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TRANSCRIPTIONAL REGULATION OF THE HUMAN SMALL
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TRANSCRIPTIONAL REGULATION OF THE HUMAN SMALL NUCLEAR RNA
GENE FAMILY

By

Gauri W. Jawdekar

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Microbiology and Molecular Genetics

2006

ABSTRACT

TRANSCRIPTIONAL REGULATION OF THE HUMAN SMALL NUCLEAR RNA
GENE FAMILY

By

Gauri W. Jawdekar

Human RNA polymerase III synthesizes small, non-translated RNAs including SS rRNA,
tRNA, and U6 snRNA that control numerous critical steps during the flow of genetic
information in biological systems. U6 snRNA genes and other related family members
are unusual because they are transcribed by either RNA polymerase II or III depending
on the arrangement of core promoter elements. Thus human snRNA genes in general
provide a good model to study polymerase preference and activity during normal and
deregulated growth.

One characteristic feature of all snRNA genes is the presence of an essential
proximal sequence element (PSE) in the core promoter region that is recognized by the
multi-subunit general transcription factor called SNAPC. DNA bindingby SNAPC is a
crucial early event during preinitiation complex assembly, and is a target for regulatory
intervention. The mechanism of DNA binding by SNAPc was investigated in this study.
The SNAPSO subunit of SNAPC plays an important role in preinitiation complex
assembly for RNA polymerase II and III transcription in a process involving an
unorthodox but highly conserved zinc finger domain. This zinc finger domain functions

directly in DNA binding and is essential for cooperative promoter recognition by SNAPc.

The Retinoblastoma (RB) tumor suppressor protein represses U6 snRNA
transcription by RNA polymerase III and interestingly can interact with the SNAPSO
subunit of SNAPC suggesting that RB could impair DNA binding by SNAPC to repress
transcription. However, studies from our lab suggest that RB does not affect DNA
binding by SNAPC during repression. Therefore, to further understand the mechanism of
RB repression of U6 snRNA transcription, I examined the role of RB co-factors such as
histone deacetylases (HDACs) and components of the ATP-dependent SWI/SNF
chromatin remodeling complex in RB repression. In this study I show that endogenous
RB co-occupies the U6 snRNA promoter with HDACs and stimulates association of
HDAC2 and Brgl with the U6 promoter during repression. Interestingly, HDAC
enzymatic activity is required for RB repression of chromatin template but not naked
DNA template, suggesting that RB repression and HDAC activity are biochemically
separable.

In a search for additional factors that associate with SNAPC and potentially
regulate human snRNA transcription, a protein called HEXIMI was identified. In this
study, I show that HEXIMI co-purifies with SNAPC and stimulates DNA binding by
SNAPC and TBP. HEXIMI positively regulates 78K transcription by potentially
enhancing preinitiation complex assembly by these factors. Interestingly, HEXIMI and
78K RNA are part of a complex that inhibits the activity of P-TEFb, which is required for
transcription of HIV-1 and cellular genes by RNA polymerase 11. Our observation that
HEXIMI stimulates transcription of its co-repressor partner suggests a mechanism by
which P-TEFb activity and hence global RNA polymerase II transcription may be

regulated.

ACKNOWLEDGEMENTS

I would earnestly like to thank my mentor R. William Henry for his guidance and
excellent mentorship. His encouragement and patience were vital for me to continue with

research and not quit graduate school.

I wish to thank my thesis guidance committee: Dr. Susan Conrad, Dr. Lee Kroos, Dr.
Richard Schwartz and Dr. Steve Triezenberg. I would like to thank Dr. James Geiger for
a fruitful collaboration. I also want to thank Dr. David Arnosti and Dr. James Geiger for

writing letters of recommendation for my post-doctoral job.

Members of the Henry laboratory, past and present: Dr. Craig Hinkley, Dr. Heather
Hirsch, Dr. Zakir Ulla, Dr. Anastasia Gridasova, Dr. LiPing Gu, Tharakeswari
Selvakumar and Xianzhou Song have been great colleagues to work with and I am glad

to have gained their friendship.

I would like to thank Dr. Shibani Mukherjee for engaging in stimulating scientific
discussions and help with new techniques. I am also thankful to Dr. Debbie Yoder-Himes

and Rim Shanna for their friendship.

Finally I would like to thank my parents, brother, sister-in law and my nephew for their
love and support. They have always encouraged me to pursue my dreams, even if it

meant living far away from them for extended periods of time.

iv

TABLE OF CONTENTS

LIST OF FIGURES .............................................................................. vii
LIST OF TABLES ................................................................................. ix
KEY TO ABBREVIATIONS .................................................................... x
CHAPTER 1:
Introduction ......................................................................................... 1
RNA polymerase III transcription ................................................................. l
1. Diverse cellular functions of genes transcribed by RNAP III ..................... 1
1.]. RNAP III and information flow in biological systems .................. 4
1.2. RNAP III transcripts and cellular homeostasis ........................... 6
2. Regulation of RNAP III transcription ................................................. 7
2.1. Transcriptional regulation during normal grth ........................ 7
2.2. Transcriptional regulation and disease ..................................... 9
3. Mechanisms of RNAP III regulation- Promoter structure ......................... 11
4. Mechanisms of RNAP III regulation- General transcription factors ............. 15
5. Mechanisms of RNAP III regulation- Regulatory factors ......................... 19
5.1. Factors that positively regulate transcription ............................. 19
5.2. Factors that positively & negatively regulate transcription ............ 23
5.3. Factors that negatively influence transcription ........................... 24
CHAPTER 2:
RB recruits HDAC activity for repression of human U6 snRNA transcription by
RNA Polymerase III .............................................................................. 46
Abstract ...................................................................................... 46
Introduction ................................................................................. 47
Materials and Methods ..................................................................... 51
Results ....................................................................................... 54
Discussion ................................................................................... 68
References ................................................................................... 76
CHAPTER 3:
The unorthodox SNAP50 zinc finger domain contributes to co-operative promoter
recognition by human SNAPc .................................................................. 79
Abstract ...................................................................................... 79
Introduction ................................................................................. 80
Materials and Methods ..................................................................... 82
Results ....................................................................................... 86
Discussion ................................................................................. l 12
References ................................................................................. 1 19

CHAPTER 4:
The HEXIMI repressor of P-TEFb stimulates transcription of its 78K snRNA co-

repressor partner by RNA polymerase III .................................................. 124
Abstract ..................................................................................... 124
Introduction ................................................................................ 125
Materials and Methods ................................................................... 128
Results ...................................................................................... 131
Discussion .................................................................................. 153
References ................................................................................. 158

CHAPTER 5:

Summary .......................................................................................... 163

APPENDIX A

Multiple subunits of SNAPC co-expressed in E. coli are active for transcription by

human RNA polymerase II and III .......................................................... 169
Results and Discussion ................................................................... 169
References ................................................................................. 176

APPENDIX B

Differential U6 snRNA promoter association by RB family members ................ 177
Results and Discussion .................................................................. 177
References ................................................................................. 1 83

APPENDIX C

RB recruitment to human U6 snRNA promoter through interactions with SNAPc

and TFIIIB ........................................................................................ 184
Results and Discussion .................................................................. 184
References ................................................................................. 193

vi

Figure 1-1.

Figure 1-2.

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.
Figure 3-7.

Figure 4-1.

LIST OF FIGURES

Schematic representation of the promoter elements of genes transcribed
by RNA polymerase III and II transcribed .................................... 13

The general transcription machinery required for RNA polymerase III
transcription ........................................................................ l7

Endogenous chromatin modifying proteins associate with
a human U6 snRN A promoter ................................................... 56

RB concurrently occupies an endogenous U6 snRN A promoter with
HDAC] , HDAC2 and RNA polymerase III ................................... 60

RB stimulates enrichment of HDACs and SWI/SNF at a U6 snRNA
promoter during repression ...................................................... 63

RB repression of U6 transcription and HDAC activity are biochemically
separable activities ................................................................ 67

RB requires HDAC activity to repress U6 transcription from a chromatin

DNA template but not from a naked DNA template ......................... 70
Model for RB repression ......................................................... 74
The C-terminal region of SNAP50 is required for DNA binding

by SNAPc .......................................................................... 88
Single amino acid changes in the SNAP50 subunit abolish DNA

binding by SNAPC ................................................................ 96
The SNAP50 zinc finger domain is required from human snRNA gene
transcription by RNA polymerase II and III ................................. 101
SNAPC recruits Ber-TFIIIB to U6 promoter DNA in vitro ............... 104
TFIIIB suppresses DNA binding defects in SNAPC ........................ 108
SNAP50 C-terminus binds zinc ................................................ 111
Model for the SNAP50 zinc finger domain .................................. 116
Endogenous HEIMI associates with SNAPC ................................ 134

vii

Figure 4-2.
Figure 4-3.

Figure 4-4.

Figure 4-5.

Figure A-l.

Figure A-2.

Figure B

Figure C-l.

Figure C-2.

HEXIMI cooperates with SNAPc and TBP for DNA binding ........... 139

HEXIMI occupies endogenous snRN A promoters ........................ 144
P-TEF b is differentially used for snRNA gene transcription by RNA

polymerase II and III in vitro ................................................... 148
HEXIM] positively regulates 7SK snRNA transcription in viva ......... 151

mSNAPcy4 is competent for DNA binding and TBP promoter
recruitment ........................................................................ 173

mSNAPcy4 supports human snRNA gene transcription in vitro
by both RNA polymerase II and III ........................................... 175

Differential U6 snRN A promoter association by human RB family
members .......................................................................... 179

Characterization of the regions in SNAP50 and de1 that are required
for interactions with RB ........................................................ 187

The A/B pocket domain and the C-terminal region of RB is
required for interactions with RNA polymerase III-general transcription
machinery ........................................................................ 191

Images in this dissertation are presented in color.

viii

LIST OF TABLES

Table 1-1 RNA polymerase III and II transcribed non—translated genes with the
known cellular function ....... . ..................................................... 3

Table 3-1. Table showing zinc and nickel analysis of SNAPC ........................... 92

ix

ATP
de1
Brfl
Brf2
CDK
CHI-I
ChIP
CK2
Co-IP
CSB
CTD
DNMTI
DSE
EDGl
EMSA
ERE
FAA
GAPDH

GST

KEY TO ABBREVIATIONS

Adenosine triphosphate

B double prime

TFIIIB related protein 1

TFIIIB related protein2

Cyclin dependent kinase
Cartilage-hair hypoplasia

Chromatin immunoprecipitation
Casein Kinase 2 I
Co-immunoprecipitation

Cokayne syndrome group B protein
Carboxy terminal domain

DNA methyl transferase 1

Distal sequence element

Estrogen down-regulated gene 1
Electrophoretic mobility shift assay
Estrogen receptor response element

F lame atomic absorption spectroscopy
Glyceraldehyde-3-phosphate dehydrogenase
Glutathione S transferase
Heamagglutinin

Histone aoetylase

HDAC
HEXIMI

HIV

ICP-MS
ICR
LTR

MBD

mSNAPc

mSNAPcy4

NLS
PCR
PNC
PSE

P-TEF b

rRNA
RT-PCR

SANT

Histone deacetylase

Hexamethylene bis-acetamide inducible 1
Human immunodeficiency virus
Heterochromatin protein 1

