly to that described previously (2). Human 184B5 or HeLa cells were grown to
75% confluency and then crosslinked with formaldehyde for 30 minutes. After cell lysis
and sonication, chromatin immunoprecipitations were performed (approximately 1 x 107
cells per immunoprecipitation) in dilution buffer containing 400 mM NaCl using 1 ug of
each antibody overnight at 4°C. Protein complexes were isolated using Protein-G Fast
Flow sepharose beads (Upstate Biotechnology) and were washed extensively as described

previously (2). Cross-links were reversed at 65°C overnight and recovered chromatin was

205

suspended in 50 pl TE buffer. PCR analysis was performed using 5 pL of
immunoprecipitated chromatin or input chromatin. The primers used for each gene are:
U6 forward — 5’-GTACAAAATACGTGACGTAGAAAG-3’,

U6 reverse — 5’-GGTGTTTCGTCCTITCCAC-3’,

Ul forward — 5’-CACGAAGGAGTTCCCGTG-3’,

U1 reverse — 5’-CCCTGCCAGGTAAGTATG-3’,

U1 upstream control forward — 5’-GAACTTACTGGGATCTGG-3’,

U1 upstream control reverse — 5’-GAGACAACTGAGCCACTTG-3’,

GAPDH forward — 5’-GGTCATCCCTGAGCTGAAC-3’, and

GAPDH reverse — 5’-GCAATGCCAGCCCCAGCGTC-3’.

PCR products were separated by 2% TBE-agarose electrophoresis and visualized using

Kodak imaging software.

Sequential Chromatin Immunoprecipitations:

Soluble chromatin fraction was prepared from 18485 cells as above. Primary IPs were
performed scaled up 5x (~ 5 x 107 cells per IP) using 5 pg Ot-RB, or-SNAP43, (x-
RNAPIII. The immunoprecipitations were performed at room temperature for 1 hour and
then incubated with protein G agarose for an additional hour at room temperature. After
extensive washing, precipitated protein-DNA complexes were eluted in elution buffer
containing 15 mM DTT + 1% SDS at room temperature for 30 minutes. One sixth of the
recovered material was used for second ChIP and material was processed exactly the

same as chromatin immunoprecipitations described in above.

206

Co-immunoprecipitation: Approximately 300 pL of HeLa cell nuclear extract (ca. 8
mg/mL) was incubated with 2 pl of antibodies directed against p70 (CS160) or 1 pg
rabbit IgG overnight at 4°C as indicated in figure legend. Reactions were diluted to lml
in HEMGT-150 buffer and stable complexes were affinity purified by incubation with
Protein-G Fast Flow sepharose beads (Upstate Biotechnology) for 4 hours at 4°C. Beads
were washed 3 times in HEMGT-150 and boiled for 3 minutes in Laernmli Buffer. Bound
proteins were separated by 10% SDS-PAGE, transferred to nitrocellulose and associated
proteins were determined by western blot analysis using antibodies directed against RB
(G3-245; Pharmingen). The same membrane was stripped and probed with antibodies

directed against Galectin 3 (Mac-2) (4).

207

References

10.

ll.

Boyer, L. A., and C. L. Peterson. 2000. Actin-related proteins (Arps):
conformational switches for chromatin-remodeling machines? Bioessays 22:666-
72.

Braunstein, M., A. B. Rose, S. G. Holmes, C. D. Allis, and J. R. Broach. 1993.
Transcriptional silencing in yeast is associated with reduced nucleosome

acetylation. Genes Dev 7 :592-604.

Chang, S. S., P. Hu, and N. Hernandez. 2001. Reconstitution of Transcription
from the Human U6 Small Nuclear RNA Promoter with Eight Recombinant
Polypeptides and a Partially Purified RNA Polymerase III Complex. J Biol Chem
276:20727-20734.

Dagher, S. F., J. L. Wang, and R. J. Patterson. 1995. Identification of galectin-
3 as a factor in pre-mRNA splicing. Proc Natl Acad Sci U S A 92:1213-7.

Henry, R. W., C. L. Sadowski, R. Kobayashi, and N. Hernandez. 1995. A
TBP-TAF complex required for transcription of human snRNA genes by RNA
polymerase II and 111. Nature 374:653-6.

Olave, I. A., S. L. Reck-Peterson, and G. R. Crabtree. 2002. Nuclear actin and
actin-related proteins in chromatin remodeling. Annu Rev Biochem 71 :755-81.

Ruppert, S. M., V. McCulloch, M. Meyer, C. Bautista, M. F alkowski, H. G.
Stunnenberg, and N. Hernandez. 1996. Monoclonal antibodies directed against
the amino-terminal domain of human TBP cross-react with TBP from other
species. Hybridoma 15:55-68.

Thomas, J. O. 2001. HMG] and 2: architectural DNA-binding proteins. Biochem
Soc Trans 29:395-401.

Zhao, X., P. Shannon Pendergrass, and Nouria Hernandez. 2001. A
positioned nucleosome in the human U6 promoter allows recruitment of SNAPc
by the Oct-l POU domain. Molecular Cell 7:539-549.

Zlatanova, J., and K. van Holde. 1998. Linker histones versus HMGl/Z: a
struggle for dominance? Bioessays 20:584-8.