Inductively coupled plasma resonance mass spectroscopy
Internal control region

Long terminal repeat

Methylated DNA binding protein
Mitochondrial RNA processing
mini-SNAPc

mini-SNAPc gamma 4

Mutant

Nuclear localization signal

Polymerase chain reaction

Perinucleolar compartment

Proximal sequence element

Positive transcription elongation factor b
Retinoblastoma tumor suppressor

RNA interference

RNA polymerase

Ribosomal RNA

Reverse transcriptase polymerase chain reaction

SWI-SNF, ADA, N-CoR and TFIIIB

xi

siRN A
SNAPC
snRNA
snRNP
SRP
STAF
SV40
TBP
TFIIA
TFIIB
TFIID
TFIIF
TFIIH
TFIIIA
TFIIIB
TFIIIC
tRNA
TSA
UV
VSMC
vRNA
WCE

Wt

Short interfering RNA

Small nuclear RNA activating protein complex
Small nuclear RNA

Small nuclear ribonucleoprotein
Signal recognition particle
Selenocysteine tRN A gene transcription activating factor
Simian virus 40

TATA- binding protein
Transcription factor II A
Transcription factor II B
Transcription factor II D
Transcription factor II F
Transcription factor II H
Transcription factor 111 A
Transcription factor 111 B
Transcription factor III C
Transfer RNA

Trichostatin A

Ultra violet

Vascular smooth muscle cells
Vault RNA

Whole cell extract

Wild type

xii

CHAPTER 1

INTRODUCTION

RNA polymerase III transcription

In most eukaryotes three major related nuclear RNA polymerases are responsible
for catalyzing the synthesis of RNA from DNA. RNA polymerase I is dedicated to
transcribing only one set of genes, the tandemly repeated ribosomal RNA (rRNA) genes.
RNA polymerase II transcribes protein coding messenger-RNA (mRNA) genes, micro
RNA (miRNA), and some non-translated small nuclear RNA (snRNA) genes. In contrast,
RNA polymerase III transcribes the well-studied SS ribosomal RNA (rRNA) and transfer
RNA (tRN A) genes, and a diverse collection of small nuclear RNA (snRNA) genes. One
common feature shared by these RNA polymerase III transcribed genes is that they

encode non-translated RNAs that are almost always shorter than ~400 base pairs (111).

I. Diverse cellular functions of genes transcribed by RNA polymerase III

The products of RNA polymerase III transcription such as U6 snRNA, SS rRNA
and tRNA have been best studied for their function in mRNA splicing and protein
synthesis, respectively. However, recent evidence suggests that RNA polymerase III
transcripts function for most, if not all, crucial steps during the flow of information in
biological systems and for maintaining cellular homeostasis. Table 1 describes the

various RNAs molecules transcribed by RNA polymerase III and their known functions.

Table 1-1. Diverse cellular functions of some non-translated RNAs transcribed by
RNA polymerase III and [1.

RNA polymerase III transcribes non-translated RNAs that are characteristically less than
400 base pairs long. These gene products play an important role in diverse cellular
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1.1. RNA polymerase III and information flow in biological systems

The central dogma is described as the sequential transfer of genetic information
from DNA to RNA by the process of transcription and from RNA to protein by the
process of translation. Recent observations show that snRNA gene products transcribed
by RNA polymerase III influence each step in these processes. For example, B2 RNA
represses RNA polymerase II-driven transcription. When mouse cells are exposed to heat
shock, RNA polymerase II transcription of genes including actin, histone H1 and
hexokinase II is repressed, while transcription of heat shock genes including hsp70
increases. In response to heat shock, B2 RNA binds to core RNA polymerase II and
forms transcriptionally inactive preinitiation complexes at specific gene promoters to
inhibit mRNA synthesis by an unknown mechanism (21). Like BZ RNA, 7SK snRNA
also inhibits RNA polymerase II transcription through its membership in the
7SK/I-IEXIM1 ribonucleoprotein complex that interacts with and inhibits the positive
elongation factor b (P-TEFb) (94, 137, 138). P-TEFb, which is composed of cyclin T1
and cdk9, phosphorylates the carboxy terminal domain (CTD) of the largest subunit of
RNA polymerase II to stimulate transcription elongation of cellular genes as well as HIV-
1 transcription and replication from the HIV long terminal repeat (LTR) (83, 84, 147). A
cellular function for 78K snRN A that might be independent of P-TEFb has been recently
described. si-RNA mediated depletion of endogenous 7SK caused a marked decrease in
cellular growth that was accompanied with increasing apoptosis by 72 hrs post-
transfection in HeLa cells (36).

Newly synthesized pre-mRN A products of RNA polymerase II transcription are

almost always spliced before they can be translated into proteins. Splicing is carried out

by the uridine rich snRNAs that are members of the snRN A gene family. Interestingly,
some snRNA genes like U6 are transcribed by RNA polymerase III, whereas other
snRNA genes such as U1, U2, U4, and US are transcribed by RNA polymerase II. Both
types of snRNA function in mRNA splicing as a part of the small nuclear
ribonucleoprotein particles (snRNPs) (48). Once the mRN A is appropriately processed by
the snRNPs, it is ready for protein translation in a process involving the RNA polymerase
III-transcribed 5S rRNA and tRNA gene products. 5S rRNA and tRNA are themselves
processed before becoming active. For example, U3 snRNA is involved in rRNA
processing (23, 33), while H1 snRNA is part of the catalytic subunit of RNase P that
functions in tRNA maturation (3). Finally 7SL RNA as a component of the signal
recognition particle (SRP) directs integral membrane and secretory proteins to the
endoplasmic reticulum during translation (19, 131). Thus numerous products of RNA
polymerase III transcription perform critical functions during transmission of information
from DNA to RNA to protein.

In addition, a role for the MRP RNA in DNA replication was described. MRP
RNA, transcribed by RNA polymerase III, was originally identified as an essential
component of a ribonucleoprotein particle with endoribonuclease activity (RNase MRP).
RNase MRP in mouse cells was shown to be involved in cleavage of mitochondrial RNA,
which functions as a primer for mitochondrial DNA replication (8). Subsequent studies
have identified a role for MRP RNA in pro 5.88 rRNA processing in the nucleolus (14,
45, 110). Interestingly, mutations in the gene encoding MRP RNA in the yeasts
Saccharomyces cerevisiae and Schizosaccharomyces pombe lead to a delay in exit from

mitosis. This delay was caused by MRP RNA mediated destabilization of the CLBZ

mRNA, which encodes the major mitotic B-cyclin (28). While the regulation of B-cyclin
by RNA is a novel result in yeast, the mechanism for reduction in B-cyclin by the MRP
RNA is yet to be elucidated. Nevertheless, this result is interesting given the fact that the

RNA and protein components of the RN ase MRP are highly conserved in all eukaryotes.

1 . 2. RNA polymerase III transcripts and cellular homeostasis

Products of RNA polymerase III transcription have been suggested to play a role
in maintaining cellular homeostasis. Although the mechanisms for understanding DNA,
mRNA, and protein quality control have been fairly well studied, not much is known
about the cellular response to misfolded, abnormal non-coding RNAs. A role for Y RNAs
that are transcribed by RNA polymerase III has been proposed in this process. Y RNA is
associated with the R0 autoantigen (74), which was originally discovered in patients
suffering from lupus erythematosis. R0 was subsequently shown to recognize misfolded
5S rRNA in Xenopus oocytes (115) and human U2 snRNA in response to UV irradiation
(l l). The Ro orthologue in the eubacteria Deinococcus radiodurans binds to several
small RNAs, including one that resembles human Y RNA and contributes to resistance to
UV irradiation thus allowing cell survival (10). Y RNA is speculated to sequester R0 in
an inactive state by causing a steric hindrance in the misfolded RNA binding site. While
the trigger for Y RNA disassociation from Ro remains to be identified, the Y RNA is
replaced by misfolded RNA that is ultimately degraded by an unknown mechanism (81 ,
116).

Vault RNA (vRNA), a small non-translated RNA transcribed by RNA polymerase

III, binds to certain chemotherapeutic compounds (31, 65, 66). vRNA is part of a large

cytoplasmic ribonucleoprotein complex called Vault. Vault particles appear to be
localized in both the nucleus as well as the cytoplasm, and hence have been implicated in
intracellular and nucleocytoplasmic transfer. Furthermore, Vault particles have been
postulated to play an important role in intracellular detoxification, thus contributing to the
multidrug resistance of cancer cells (92). For example, the interaction of vRNA with
certain chemotherapeutic compounds has been suggested to play an important role in

Vault function by participating in the detoxification process (31, 65, 66).

2. Regulation of RNA polymerase III transcription

For a long time the transcription of RNA polymerase III genes was believed to be
constitutive and not subject to regulation. However, recent data have clearly shown that
transcription of RNA polymerase III is controlled via several regulatory pathways in the

cell during both normal as well as disease conditions.

2.1. Transcriptional regulation during cellular proliferation and difl'erentiation

RNA polymerase III synthesizes structural and catalytic RNAs that are essential
components of the RNA processing and protein synthesis machinery. The demand for
these RNAs fluctuates depending on the metabolic state of the cell, and therefore RNA
polymerase III transcription is regulated during cell growth and cell cycle progression
(97). The oncogene c-Myc plays an important role in positively influencing cellular
growth (109). Recent findings that Myc is a direct activator of SS rRNA and tRNA (29)
suggest that increasing products of RNA polymerase III transcription might be a way by

which c-Myc can increase the biosynthetic capacity of a cell and therefore increase cell

growth. Once cells exit the cell cycle they either arrest in the G0 phase or differentiate
terminally. Recent studies show that RNA polymerase III transcription is regulated in
terminally differentiated mammalian cardiomyocytes. When serum starved
cardiomyocytes were stimulated to differentiate in response to hypertrophic signals such
as fetal calf senun (F CS), levels of RNA polymerase III-transcribed 5S rRNA, tRNA, and
U6 snRNA were elevated. Additionally, whole cell extracts from FCS treated
cardiomyocytes stimulated RNA polymerase III-transcribed templates more actively as
compared to extracts from un-stirnulated cells. Consistent with a positive role of c-Myc in
terminally differentiated cells, the authors of this study show that the increase in RNA
polymerase III transcripts is mediated by c-Myc (30). Interestingly, RNA polymerase III
transcription is most active during the S and G2 phase of the cell cycle and is repressed
during the M and G0/early GI phases (22, 32, 73, 132). The oncoprotein protein kinase
CK2 represses RNA polymerase III transcription in the M phase, but stimulates
transcription in the S phase (58, 62). Thus, CK2 can both positively and negatively
influence RNA polymerase III transcription. What then is the mechanism for repression
of RNA polymerase III transcription during the GO/early G1 phase of the cell cycle? One
obvious possibility is that the Retinoblastoma (RB) tumor suppressor protein contributes
to the silencing of RNA polymerase III transcription at the G0/early G1 stage of the cell
cycle. This link is made because hypo-phosphorylated RB is active in the early G1 phase,
and is switched off by increased phosphorylation as cells progress through middle and
late G1 into the S phase. That RB activity correlates with an increase in RNA polymerase
III transcription from the early G1 to S phase suggests a role for RB in regulating these

genes (114, 132). Indeed, RB represses global RNA polymerase III transcription (131).

2. 2. RNA polymerase III transcription and disease

Products of RNA polymerase III transcription contribute to the cellular growth
and are elevated in many cancers. The observation that RNA polymerase III transcription
is regulated by two cardinal tumor suppressor proteins, p53 and RB, strongly suggests
that deregulation of transcription due to inactivating mutations in p53 and RB, that are a
common feature of almost all human cancers, might contribute to cancer initiation and/or
progression. Indeed, elevated levels of RNA polymerase III transcripts were observed in
primary fibroblasts cells from Li-Fraumeni patients. Li-Fraumeni syndrome is
characterized by a predisposition to cancer due to inherited mutations in p53 (117). This
result suggests that endogenous p53 regulates RNA polymerase III transcription.
Moreover, RNA polymerase III activity is elevated in p53-/- and RB -/- tumor cells,
further supporting the idea that deregulation of RNA polymerase III transcription may be
an important component of the growth control function by these proteins (7, 12, 34, 52,
131, 133). While it has not yet been established that p53 and RB repress RNA
polymerase III transcription to limit growth, it is widely accepted that the rate of RNA
polymerase III is linked to cell growth. Thus it was proposed that this arm of the p53 and
RB pathways functions to contribute to tumor suppression by restricting the biosynthetic
capacity of the cell (130, 133).

Levels of the general transcription factors required for RNA polymerase III
transcription are elevated in tumors. For example, RT-PCR analysis showed increased
levels of mRNAs encoding all five subunits of TFIIIC2 in ovarian tumors analyzed
compared to normal control mRN As. The penta-protein TFIIIC2 complex is required for

SS rRNA and tRNA transcription. This was the first example showing overexpression of

RNA polymerase III transcription factors in cancer (134). Furthermore, increased RNA
polymerase III transcription during simian virus 40 (SV40) induced transformation of
mouse cells was ascribed to an increase in the level of the de1 component of TFIIIB
(24). Also, the SNAP43 and SNAP45 subunits of the snRNA specific SNAPc are
overexpressed in certain tumors, indicating that snRNA genes with a requirement for
SNAPC might also be deregulated in these cancers (102).

Elevated levels of RNA polymerase III transcripts have recently been associated
with the perinucleolar compartment (PNC). The PNC is usually enriched in a subset of
RNA polymerase III transcribed genes such as the 7SL RNA, H1, MRP, and Y RNA (27,
71, 85, 128) but not tRNA, SS rRNA, or U6 snRNA (85). Although the exact ftmction of
the PNC is not clear, it is possible that the PNC functions in trafficking of a subset of
newly synthesized RNA. Moreover, continuous transcription of these RNA polymerase
III products is necessary for maintaining the structural integrity of the PNC. Interestingly,
an increase in the number of PNCs was observed in transformed cells with metastatic
advantage (64). As an increase in RNA polymerase III transcription is observed during
transformation (131), it is possible to use these transcripts and PNCs as a prognostic
marker to assess the stage of the cancer.

Several reports link the function of RNA polymerase III transcribed genes to
many disease conditions. For example, mutations in the MRP RNA gene have been
implicated as the cause of human cartilage-hair hypoplasia (CHH). CHH is a recessively
inherited disorder that is characterized by disproportionate short stature, defective cellular
immunity, and a predisposition to several cancers (104). The MRP RNA normally

fimctions in 5.88 rRNA processing and in yeast also controls exit from mitosis. Real-time

10

PCR analysis showed a reduction in the expression of MRP RNA in CHH patients, in
some cases due to mutations located in the promoter region. Other mutations located in
the transcribed region might cause decreased RNA stability. Nonetheless, the ratio of
unprocessed 5.8S rRNA compared to processed 5.8S rRNA was altered when MRP
mutations mimicking those found in CHH patients were introduced into the yeast
orthologue of MRP. Moreover, micro-array analysis of leukocytes obtained from CHH
patients showed an increase in the levels of several cytokines and cell cycle regulatory
genes (46). Thus, these results suggest that CHH symptoms might be caused by a

compromise in MRP RNA function.

3. Mechanism of regulation of RNA polymerase III transcription- Promoter structure
Because the products of RNA polymerase III transcribed genes function in
numerous cellular processes, RNA polymerase III regulation is critical to maintain
cellular homeostasis. The first level of regulation is dependent upon the unique promoter _
structures of RNA polymerase III transcribed genes. As illustrated in Figure 1-1, these
genes are grouped into four different classes. 5S rRNA genes (class 1) as well as tRNA
and the Adenovirus (Ad) VAI genes (class 2) use intragenic promoter elements. 5S rRNA
promoters contain an A box, an intermediate element (IE), and a C box that is conserved
in the 5S promoters across species. Together, these sequence elements constitute the
internal control region (ICR) that is required for transcription (48, 98, 111). tRNA

promoters consists of an A and B box (26, 48, 111).

ll

Figure 1-1. Schematic representation of the promoter elements of genes transcribed
by RNA polymerase III and 11.

RNA polymerase III transcribed genes can be grouped into four classes. Class 1 and class
2 genes such as SS rRNA and tRNA, respectively contain intragenic promoter elements.
Class 1 genes contain the A and C boxes that are separated by an internal element (IE).
Together these three elements constitute the internal control element (ICR). Class 2 genes
contain an A box and a B box. Class 3 genes exemplified by U6 and 7SK snRNA
contain extragenic promoter elements including the distal sequence element (DSE), a
proximal sequence element (PSE) and a TATA box. The promoter architecture of these
genes is similar to other snRNA genes that are transcribed by RNA polymerase 11 such as
U1, and U2 although these genes lack a TATA box, unlike the prototypical mRN A genes
also transcribed by RNA polymerase II. The class 4 genes exemplified by Vault RNA
contain both intragenic as well as extragenic promoter elements.

12

 

 

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<zmh mm II. glusl <ti
m9

 

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=_ 22:

13

In contrast to the class 1 and 2 gene promoters, the class 3 promoters contain
extragenic promoter elements. Examples of class 3 genes include U6 and 7SK snRNA
genes. These genes are grouped into a family of genes called the snRNA gene family.
Interestingly, some human snRNA genes such as U6 are transcribed by RNA polymerase
III while other human snRNA genes such as U1 are transcribed by RNA polymerase II,
although both function in the same spliceosomal complex. snRNA gene promoters
contain a distal sequence element (DSE) and a proximal sequence element (PSE),
regardless of RNA polymerase specificity. The DSE contains an octamer motif, which
activates transcription from the core promoter, and is located in the regulatory region
around -220 bp upstream of the transcription start site. An adjacent SphI postoctamer
homology (SPH) motif also stimulates snRNA transcription (68). The essential PSE is
located within the core promoter region at approximately -45 bp upstream of the
transcription start site. Those snRNA genes transcribed by RNA polymerase III also
contain a TATA box at position -25 bp (49, 69, 77). Interestingly, in vertebrate snRNA
promoters, the TATA box is a crucial element for determining RNA polymerase III
specificity (77). Thus, due to their similar promoter elements the snRNA gene family is a
good model system to compare and contrast the mechanisms of RNA polymerase
preference and activity during normal and deregulated growth.

RNA polymerase III transcribes yet another category of genes with hybrid
promoter elements. These class 4 genes contain intragenic as well as extragenic promoter
elements. In the case of vault RNA, selenocysteine tRNA, and the Epstein-Barr viral
product EBER2 the promoter contains gene-external PSEs as well as gene-internal

control elements (56, 57, 65, 96, 124, 126). Additionally, the U6 snRNA-like H1 RNA

14

gene promoter contains an internal A-box but not B-box homology region (1). Like H1
RNA, the promoters for the MRP, Y1 and Y3 genes also contain DSE, PSE, and a
TATA-box elements, in addition to gene-internal A box. However, mutational studies
indicate that the upstream sequences of the gene encoding MRP RNA are required for

transcription in HeLa cell extracts, whereas internal sequences are not (123, 142).

4. Mechanisms of regulation of RNA polymerase III transcription- General
transcription machinery

The different classes of RNA polymerase III gene promoters recruit distinct sets
of well characterized basal transcription factors as illustrated in Figure 1-2. These factors
specialize in nucleating unique preinitiation complexes that ultimately converge on RNA
polymerase III recruitment.

In class 1 promoters (SS rRNA), the ICR is recognized by TFIIIA, which is a
founding member of the C2H2 zinc finger family of DNA binding proteins (88, 122). The
binding of TFIIIA is followed by recruitment of TFIIIC (48, 111). In contrast to class 1
promoters, the A and B boxes of class 2 genes (tRNA) are directly recognized by TFIIIC
without prior binding by TFIIIA (48, 111). Thus, TFIIIA acts as a specificity factor that
influences promoter recognition function of TFIIIC, and directs TFIIIC to the 5S
promoter. In both class 1 and class 2 promoters, TFIIIC binding is followed by
recruitment of the TBP containing Brfl -TFIIIB complex, which in addition to TBP,
consists of Brfl and del (111). Thus, TFIIIA and TFIIIC can be considered as
recruitment factors that function to engage TFIIIB to promoters of different structures

allowing the subsequent recruitment of RNA polymerase III.

15

Figure 1-2. The general transcription machinery required for RNA polymerase III
transcription.

RNA polymerase III transcribed Class 1 and 2 genes both require TFIIIC and TFIIIB.
The TFIIIB used by these genes is composed of Brfl, del, and the TATA-box binding
protein (TBP). In addition, Class 1 genes additionally require TFIIIA. For Class 3 genes,
the transcriptional activator protein, Oct-l, binds the DSE. The PSE is recognized by
SNAPC, which consists of at least SNAP19, SNAP43, SNAP45, SNAP50 and SNAP190.
The TATA-box is recognized by TFIIIB, which is composed of Ber, del, and TBP.
The snRNA genes transcribed by RNA polymerase II, also require Oct-1 and SNAPC.

16

SS rRNA
(Class 1)

 

tRNA
(Class 2)

 

TFIIIB

U6 snRNA _
(Class 3)

 

SNAPc TFIIIB

U1 snRNA

 

The PSE of class 3 RNA polymerase III transcribed as well as RNA polymerase II
transcribed snRNA genes is recognized by the small nuclear activating proteincomplex
(SNAPC) (108), also known as the PSE transcription factor (PTF) (93). SNAPC is
composed of at least five subunits, SNAPI9 (43), SNAP43 (44, 140), SNAP45 (107,
140), SNAP50 (2, 42), and SNAP190 (135). SNAP190 interacts with all the other SNAPC
subunits and acts as a central scaffold for the complex (79). Promoter recognition by
SNAPC is a crucial early event during preinitiation complex assembly and therefore it
might be a target for regulatory proteins. Several studies have focused on understanding
the mechanism of DNA binding by SNAPC. The SNAP190 subunit of SNAPC was
initially shown to be in close contact with DNA in UV cross-linking experiments (139).
Indeed, subsequent experiments showed that SNAP190 mediates specific DNA binding
by SNAPC via a Myb- like DNA binding domain within its N-terminal region (135). A
minimal SNAPC (mini-SNAPC) consisting of SNAP190 (1-505) that includes its DNA
binding domain, along with SNAP50, and SNAP43 has been shown to be sufficient for
specific DNA binding (90) and transcription by RNA polymerase III and II in vitro (37).
However, DNA binding by mini-SNAPC requires all three subunits (37, 51), suggesting
that the SNAP190 DNA binding domain is required, but not sufficient. Consistent with
the requirement of other subunits for DNA binding, SNAP50 was shown to be close to
DNA in UV cross-linking experiments, indicating that SNAP50 most likely provides
additional DNA contacts (42). Further evidence supporting a role for SNAP50 in DNA
binding by SNAPC is discussed in Chapter 3. Interestingly, unlike the mini-SNAPC that
binds DNA efficiently, the endogenous SNAPC binds poorly to DNA on its own. On a

TATA-box containing U6 promoter, SNAPC is efficiently recruited to the DNA via at

18

least two cooperative binding interactions; one with Oct-1 and the other with TBP, such
that each factor binds to its cognate promoter element [reviewed in (111)]. SNAPC aids
recruitment of the TBP containing Bra-TFIIIB complex, which in addition to TBP
consists of Ber and de1, thus nucleating RNA polymerase III- specific preinitiation
complex assembly (108, 112). In contrast, on TATA-less snRNA promoters (U1
snRNA), SNAPC participates in RNA polymerase II-specific preinitiation complex
assembly with TBP and other general transcription factors including TFIIA, TFIIB,
TFIIE, and TFIIF (67). Thus, in contrast to most promoter recognition complexes that
function in transcription by a single class of RNA polymerase, SNAPC is the most

functionally versatile due to its role in both RNA polymerase III and II systems.