Zwilling, S., H. Konig, and T. Wirth. 1995. High mobility group protein 2
functionally interacts with the POU domains of octamer transcription factors.
EMBO J 14:1198-208.

208

Appendix C

Subcellular localization of SNAPc and TFIIIB components

To determine whether components of SNAPc and TFIIIB localize to specific parts of the
cell, indirect immunofluorescence was performed using antibodies to SNAP43, TBP and
deI. As shown in figure C-IA, SNAP43 does in fact localize to discrete subnuclear
locations. Two distinct types of subnuclear particles that appear contain SNAP43.
SNAP43 appears to be present in are smaller speckles that number between 15 and 20 per
nucleus. SNAP43 also appears to be present in larger dots in the nucleus that number
from 1 to 3 copies in the nucleus. These dots do not appear to nucleoli which are visible

under phase contrast light microscopy.

The subcellular localization of del was also investigated. As shown in Figure C-lC,
deI seemed to localize diffusely in the cytoplasm and not at all in the nucleus. This
was an unexpected result since transcription factors are expected to be in the nucleus and
this protein has been shown to occupy the U6 snRNA promote in viva (3). As a positive
control for nuclear staining, a—TBP antibodies (Figure C-lB) were also used indicating

that the perrneabilization was sufficient to detect nuclear proteins.

209

Figure C-l: Subcellular Localization of components of SNAPc and TFIIIB.

210

 

 

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211

One possibility to account for the lack of de1 nuclear staining is that the cells used in
this experiment were very nearly 100% confluent and most likely growth arrested by
contact inhibition. Potentially, if RNA polymerase 111 genes were not being actively
transcribed due to growth arrest, there would be no need for an RNA polymerase III
specific transcription factor in the nucleus. To directly test this hypothesis, coverslips
were prepared such that the cell densities were approximately 50%, 75, or 100% and then
analyzed for del localization. As shown in Figure C-2, cells at 50% exhibit del
localization around the periphery of the nucleus although from the staining in this
experiment it is not possible to determine whether this staining is perinuclear or
associated with the inside of the nuclear membrane. Cells at 100% confluency show a
similar pattern to the previous figure and seem to perhaps have less de1 than the other
two cell densities. Interestingly, at 75% confluency, when the cells are growing
efficiently, there is more staining of the nuclear periphery perhaps indicating an increase
in protein expression and also the emergence of one to two large dots in the nucleus
itself. These dots may indicate a sub-compartment of nuclease dedicated to RNA

polymerase III transcription.

Materials and Methods:

Tissue culture: Human mammary epithelieal cells (184B5) were a gift from Susan
Conrad. Cells were maintained in Dulbecco’s minimum essential media (DMEM -
Gibco) plus 10% fetal bovine serum (Gibco), 200 mM Glutamine, and penicillin-

streptomycin in 37°C incubator with 5% CO;. For the purpose of immunofluorescent

212

Figure C-2: Cell density affects subcellular localization of del.

213

 

 

50% Confluent

 

   

, 75% Confluent

100% Confluent

214

studies, these cells were grown directly on poly-lysine treated coverslips (Thermonox —

Nunc).

Antibodies: Anti-SNAP43 (CS48) and polyclonal anti-TBP antibodies have been
described previously (1, 2). Anti-thpl (CS913) antibodies were a gift from Nouria
Hernandez (3). Secondary goat a-rabbit antibodies conjugated to Alexa 488 chromaphore

were purchased from Molecular Probes.

Indirect Immunofluorescence: Cells on coverslips were fixed for 15 minutes at —20°C
in 50:50 acetone-methanol and then washed three times in PBS. The coverslips were
then pre-blocked in 10% BSA for 15 minutes at room temperature and washed two times
in PBS. Coverslips were incubated in primary antibodies inverted on parafilm for 30
minutes at 37°C. Primary antibodies were used at a 1:100 dilution in 1% BSA in PBS.
The coverslips were washed three times with PBS for 10 minutes at room temperature.
Coverslips were incubated in secondary antibodies inverted on parafilm for 30 minutes at
37°C in the dark. Secondary antibodies were used at a 1:200 dilution in 1% BSA in PBS.
The coverslips were washed three times with PBS for 10 minutes at room temperature.
The coverslips were then washed three times with water for 10 minutes at room
temperature. Coverslips were drained and mounted on microscope slides in Permafluor
(Fisher). Immunofluorescent images were taken using computer assisted fluorescent

laser scanning confocal microscopy on a Meridian-Insight microscope.

215

Acknowledgments:

We would like to thank Drs. Shirley Owens and Joanne Whalon for assistance with the

confocal microscopy.

216

References

1. Henry, R. W., C. L. Sadowski, R. Kobayashi, and N. Hernandez. 1995. A
TBP-T AF complex required for transcription of human snRNA genes by RNA
polymerase II and 111. Nature 374:653-6.

2. Ruppert, S. M., V. McCulloch, M. Meyer, C. Bautista, M. F alkowski, H. G.
Stunnenberg, and N. Hernandez. 1996. Monoclonal antibodies directed against
the amino-terminal domain of human TBP cross-react with TBP from other
species. Hybridoma 15:55-68.

3. Schramm, L., P. S. Pendergrast, Y. Sun, and N. Hernandez. 2000. Different
human TFIIIB activities direct RNA polymerase III transcription from TATA-
containing and TATA-less promoters. Genes Dev 14:2650-63.

217