5. Mechanism of regulation of RNA polymerase III transcription- Regulatory factors
While the general transcription factors required for RNA polymerase III
transcription are well characterized much less is known about the factors that modulate
RNA polymerase III transcription. This section describes some of the transcription
factors, kinases, and tumor suppressor proteins that have been shown to regulate RNA

polymerase III transcription.

5.1. Factors that positively regulate transcription

Oct-1 & STAF- As mentioned earlier, all human snRNA genes contain a DSE,
which is recognized by the transcriptional activator protein Oct-l (47, 48, 111).
Invariably the DSE contains a SPH element, which is recognized by a C2H2 type zinc

finger containing transcription factor called STAF (also known as the SPH binding factor

19

or SBF) (l l l). Chromatin immunoprecipitation (ChIP) experiments provide evidence that
STAF binds to the DSE elements of both RNA polymerase II and III transcribed snRNA
genes (80). Several experiments have established a role for Oct-l as an activator of
snRNA gene transcription. Oct-1 has been localized to snRNA promoter sequences in
vivo by ChIP (145). The C-terminal region of SNAP190 inhibits SNAPC DNA binding
whereas the Oct-1 POU domain relives this auto-inhibition through direct protein-protein
interactions with SNAP190. This interaction allows the cooperative binding of Oct-1 and
SNAPC to the DSE and PSE, respectively during activated transcription of human snRNA
genes by both RNA polymerase III and II (25, 55, 89, 90). Interestingly, mapping of
DNase I and micrococcal nuclease cleavage sites in chromatin suggested that the natural
U6 and U1 promoters harbor a positioned nucleosome between the DSE and the PSE.
Positioning of the nucleosome at this location was further confirmed using an in vitro
chromatin assembly assay (4, 119, 145). The correct positioning of the nucleosome is
suggested to place these two promoter elements into proximity such that Oct-1 and
SNAPC can cooperatively bind to the DSE and PSE, respectively, to facilitate
transcriptional activation (145). Thus, this is an example where a nucleosome is a
functional component of the transcriptional activation process instead of causing

repression.

Myc- The Myc oncoprotein is a transcription factor that is upregulated in many
types of cancers. Among other functions, Myc regulates cell growth and division (17,
18). Endogenous Myc occupies SS rRNA, tRNATy', and tRNALen genes, and stimulates

RNA polymerase III transcription of these genes. These genes lack the E-box that is

20

characteristically recognized by Myc in its RNA polymerase I- and II-transcribed target
genes, and thus it is thought that Myc is recruited instead via direct protein-protein
interactions with Brfl -TFIIIB. It is postulated that the Myc-TFIIIB interaction recruits
coactivators and chromatin-modifying activities, or abrogates the effects of negative
regulators of RNA polymerase III transcription to stimulate transcription (29). In
subsequent experiments the same group of researchers used an inducible Myc construct
to show that a Myc/T F IIIB complex, that also contained the HAT GCNS and the cofactor
TRRAP, is recruited rapidly to the tRNA promoter. Concurrently, they observed
hyperacetylation of promoter-proximal histone H3, but not H4, followed by RNA
polymerase III recruitment. This observation is in contrast to the c-Myc activation of
RNA polymerase II transcription, during which histone H4 acetylation is predominant
(Robert J. White, personal communication). It should be noted that the U6 snRNA gene
transcribed by RNA polymerase 111 does contain an E-box sequence upstream of the
DSE; however, the role of the E-box sequence or Myc for U6 snRN A transcription is not

yet known.

HEXIMI- Hexamethylene bis-acetamide inducible 1 protein (HEXIMI) was
recently shown to interact with the RNA polymerase III transcribed 7SK snRNA in
transient transfection experiments. 7SK RNA containing various deletions was expressed
in human cells and the bound protein was identified by co-immunoprecipitation with a
I-IEXIMl specific antibody (20). Interestingly, HEXIMI was also found to co-purify with
endogenous SNAPC, which is a transcription factor that recognizes the PSE in the 7SK

snRNA gene promoter. Moreover, HEXIMI was cross-linked to DNA in a PSE specific

21

manner (R. William Henry, unpublished results). These two observations suggested that
HEXIMI influences the DNA binding properties of SNAPC to potentially regulate
transcription of its interacting partner 7SK snRN A. The role of HEli in the regulation
of 7SK snRNA transcription was further investigated and the results are discussed in
Chapter 4.

The HEXIM1/7SK snRNA ribonucloprotein complex can inhibit RNA
polymerase II elongation by targeting the P-TEFb complex (87, 138). P-TEFb consists of
cyclin T1 and cdk9 that phosphorylates the CTD of RNA polymerase II thus facilitating
promoter clearance and subsequent transcription elongation of most mRN A genes and
HIV-1 transcription (101). In the current model for P-TEFb inhibition, phosphorylation
of the cdk9 component of P-TEF b at a key tyrosine residue at position 186 within the T-
loop, that is required for P-TEF b kinase activity, is also required to associate with the
HEXIM1/7SK RNP. Thus, the primed active form of P-TEFb is held inactive by the 7SK
snRNP. The association between HEXIMl/7SK snRNP and P-TEFb is disrupted by
various stress inducing agents including DNA damaging UV irradiation thereby allowing
transcription of genes regulated by P-TEFb. In addition to transcription of cellular genes,
P-TEF b is also required for HIV-1 transcription and replication. Interestingly, recent
evidence suggests that HEXIMI competes with the HIV-1 Tat protein for binding to the
cyclin T1 component of P-TEFb thus decreasing levels of the active P-TEFb and

interfering with HIV-1 transcription (113).

22

5. 2. Factors that positively and negatively regulate transcription

Protein Kinase CK 2- Casein kinase 2 (CK2) is a highly conserved
serine/threonine protein kinase which is implicated in the regulation of cellular growth
(86, 99, 100), and deregulated expression of CK2 may lead to tumor progression (121).
Several studies have indicated a role for CK2 in regulating RNA polymerase III
transcription. For example, inhibition of CK2 in S phase extracts debilitated transcription,
while inhibition of CK2 in M phase extracts restored transcription, suggesting that
depending upon the phase of the cell cycle, CK2 is can activate transcription (S phase) or
inhibit transcription (M phase). A positive role for CK2 was indicated due to the
observation that both AdVAI, as well as U6 snRNA transcription were abolished in the
presence of a CK2 substrate peptide, when extracts from S-phase cells were used to
support transcription, indicating that CK2 is essential for transcription (58). The exact
mechanism of CK2 regulation in the S phase is not yet known. However, CK2
phosphorylation of purified RNA polymerase III was essential for active transcription
(59). Taken together these observations suggested a mechanism in which CK2 is
proposed to target RNA polymerase III or a tightly associated factor to activate
transcription. In contrast, AdVAI as well as U6 snRN A transcription was restored only in
the presence of del and a CK2 substrate peptide when mitotic cell extracts were used to
support transcription. The restoration of transcription was not observed by other
components of the RNA polymerase III preinitiation complex, such as TBP or by the
polymerase itself, indicating that CK2 targets de1 specifically to inhibit transcription
during the M phase. Furthermore, CK2 phosphorylation of del was shown to cause

disassociation of de1 from both tRNA as well as U6 snRNA promoters (22, 58). In yet

23

another report, CK2 also had a positive role in RNA polymerase III transcription by
enhancing preinitiation complex assembly between Brfl -TFIIIB and TFIIIC at tRNA
promoters (62). In addition to regulating TFIIIB, CK2 has recently been shown to
regulate SNAPC, a transcription factor that is required for snRN A gene transcription.
Phosphorylation of SNAP190 by casein kinase 2 (CK2) abrogates DNA binding by
SNAPC; however, promoter recognition can be restored by cooperative binding of TBP to
RNA polymerase III transcribed U6 snRNA promoter sequences containing both a PSE
and a TATA-box, but not to RNA polymerase II transcribed U1 snRNA promoter
sequences lacking a TATA-box (35). This observation suggests that post-translational
modification of SNAPC by CK2 alters its DNA binding properties even though SNAPC is
required for transcription by both RNA polymerase III and II. Thus, the snRNA gene
family is a good model to compare and contrast the mechanism(s) of CK2- mediated

regulation of gene transcription.

5.3. Factors that negatively influence transcription

p53 tumor suppressor protein- p53 is one of the most intensively studied tumor
suppressor proteins due to its role in tumor suppression and the fact that the normal
function of p53 is lost in almost all cancers in vertebrates (54). p53 plays a pivotal role in
the DNA damage response pathway. RNA polymerase III transcription is regulated in
response to DNA damage caused by UV irradiation. For example, transcription of U6
snRNA (and RNA polymerase II transcribed U1 snRNA) decreases in response to UV
irradiation as measured by nuclear run-on assay. Interestingly, this effect was observed in

MCF-7 cells that contain functional p53, but not in HeLa cells in which p53 is inactivated

24

by papillomavirus E6 protein. This observation suggested a role for p53 in regulation of
these genes (7, 12, 34). Indeed, p53 can repress in vitro transcription of both U6 snRNA
and SS rRNA genes by RNA polymerase III (127). A direct role of p53 in repressing
RNA polymerase III transcription is indicated by the observation that p53 can interact
with the general transcription factors, TFIIIB and SNAPC, required for RNA polymerase
III transcription (15, 34, 118). p53 interaction with the TBP component of TFIIIB
interferes with Brfl -TFIIIB promoter association at SS rRNA and tRNA promoters
resulting in a failure to assemble the preinitiation complex. However, the consequence of
p53 interaction with SNAPC for preinitiation complex formation on U6 snRNA promoter
is not yet known. Nonetheless, p53 can repress snRNA gene transcription by RNA
polymerase II and III in response to DNA damage (34)

Chromosomal breakpoints referred to as fiagile sites, often found in cancers, are
formed at the RNA polymerase III transcribed 5S rRNA gene loci in addition to the RNA
polymerase II transcribed U1 and U2 snRNA gene loci during Adenovirus serotype 12
(Ad12 infection). Interestingly, while p53 is required for Ad12-induced fragility at these
sites (75), Cockayne syndrome group B (CSB) cells that fail to express functional CSB
protein also contain these same fragile sites (141), thus suggesting an interplay between
p53 and CSB. CSB is a DNA repair protein shown to play a role in the transcription
coupled repair (TCR) pathway by recruiting the nucleotide excision repair apparatus in
response to DNA damage (76). In addition, CSB is also thought to play a role in
elongation by permitting resumption of elongation by polymerase that had been stalled
due to highly structured RNA, such as RNA polymerase II-transcribed U1 and U2

snRNAs (141). A role for CSB in RNA polymerase III transcription is suggested by

25

recent ChIP experiments indicating that CSB occupies a variety of RNA polymerase 111
target genes, including 5S rRNA, U6 snRNA, 7SK snRNA, MRP, and Y RNA
(Gridasova A.A. et. al. unpublished results). Interestingly, CSB dissociates from these
gene promoters upon UV irradiation concomitant with an increased p53 promoter
association. Moreover, in vitro recruitment assays show that p53 might displace CSB
from promoters during repression of RNA polymerase III transcribed genes. These
observations suggest an antagonistic relationship between p53 and CSB for regulating

RNA polymerase III transcription (Gridasova A.A. et. al. unpublished results).

Retinoblastoma tumor suppressor protein- The Retinoblastoma (RB)
susceptibility gene encodes a nuclear phosphoprotein that was originally isolated due to
its frequent mutations observed in retinoblastoma, a rare pediatric tumor of the retina
(72). Subsequently, mutations in the gene encoding the RB protein have been implicated
in approximately 30% of all human tumors including osteosarcomas, small-cell lung
carcinomas, cervical carcinomas, prostate carcinomas, breast carcinomas, and some
forms of leukemia (91). Ectopic expression of RB in RB -/- cells obtained from various
tumors suppressed growth and proliferation, soft agar colony formation, and
tumorigenicity in nude mice (60, 103). Thus, RB has the credentials of a tumor
suppressor protein. It is suggested that RB regulation of RNA polymerase III
transcription may contribute to the growth suppressive function of RB.

The growth-suppressive function of RB is dependent on the pocket region, which
extends from amino acids 379-792 and can be further subdivided into the A and B

pocket. The A/B pocket is required but not sufficient for tumor suppression whereas the

26

C-terminal region of RB provides additional functions that are critical for tumor
suppression. Indeed, a larger region of RB containing the NB pocket and the C-terminal
region is sufficient for regulation and tumor suppressor function because this same region
rescued the lethal phenotype when reintroduced into RB-l- mice, thus allowing the mice
to develop normally (103, 136). The activity of RB as a transcription factor is critical for
its role in tumor suppression function. Interestingly, the same A/B pocket and the C-
terminal region of RB that is required for its tumor suppressor function is also required to
repress RNA polymerase III transcription in vitro (53, 133). In contrast, the NB pocket
alone is sufficient for RB repression of prototypical E2F-regulated RNA polymerase II
transcribed target genes (13). These observations indicate that the tumor suppressor
function of RB might be linked to its ability to regulate products of RNA polymerase IH
transcription. Indeed, RNA polymerase III transcription was elevated in RB -/- SAOS2
osteosarcoma cells compared to RB +/+ U2OS cells, while RNA polymerase II
transcription remained largely unaffected. In a more rigorous test of endogenous RB
function, a 5 fold increase in transcription of some RNA polymerase 111 genes was
observed in nuclear run-on assays performed using primary embryonic fibroblast cells
derived from RB-knockout mice compared to equivalent cells from wild type mice (70,
133). RB has been shown to inhibit the synthesis of 5S rRNA, tRNA, and U6 snRNA
transcription by RNA polymerase III both in vivo and in vitro (52, 133). Thus, RB

appears to be a global repressor of RNA polymerase III-transcribed genes.

27

Models of RB repression
Although RB is a global repressor of genes transcribed by RNA
polymerase III, these genes contain diverse promoter elements and distinct general
transcription machinery. How then does RB bring about repression of RNA polymerase
III transcription? As discussed in this section, RB utilizes distinct mechanisms to repress
RNA polymerase III transcription.

RNA polymerase III transcription of SS rRNA (classl) and tRNA (class 2) genes
utilize the Brfl -TFIIIB general transcription factor. TFIIIB is an initiation factor that is
recruited to the promoter of these genes by the TFIIIC complex, which directly
recognizes these promoters (112). RB targets the Brfl component of TFIIIB complex,
thereby disrupting the TFIIIB-TFIIIC interactions. Thus, RB prevents preinitiation
complex assembly and subsequent recruitment of RNA polymerase III to the promoter
(70, 120). This mechanism of RB repression is similar to that used by RB to inhibit
transcription of RNA polymerase II transcribed, E2F-stimulated target genes such as
DHFR, TK, cyclin A, cyclin B, cdk2, cdc2 that are important for cell cycle progression.
In the E2F model, RB stably associates with the promoter via protein-protein interactions
with E2F and represses transcription by disrupting the TFIID-TFIIA complex thereby
blocking preinitiation complex assembly and subsequent RNA polymerase II recruitment
(5, 39, 40, 50, 106).

In contrast to the mechanism used by RB to repress RNA polymerase III
transcribed class land 2 genes, RB utilizes a novel mechanism to repress U6 snRNA
(class 3) transcription by RNA polymerase III. RB specifically accesses the U6 snRNA

promoter via protein-protein interactions with components of SNAPC and TFIIIB and

28

stably associates with the U6 snRNA promoter both in vitro and in vivo. This aspect of
repression is similar to E2F where RB targets the RNA polymerase II transcription factor,
E2F, for stable promoter association. RB, SNAPC and TFIIIB co-occupy the same U6
snRN A promoter in vivo as demonstrated by sequential ChIP experiments. Interestingly,
RB and RNA polymerase III concurrently occupy a human U6 snRNA promoter during
repression (53), in contrast to the E2F model wherein RB disrupts RNA polymerase II
recruitment (9, 41, 61, 129). Our results suggest that RB inhibits steps subsequent to
RNA polymerase III recruitment during U6 repression. For example, RB might target
promoter escape, open complex formation, first dinucleotide bond formation, or
elongation. In vitro KMnO4 sensitivity assays performed in our laboratory indicate that
RB might trap RNA polymerase III in an open complex and facilitate a DNA
modification event, presumably methylation, to inhibit transcription (T. Selvakumar,
unpublished results). Thus, it is possible that RB recruits corepressor(s) to the U6 snRNA
promoter to repress transcription. It is intriguing to note that despite the same cohort of
transcription factors, p53 disrupts RNA polymerase III recruitment during U6 repression
(Gridasova, AA et. al. unpublished data). Thus, more insight into the tumor suppression
function of RB and p53 can be obtained by comparing and contrasting the mechanisms
used by these proteins to repress human U6 snRN A transcription.

While not much is known about the RB co-repressor proteins for regulating RNA
polymerase III transcription, several corepressor protein partners have been associated
with RB mediated repression of the E2F-stimulated RNA polymerase 11 target genes. RB
interacts with co-repressors that can control the chromatin structure, and therefore,

repress transcription by altering the state of chromatin surrounding a gene promoter. The

29

altered state of chromatin in turn might further limit access of transcription factors to
gene promoters and ablate transcription. Emphasis herein is given only to a subset of
these proteins that are pertinent to this document. Among other proteins, RB interacts
with the ATP-dependent chromatin remodeling complex SWI/SNF that repositions
promoter proximal nucleosomes to create repressive barriers within gene promoters, or to
prevent DNA unwinding required for transcription factor binding (38, 144). RB has also
been shown to associate with chromatin modifying proteins, including the class 1 histone
deacetylases (HDACs) consisting of HDACl, HDAC2, and HDAC3 (16). Deacetylation
of histones was observed on EZF-regulated target gene promoters in correlation with RB
repression. Furthermore, RB-mediated repression was inhibited when TSA, a HDAC-
specific inhibitor, was used in the transcription assays (6, 78, 82, 143, 144). HDAC3
remove acetyl groups from lysine residues in histone tails resulting in condensation of
chromatin. This altered state might obstruct transcription factors from accessing promoter
elements or inhibit DNA unwinding required for transcription. Interestingly, RB has been
associated with both HDAC and SWI/SNF activities as part of the same complex during
repression. For example, studies indicate that RB recruits both HDACs and SWI/SNF as
part of the same complex to regulate exit from G1. Subsequent disassembly of HDAC
activity is then thought to allow the RB-SWI/SNF containing complex to regulate exit
from S phase of the cell cycle (144). Interestingly, in addition to causing chromatin
condensation to hinder transcription factor access to promoter DNA, RB recruited HDAC
proteins may initiate a relay of histone modifications leading to transcriptional repression.
For example, deacetylation of histone H3 at lysine residue 9 (H3K9) would facilitate the

subsequent methylation of H3K9 by the histone methyl transferase SUV39H1. The

30

methylated H3K9 would in turn facilitate binding of the heterochromatin protein 1 (HPl)
(146). Indeed, RB has been shown to associate with SUV39H1 and the methyl lysine
binding protein HPl in co-immunoprecipitation experiments, and use the concerted
activities of SUV39H1 and HP] to repress cyclin B expression (95, 125). Contrary to its
role in promoting heterochromatic silencing in the context of RB-mediated repression,
HP] is thought to mask the methylated H3K9 such that it cannot be demethylated and
subsequently acetylated. Additionally, RB was also associated with DNA methyl
transferase 1 (DNMTl) in a complex that also included HDACl and E2F 1, during the
chromatographic purification of DNMTl. DNMTl methylates cytosines in CpG islands
in the promoter region of target genes to impede transcription factor access. Interestingly,
the methyltransferase activity of DNMTl was partially abolished in the presence of
TrichostatinA (TSA), which is a HDAC inhibitor, suggesting that RB can engage the
concerted activities of DNMTl and HDACs for repression (105). Indeed it is suggested
that the methylated Cst are bound by methyl DNA binding protein MBD, which in turn
can help coordinates HDAC activity to repress transcription (63). While RB can utilize a
constellation of co-repressor proteins as part of a repressor complex, a common theme

that emerges is that HDACs are almost always present in these complexes.

Although not much is known about corepressor proteins that RB might engage to
repress RNA polymerase III transcription, human U6 snRN A gene transcription is a good
model system to investigate the putative RB corepressor protein partners. As RB can
stably occupy a U6 snRN A promoter (53), it allows us to study the mechanistic order of

events that occur during RB repression. Chapter 2 describes the studies I performed to

31

investigate the role of RB co-factors in RB-mediated repression of U6 snRNA

transcription.

32

References

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45

CHAPTER 2

RB RECRUITS HDAC ACTIVITY FOR REPRESSION OF HUMAN
U6 snRNA TRANSCRIPTION BY RNA POLYMERASE IIIl

Abstract

The Retinoblastoma (RB) tumor suppressor protein is a global repressor of RNA
polymerase III transcription. We have previously shown that RB, components of the
preinitiation complex and RNA polymerase III co-occupy a U6 snRNA promoter during
repression. To further elucidate the molecular mechanism underlying RB mediated
repression of U6 snRNA transcription, the role of putative RB co-factors was examined.
In this study I show that endogenous RB, HDACs and SWI/SNF chromatin remodeling
proteins occupy a human U6 snRN A promoter. Interestingly, RB stimulates the binding
to HDAC2 and Brgl to a U6 promoter during repression. Importantly, HDAC enzymatic
activity is required for RB repression of U6 transcription initiated from chromatin-
assembled DNA but not from naked DNA templates. These results suggest that RB
repression of U6 transcription is a multi-step process in which a HDAC2-dependent
step(s) is required during repression of chromatin templates but not during the

establishment of repression on naked DNA template. This biochemical separation of RB

 

1 Figure 4-1A used in this document was published in the following manuscript: Heather A. Hirsch, Gauri
W. Jawdekar, Kang-Ae Lee, LiPing Gu, and R. William Henry (2004) Distinct mechanisms for repression
of RNA polymerase III transcription by the Retinoblastoma tumor suppressor protein. Molecular and
Cellular Biology Vol.24; pp.S989-5999

46

repression and HDAC activity will be a useful tool to understand the mechanistic order of

events involved in this process.

Introduction

The gene encoding the Retinoblastoma (RB) tumor suppressor protein was
identified due to its frequent mutation in the rare pediatric eye tumor, retinoblastoma.
Subsequently, mutations in the retinoblastoma gene were also found in approximately
30% of all adult onset tumors including osteosarcomas, small-cell lung carcinomas,
cervical carcinomas, prostate carcinomas, breast carcinomas, and some forms of
leukemia (3, 7). Many studies over the years have established the credentials of RB as a
tumor suppressor protein, and most support a central role for RB in constraining growth
and proliferation (29). For example, overexpression of RB in RB -/- tumor cells
suppressed growth and proliferation, soft agar colony formation, and tumorigenicity in
nude mice, showing that RB restricts growth (12, 20). RB overexpression induces G1
arrest while the lack of RB in cells was correlated with accelerated G1 to S phase
transition, suggesting that RB regulates cell cycle progression. RB is also implicated in
other cellular functions such as DNA replication, DNA repair, apoptosis, and
differentiation through its ability to repress E2F-stimulated transcription of target genes
whose products influence these processes (2, 6, 7). Thus, RB function as a transcription

factor is central for its tumor suppressor activity.

RNA polymerase III transcribed gene products are essential for cell growth and
proliferation (8). Moreover, RB has been shown to be a global repressor of RNA

polymerase III transcription. Indeed, we and others have shown that RB represses human

47

RNA polymerase III transcribed genes including SS rRNA, tRNA, and U6 snRNA both
in vivo and in vitro (10, 15, 30). For example, RNA polymerase III transcription of these
genes was elevated in RB -/- SAOSZ human osteosarcoma cell line compared to RB +/+
U2OS human osteosarcoma cell line, while RNA polymerase II transcription remained
largely unaffected (15, 30). As elevated RNA polymerase III activity and loss of RB
function are both common features of transformed and tumor cells, it was proposed that
RB regulates RNA polymerase III transcription to restrict growth. Recent studies have
demonstrated that the NB pocket domain and C-terminal region of RB (extending fi'om
amino acids 379-928), is required for efficient repression of RNA polymerase III
transcription (11, 30). Interestingly, this same region of RB is also required for tumor
suppressor function (20, 31). These observations indicate that repression of RNA

polymerase III transcription by RB could contribute to its tumor suppressive potential.

While RB is a global repressor of RNA polymerase III transcription, RB utilizes
distinct mechanisms of repression for different classes of genes. RNA polymerase III
transcribed genes can be divided into four different classes. Class 1 and class 2 genes
contain intragenic promoter elements exemplified by SS rRNA and tRNA genes,
respectively. In contrast, class 3 genes contain extragenic promoter elements exemplified
by U6 snRN A gene. Class 4 genes contain both intragenic as well as extragenic promoter
elements exemplified by the Vault RNA. The model for RB regulation 0f class 2 (tRNA)
genes suggests that RB disrupts preinitiation complex assembly at the promoter. In this
model, RB targets the Brfl-TFIIIB complex for repression. Brfl-TFIIIB complex
contains the TATA-box binding protein (TBP) and del in addition to Brfl, and is

recruited to the promoter of these genes by the TFIIIC complex, which directly binds to

48

the promoter (23). RB-mediated disruption of the Brfl -TFIIB complex is proposed to
abrogate recruitment of RNA polymerase III thereby repressing transcription (15, 26).
RB utilizes a similar mechanism to repress RNA polymerase II transcribed genes, where
RB interacts with the activator protein, E2F, and inhibits polymerase II recruitment (6, 7,

9, 22).

Unlike tRNA genes and E2F-regulated genes where RB prevents assembly of the
preinitiation complex, RB utilizes a novel mechanism to repress U6 snRNA gene
transcription by RNA polymerase III. At these genes, RB stably associates with the
promoter, like E2F-mediated repression, but instead does not abrogate association of the
general transcription machinery consisting of SNAPC and Ber-TFIIIB, with the U6
promoter. Interestingly, RB and RNA polymerase III also simultaneously occupy a
human U6 snRNA promoter in vivo and in vitro during repression (11). This last
observation indicates that a third model of RB repression is required to explain RB
repression at these genes wherein RB inhibits a step after RNA polymerase III
recruitment, possibly affecting promoter escape, open complex formation, first di-
nucleotide bond formation, or elongation. RB could inhibit these processes directly by
interacting with RNA polymerase III; alternatively, RB could act indirectly by occluding
binding of a critical transcription factor required for RNA polymerase III elongation or

by recruiting a co-repressor(s) that inhibit RNA polymerase III fimction.

Several RB co-repressor proteins have been identified for RB repression of E2F-
regulated genes transcribed by RNA polymerase II. For example, RB interacts with
histone deacetylases (HDACs), and the SWI/SNF ATP-dependent chromatin remodeling

complex that may reposition promoter proximal nucleosomes and affect access of

49

additional transcription factors (1, 5, 16, 17, 32). RB has been suggested to recruit
multiple co-repressors as components of a single co-repressor complex. For example, a
RB-HDAC-SWI/SNF complex regulates exit from the G1 phase of the cell cycle, while a
RB-SWI/SNF complex regulates exit from the S phase (32). In addition to HDAC3, RB
also recruits methyltransferases, for example the histone methyltransferase SUV39H1,
and DNA methyltransferase (DNMT) to E2F target genes (13, 18, 21, 28). RB is thought
to utilize both histone deacetylase activity and SUV39H1 histone methylase activity
sequentially to inhibit transcription of the cyclin B gene (34). These RB cofactors are also

candidates as RB co-regulatory factors for RNA polymerase III transcription.

In this study, I have examined the role of RB co-factors in regulation of RNA
polymerase III transcription. I found that the bona fide RB co-factors including HDAC2
and the Brgl component of the SIW/SNF ATP-dependent chromatin remodeling complex
occupy a human U6 promoter in cells that retain an intact RB pathway. Furthermore, RB
stimulates association of HDAC2 and the Brgl component of SWI/SNF with promoter
DNA during repression of U6 transcription. Although RB can repress U6 transcription
from both naked and chromatin DNA templates, the HDAC enzymatic activity is
required for RB repression only when the U6 transcription template is assembled into
chromatin, but not when the template is non-nucleosomal. These results suggest that RB
repression of U6 transcription may be a multi-step process in which a HDAC2 dependent
step(s) is not required during the establishment of repression on naked DNA template but

is required during repression of chromatin templates.

50

Materials and Methods

Chromatin immunoprecipitation.

Human 184BS cells and HeLa cells were grown to approximately 75% confluence
and were then crosslinked with formaldehyde for 30 minutes. Chromatin was harvested
as described before (11). 100 uL of soluble chromatin corresponding to approximately IX
X 10 7 cells was used per immunoprecipitation reaction (total volume 1 mL) along with
the following antibodies: 2 uL of anti-SNAPC (C8143 Term Bleed), anti-TBP (SL2), or
preimmune serum, or 1 ug of anti-RB (SC-7872), anti-HDAC] (SC-7899), anti-HDAC2
(SC-7872), anti-Brgl (SC-10768), anti-BRM (SC-6450), anti-CBP (SC-A22),a-p300
(SC-N15), anti-H3 (Abcam-abl791), anti-AcH3 (Lys9/Lysl4, Upstate) or 2 ug goat
irnmunoglobin (IgG). After overnight incubation at 4°C, proteins G agarose beads were
added for 3 hours at 4°C. After several washes, protein complexes were eluted in 300 ul
of 0.1 M NaHCO; + 1% SDS for 30 minutes at room temperature. Cross-links were
reversed overnight at 65°C and the recovered chromatin was suspended in 50 uL of TE
buffer.

Sequential ChIPs were performed as described previously (11). Soluble chromatin
was prepared from 18485 cells as described above. 500 uL of chromatin, which is
equivalent to 5 x 10 7 cells was used in a total volume of 5 mL for each of the primary
immunoprecipitation reactions done with S uL of a-RB (M1170 Test Bleed 2), a-RNA
polymerase III (M1172 Test Bleed 2) or preimmune sera. After 1 hour at room
temperature, the immunoprecipitated material was collected using Protein G agarose
beads for an additional hour at room temperature. The beads were then washed as

described above. Next, immunoprecipitated material was released from the beads by

51

elution in a buffer containing 15 mM DTT. Subsequently, one-sixth of the eluted material
was used in the second round of immunoprecipitation reaction. The immunoprecipitated
material was then bound to protein G agarose beads and subsequently eluted in a buffer
containing 0.1M NaHC03 + 1% SDS. After reversing the cross-links, the
immunoprecipiated material was treated with Proteinase K, phenol extracted and ethanol
precipitated. The recovered DNA was suspended in 50 uL of TE buffer. PCR analysis
was performed using 5 uL of each immunoprecipitated sample or input chromatin. PCR
products were separated by 2% TEE-agarose electrophoresis, stained with ethidium
bromide, and visualized using Kodak imaging software.

The primers used to amplify each gene were:

U6 forward - S’-GTACAAAATACGTGACGTAGAAAG-3’

U6 reverse - 5’-GGTGTI‘TCGTCCTTTCCAC-3’

U2 forward — 5’-AGGGCGTCAATAGCGCTGTGG-3’

U2 reverse — 5’-TGCGCTCGCCTTCGCGCCCGCCG-3’
GAPDH forward — 5’-AGGTCATCCCTGAGCTGAAC-3’
GAPDH reverse - 5’-GCAATGCCAGCCCCAGCGTC-3’
tRNA-Lys forward - S’-GGTTTCCCTCAAGGAGGGGG-3’ and

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

Protein purification and expression.

Recombinant GST-RB (379-928) was expressed and purified as described before

(10).

52

Immunoprecipitationfiom an in vitro transcription reaction mixture.

In vitro transcription repression of a U6 promoter plasmid was performed as
described before in the presence of GST-RB (379-928) or GST (10). One-third of the
reaction was processed for RNase T1 protection to measure transcription. The remaining
two-thirds was diluted to 1 mL with ChIP dilution buffer, cross-linked in 1%
formaldehyde for 15 min at room temperature and subsequently quenched with 0.125 M
glycine for 10 min at room temperature. Ten uL of the cross-linked material was used for
immunoprecipitation reactions with preimmune sera or anti-RB (SC-7872), anti-HDACI,
anti-HDAC2, anti-BRM, and anti-Brgl antibodies as above. Recovered plasmid DNA
was analyzed by PCR using primers specific to the U6 promoter region. The sequence of

the primers was described before (10).

In vitro repression assay of naked and chromatinized U6 template.

In vitro transcription repression of a naked U6 promoter plasmid was performed
as described before in the presence of GST-RB (379-928) or GST (10), except 6 uL of
HeLa cell nuclear extract was used to program transcription. U6 template DNA was
chromatinized using the protocols described before (14, 33). For the experiment shown in
Figure 2-4A, 1000 ng of GST-RB or GST was used in the absence or presence of 330 nM
TSA or methanol control. For the experiment shown in Figure 2-5, 150 ng, 300 ng, 600

ng, or 1000ng of GST-RB was used in the absence or presence of 500 nM TSA.

S3

Results

Chromatin remodeling proteins occupy the endogenous human U6 snRNA

promoter

Human RNA polymerase III transcription of SS rRNA, tRNA, and U6 snRNA
genes is inhibited by RB (10). To further understand the mechanism of RB repression of
endogenous class 2 (tRNA) and class 3 (U6 snRNA) genes, and whether RB can stably
associate with these promoters, the in vivo occupancy of RB at a tRNALys and a U6
snRNA promoter was examined by ChIP assays. As shown in Figure 2-1A, the anti-RB
immunoprecipitation was enriched in U6 snRN A promoter DNA but not U2 snRNA or
tRNALys promoter DNA (lane 8). The immunoprecipitation performed with anti-SNAP43
antibody was significantly enriched for U6 snRNA and U2 snRNA promoter DNA but
not for tRNALys or GAPDH exon 2 DNA (lane 6) as compared to the negative control
IgG precipitation (lane 5). This result is expected, as SNAPC is required for snRNA
transcription but not tRN A transcription. Furthermore, anti-TBP immunoprecipitation
was also enriched for U6 snRNA, U2 snRNA, and tRNALys promoter DNA, consistent
with a role for TBP in transcription of these genes (lane 7). That endogenous RB
occupies the U6 but not the U2 snRNA gene promoter is consistent with our previous
observation that RB preferentially regulates RNA polymerase III transcribed U6 snRNA
but not RNA polymerase II transcribed U2 snRNA even though both genes contain
similar promoter elements. Moreover, in the model for RB repression of tRNA genes
wherein RB disrupts preinitiation complex formation, RB is not expected to be present at
the promoter (11). These results show that endogenous RB can stably associate with U6

snRN A promoter DNA.

54

Figure 2-1. Endogenous chromatin modifying proteins associate with a human U6
snRNA promoter.

(A) RB occupies a U6 snRNA gene promoter in vivo. ChIP experiments from human
184B5 cells were performed using anti-SNAP43 (lane 6), anti-TBP (lane 7), and anti-RB
(lane 8) antibodies and nonspecific IgG (lane 5). Precipitated DNA was analyzed by PCR
amplification for enrichment of U6 snRNA, U2 snRNA, and tRNA promoters and
negative control GAPDH exon 2.

(B) Endogenous HDACl and HDAC2 as well as the BRM and Brgl subunits of the
ATP-dependent chromatin remodeling complex occupy a U6 snRNA promoter. ChIP
experiments were performed, using chromatin harvested from RB containing 184BS cells
with anti-SNAP43 (lane 6), anti-BRM (lane 7), anti-Brgl (lane 8), anti-HDAC] (lane 9),
and anti-HDAC2 (lane 10) antibodies or nonspecific IgG (lane 4).

(C) Acetylation levels of endogenous histone H3 at U6 snRN A promoter are affected by
RB status. Chromatin was harvested from RB containing normal human mammary
epithelial cells (184BS) and HeLa cervical carcinoma cells containing RB lacking RB
function, was used for ChIP with anti-SNAP43 (lane 6), anti-H3 (lane 7), and anti-AcH3
that recognizes acetylated histone H3 at lysine 9/ 14 (lane 8) and negative control IgG
(lane 5). Enrichment of human U6 snRNA promoter DNA and GAPDH exon 2 DNA in
the immunoprecipitated DNA was analyzed as above. In all the above experiments, lanes
1-4 show a 10-fold serial dilution of input chromatin from 10% to 0.01%.

55

 

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56

It was recently shown that a positioned nucleosome is located between the DSE
and the PSE in the natural U6 snRN A gene promoter region and the correctly positioned
nucleosome is suggested to bring these snRN A promoter elements into close proximity to
facilitate cooperative binding by Oct-1 and SNAPC to the DSE and PSE, respectively,
which is required for activated transcription (33). Thus, it is possible that RB recruits one
or more chromatin modifying activities to influence nucleosome structure or position
during U6 repression. As RB can recruit chromatin modifying proteins such as HDACl
and HDAC2, as well as the Brgl and BRM components of the ATP-dependent SWI/SNF
chromatin remodeling complex, to repress the E2F-stimulated transcription of target
genes (1, 16, 17, 32), the possibility that RB recruits these co-repressor proteins to the U6
snRNA promoter was examined. As a first step whether endogenous HDACI and
HDAC2 as well as the Brgl and BRM subunits of the SWI/SNF ATP-dependent
chromatin remodeling complex occupy a human U6 snRNA promoter in vivo was
examined using chromatin harvested from normal mammary 184B5 epithelial cells. As
shown in Figure 2-lB, U6 promoter DNA, but not GAPDH exon 2 DNA, was
significantly enriched in immunoprecipitations performed with anti-BRM (lane 7), anti-
BRGl (lane 8), anti-HDAC] (lane 9), and anti-HDAC2 (lane 10) antibodies.
Interestingly, no detectable or modest enrichment of U2 snRNA promoter DNA was
observed in these immunoprecipitations, even though significant enrichment was seen in
the anti-SNAPC immunoprecipitation (lane 6). This correlates with the lack of RB
occupancy on the U2 snRNA promoter as shown in Figure 2-1A. These observations
suggest that HDAC], HDAC2, Brgl, and BRM proteins associate with the U6 snRNA

promoter and may be involved in RB repression of U6 transcription.

57

Next, ChIP experiments were performed to detect the presence of unmodified-
histone H3 or acetylated (Ac) histone H3 at a human U6 snRNA promoter in vivo and
whether acetylation status was affected by RB status. For this experiment chromatin was
harvested from normal mammary 184BS epithelial cells containing RB, and HeLa
cervical carcinoma cells lacking functional RB. RB occupies the endogenous U6
promoter in 18435 cell but not in HeLa cells, suggesting that U6 regulation may be lost
in the HeLa cancer cells (11). As shown in Figure 2-1C, both total H3 and acetylated H3
occupy an endogenous U6 promoter in 18435 cells. Very little detectable total H3 as
compared to H3 that is acetylated occupied U6 promoter DNA in HeLa cells (lanes 7 and
8). Significant enrichment of the U6 promoter DNA, but not GAPDH exon2 DNA, is
seen in the anti-SNAP43 immunoprecipitation from both 184BS and HeLa cells (lane 6).
That total H3 is seen at the U6 promoter in 18485 cells, but not in HeLa cells, indicates
that the presence of total H3 and post-translationally modified H3 may be contingent
upon a functional RB pathway and associated RB-dependent co-factors that influence

histone modification.

RB recruits HDAC proteins during repression

As endogenous HDACs occupy a human U6 snRNA gene promoter in cells
containing functionally active RB, the hypothesis that RB and HDAC proteins associate
simultaneously on the U6 promoter during repression was tested. Sequential ChIP was
performed using chromatin harvested from 184B5 cells with either anti-RB or
preimmune antibodies in the first round of immunoprecipitation. Associated factors were

then eluted from protein G agarose beads and a second round of immunoprecipitation

58

Figure 2-2. RB concurrently occupies an endogenous U6 snRNA promoter with
HDAC], HDAC2 and RNA polymerase III.

Sequential ChIP was carried out from chromatin harvested from 184B5 cells using anti-
RB antibodies (top panel) or nonspecific preimmune serum (bottom panel) in the primary
immunoprecipitation reaction. Subsequently, secondary immunoprecipition reactions
were carried using anti-RB (lane 6), anti-RNA polymerase III (lane 7), anti-HDAC]
(lane8) or anti-HDAC2 (lane 9). DNA precipitated after the secondary
immunoprecipitation was analyzed for enrichment of U6 snRNA promoter DNA and

GAPDH exon 2 DNA.

59

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60

was performed using the indicated antibodies. As shown in Figure 2-2 (top panel),
significant enrichment of U6 promoter DNA was observed in the anti-RB/anti-RB (lane
6) and anti-RB/anti-RNA polymerase III (lane 7) immunoprecipitation reactions, but not
in the negative control anti-RB/preimmune precipitation reaction (lane 5), consistent with
previous data from our lab (11). Interestingly, U6 promoter DNA was enriched in the
immunoprecipitated reactions performed using anti-RB antibodies followed by anti-
HDAC2 antibodies (lane 8), but not by the anti-HDAC] antibodies (lane 9). The lack of
U6 promoter recovery with anti-HDAC] antibodies in this experiment may be because
the HDAC] epitope is occluded; however, these anti-HDACI antibodies did recover
HDACl in single round ChIP reactions (Figure 2-1B). Therefore, we favor the idea that
RB and HDAC2, but not HDAC], can simultaneously occupy an endogenous U6
promoter. As expected, no significant amount of U6 promoter DNA was obtained when
the negative control preimmune sera was used in the first round of immunoprecipitation
(Figure 2-2 bottom panel).

Next, whether HDACs and/or SWI/SNF proteins are recruited to the U6 promoter
when RB is actively repressing transcription was examined. To test this idea, in vitro U6
transcription was performed in the presence or absence of GST-RB (379-928). A portion
of the transcription reaction was tested for RB repression in a riboprobe protection assay.
As shown in Figure 2-3A, GST-RB repressed U6 transcription relative to the untreated
extract or extract treated with a comparable amount of GST (compare lane 2 to lanes 1
and 3). The remainder of each transcription reaction was cross-linked with formaldehyde

for subsequent immunoprecipitation reactions using non-specific preimmune sera or

61

Figure 2-3. RB stimulates enrichment of HDACs and SWI/SNF at a U6 snRNA
promoter during repression in vitro.

(A) U6 snRNA in vitro transcription was carried out in the in the absence of any added
proteins (lanel), was effectively repressed by 1000 ng of recombinant GST-RB (379-
928) (lane 2) but not by an equivalent amount of GST (lane 3). Transcription was also
carried out in the absence of nuclear extract (lane 4). Portions of the U6 transcription
reaction were cross-linked with formaldehyde as described before (11).

(B) Cross-linked in vitro transcription reactions were subjected to immunoprecipitation
reactions using anti-RB (lane 3), anti-HDAC] (lane 4), anti-HDAC2 (lane 5), anti-BRM
(lane 6), or anti-BRGl (lane 7) antibodies or nonspecific preimmune serum (lane 2). The
recovery of immuoprecipitated material was analyzed by PCR for the presence of the U6
reporter construct (pU6/Hae/Ra.2) with primers specific to the promoter region in the
plasmid.

Part A of this figure was performed by Heather A. Hirsch.

62

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Figure 2-3. RB stimulates enrichment of HDACs and SWI/SNF at a U6 snRNA
promoter during repression in vitro.

(A) U6 snRNA in vitro transcription was carried out in the in the absence of any added
proteins (lanel), was effectively repressed by 1000 ng of recombinant GST-RB (379-
928) (lane 2) but not by an equivalent amount of GST (lane 3). Transcription was also
carried out in the absence of nuclear extract (lane 4). Portions of the U6 transcription
reaction were cross-linked with formaldehyde as described before (1 l).

(B) Cross-linked in vitro transcription reactions were subjected to immunoprecipitation
reactions using anti-RB (lane 3), anti-HDAC] (lane 4), anti-HDAC2 (lane 5), anti-BRM
(lane 6), or anti-BRGI (lane 7) antibodies or nonspecific preimmune serum (lane 2). The
recovery of immuoprecipitated material was analyzed by PCR for the presence of the U6
reporter construct (pU6/Hae/Ra.2) with primers specific to the promoter region in the
plasmid.

Part A of this figure was performed by Heather A. Hirsch.

62

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antibodies directed against RB, HDACl, HDAC2, BRM, and BRGl. U6 promoter DNA
was immunoprecipitated with anti-RB antibodies only when GST-RB was included in the
transcription reaction (Figure 2-3B, lane 3). Similar experiments performed previously
demonstrated that exogenously added GST-RB (379-928) does not associate with a
promoter-less plasmid ((11), and data not shown). In the absence of added GST-RB, the
anti-HDAC] and anti-Brgl immunoprecipitation resulted in no U6 promoter DNA
recovery, whereas the anti-HDAC2 and anti-BRM immunoprecipitation led to weak U6
promoter recovery. Interestingly, U6 promoter DNA recovery in the anti-HDAC2 and
anti-Brgl immunoprecipitation was noticeably increased in the presence of GST-RB
(lanes 5 and 7, compare the top and middle panels). Whereas GST-RB only modestly
stimulated U6 promoter DNA recovery from the anti-HDAC] and anti-BRM
immunoprecipitations as compared to DNA recovery from the mock
immunoprecipitation reaction carried out using preimmune sera (lanes 4 and 6, middle
panel). Together, these results suggest that RB recruits HDAC2 and Brgl to the U6

promoter either directly or indirectly.

HDAC activity is required for RB repression of U6 snRNA transcription from a

chromatin template

The observation that RB stimulates promoter association of HDAC2 at the
U6 promoter during repression suggested that HDAC activity is required for RB
repression of RNA polymerase III transcription. To test this idea, RB repression assay

was carried out in the absence or presence of Trichostatin A (TSA), which is a potent

inhibitor of HDAC activity. The deacetylase activity of I-IDACs is mostly associated with
removing acetyl groups from histone tails that are part of a nucleosome. However, RB
potently represses U6 transcription initiated from a non-nucleosomal naked DNA
template, and the use of HDAC activity in this context would suggest that HDAC2
targets proteins other than nucleosomes, for example, U6 snRNA- specific transcription
factors such as SNAPC or Brf2-TFIIIB, to exert repression. In contrast, if HDAC activity
was required for RB repression of chromatin templates but not naked DNA templates,
then it would indicate that HDAC activity targets nucleosomes to inhibit transcription. As
shown in the Figure 2-4 top panel, U6 transcription carried out in the presence of
methanol + TSA (lane 2) or methanol, which was used as a vehicle for TSA (lane 3), was
not significantly affected as compared to the levels of transcription seen in lane 1.
However, this same amount of TSA was sufficient to inhibit transcription of a tRNA-like
(class 2) AdVAI gene (Figure 2-4, see lane 6 bottom panel). Thus, HDAC activity is not
essential for U6 transcription, permitting testing of the hypothesis that HDAC activity is
required for RB repression of naked DNA templates. Interestingly, GST-RB (379-928)
repression of U6 transcription was comparable both in the absence or presence of TSA
(compare lanes 4 and 5). Similar results were observed when sodium butyrate, a different
HDAC inhibitor was used (data not shown). Thus, HDAC activity is not required for RB
repression of U6 transcription initiated from a naked DNA template and altering the
acetyation status of U6-specific transcription factors is not critical for repression.

As RB repression of naked U6 DNA template was refractory to HDAC inhibition,
we next considered that RB directs HDAC activity towards nucleosomes during

repression. To directly test the hypothesis that RB requires HDAC activity for inhibition

65

Figure 2-4. RB repression of U6 transcription and HDAC activity are biochemically
separable activities.

RB repression of U6 transcription is not affected by concentrations of TSA that inhibit
AdVAI transcriptioin.

(A) U6 transcription was carried out in the presence of 200 nM Trichostatin A (TSA)
(lane 2) or methanol, which was used as a carrier for TSA (lane 3). The ability of 1000 ng
GST-RB (3 79-928) to repress U6 transcription in the absence (lane 4) or presence of TSA
(lane 5) was tested. As a control 1000 ng GST was added to the transcription reactions in
the absence (lane 6) or presence (lane 7) of TSA. Lane 1 represents U6 transcription
supported with untreated extract, and lane 8 shows transcription reactions that were
carried out in the absence of any nuclear extract.

(B) AdVAI transcription was carried out in the presence of increasing amounts of TSA
(25 nM, 50 nM, 100 nM, 165 nM, and 330 nM) (lanes 2 to 6). Methanol (330 nM) was
added to the transcription reaction in lane 7. Lane 1 shows AdVAI transcription
supported by untreated extract.

66

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67

of U6 transcription from nucleosomal templates, I established an in vitro transcription
assay initiated from a chromatin template plasmid harboring the U6 snRNA promoter
elements. The chromatin template was initially tested for its ability to support U6
transcription. As shown in Figure 2-5, comparable levels of U6 transcription were
obtained from naked DNA and chromatin templates (lane 1, compare the first and third
panel). When increasing amounts of GST-RB were added to the transcription reaction in
the absence or presence of TSA, GST-RB repressed U6 transcription of naked DNA
template to similar levels (compare lanes 2-4 in the first and second panels).
Interestingly, in the absence of TSA lesser amounts of GST-RB were needed for efficient
repression of chromatin templates as compared to naked DNA templates. However, in the
presence of TSA, GST-RB did not repress U6 transcription to a similar extent from a
chromatin template as it did from naked DNA templates. Thus, this result suggests that
RB requires HDAC activity to repress U6 snRNA transcription in the context of

chromatin.

Discussion

RE is a global repressor of RNA polymerase III transcription. Further
understanding the mechanism(s) of RB repression of these genes will provide insights
into RB function. The data presented herein demonstrates a role for HDACs and
components of the SWI/SNF ATP-dependent chromatin-remodeling complex in RB-

mediated repression of U6 snRNA transcription.

On other target genes, RB recruits these co-factors to prevent transcription factor

binding and polymerase recruitment. For example, RB recruits I-IDACs to deacetylate the

68

Figure 2-5. HDAC activity is required for RB repression of U6 transcription from
chromatin DNA templates but not from naked DNA templates.

Approximately 6 uL of HeLa cell nuclear extract was incubated with a U6 promoter
containing plasmid, or plasmid that had been assembled into chromatin. Increasing

amounts; 300 ng (lane 2), 600 ng (lane 3), and 1000 ng (lane 4) of GST-RB (379-928)
were tested for U6 repression in the presence of 500 uM Trichostatin A (TSA) or in

reaction without any added TSA. 1000 ng GST (lane 5) was used as a negative control.
Lane 1 shows the level of transcription supported by untreated extracts.

69

- TSA

-+TSA

- TSA

+ TSA

+GST-RB
(379-928)

  

Untreated
+GST

 

 

 

 

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70

 

naked
DNA

chromatin

upstream binding factor (UBF), which is a RNA polymerase I-specific transcription
factor. It is thought that deacetylation of UBF results in its inability to recruit the TBP-
containing SL-l complex (4, 19). Alternatively, RB utilizes HDACs and SWI/SNF to
repress E2F-regulated genes by mainly altering the chromatin structure (1, 16, 17, 24,
27). Our observation that HDAC activity is required for RB repression of U6
transcription from a chromatin template, that is presumably reflective of its endogenous
state but not from a naked DNA template, suggests that HDACs may influence the
chromatin structure at a U6 promoter. Recent studies have indicated a role of chromatin
structure in U6 snRNA transcription. For example, in vivo mapping studies have
demonstrated the presence of a positioned nucleosome between the distal sequence
element (DSE) and the proximal sequence (PSE). Correct positioning of the nucleosome
is thought to bring these two promoter elements close to each other, thus facilitating
cooperative binding of Octl and SNAPC to the DSE and PSE, respectively, to activate
transcription (33). It is possible then to imagine that RB recruites HDAC and SWI/SNF
to alter the nucleosome structure to repress transcription. For example, misaligning the
nucleosome may disrupt the Octl-SNAPc interaction and/or prevent the interaction of
Oct] with an unknown transcription factor required for promoter escape. Alternatively,
RB could remodel nucleosomes that are near the start site of transcription; however,
whether any nucleosome is located near the start site is not known. Moreover, RNA
polymerase III is capable of transcribing through a nucleosome (25) and hence the
positioned nucleosome in the U6 promoter region is the most likely RB target.

RB has also been shown to associate with multiple enzymatic activities in the

same complex. For example, RB can form a repressor complex that contains either

71

SWI/SNF or HDAC3 and SWI/SNF. The RB-HDAC-SWI/SNF complex and the RB-
SWI/SNF complex have been shown to repress cyclin E and cyclin A, respectively,
whose genes products are required for cell cycle progression (32). The sequential
disassembly of the RB-HDAC-SWI/SNF complex to one lacking HDAC activity is
proposed as one mechanism governing RB repression of different genes at distinct phases
of the cell cycle. Interestingly, we see that RB stimulates U6 promoter occupancy by
HDAC2 and Brgl. Whether RB, HDAC2 and Brgl are part of the same complex is not
known.

Previous studies investigating the mechanisms of RB repression of U6
transcription have indicated that RB and the general transcription machinery, including
SNAPC and TFIIIB, occupy a U6 snRNA promoter simultaneously (11). Furthermore,
RB and RNA polymerase III concurrently occupy a U6 snRNA promoter during
repression both in vitro and in vivo (11). These studies suggest that RB does not
necessarily exclude RNA polymerase III from the promoter, but may prevent subsequent
steps in transcription such as promoter escape by recruiting co-regulatory factors
discussed in this current study. While RB repression naked U6 DNA templates is
efficient, and RB stimulates association of HDAC2 and Brgl with naked DNA templates,
HDAC activity is invoked only when the U6 templates is nucleosomal. These
observations suggest that RB repression may be a multi-step process, as illustrated in a
speculative model depicted in Figure 2-6. In this model, RB establishes repression in the
absence of nucleosomes and maintains repression by invoking chromatin-remodeling
activity. RB establishes repression on naked DNA by interacting directly with RNA

polymerase III, possibly to tether the polymerase to the general transcription machinery

72

Figure 2-6. Model for RB repression of human U6 snRNA gene transcription

RB co-factors such as HDACs and components of the SWl/SNF ATP-dependent
chromatin remodeling complex are recruited to a U6 snRNA promoter during RB
repression. These activities may inhibit transcription by altering the positioned
nucleosome located between the distal sequence element (DSE) and the proximal
sequence element (PSE). Oct-1 and SNAPC cooperatively bind to the DSE and PSE,
respectively to activate transcription, which is facilitated in part by the positioned
nucleosome. Alternatively, RB may recruit other co-repressor proteins such as DNMTl
as part of the HDAC-containing complex to modify the DNA at the start site of
transcription, thus preventing polymerase escape. Direct interactions with RNA
polymerase III allow RB to tether the polymerase to the general transcription machinery.

73

Nucleosome

    

f‘rDNMT1 7
WVSN '3. 9
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TATA

74

thus preventing promoter escape. Preliminary data from our lab indeed shows that RB
interacts with RNA polymerase III both in vitro and in vivo (X Song, unpublished data).
Subsequently, RB recruits a complex that contains HDACs and/or SWI/SNF that may
alter the positioned nucleosome and prevent Oct-l from binding to the PSE and/or
causing disruption of the Octl-SNAPC contacts. Whether Oct-lzDNA contacts are
severed in a RB/HDAC-dependent manner is not yet known. Alternatively, RB may
modify DNA around the start site of transcription, thus preventing polymerase escape.
Data from our laboratory indicates that DNA near the start site of transcription is indeed
modified during RB repression, and this modification is most likely methylation (T
Selvakumar, unpublished data). Moreover, association of de novo DNA methyl
transferase 1 (DNMTl) with the U6 snRNA gene promoter increases when RB is over-
expressed in HeLa cells in transient transfections assays (G. Jawdekar unpublished data)
raising the hypothesis that RB recruits DNMTl to modify DNA. While RB, HDAC3, and
DNMTI have been implicated in a repressor complex for E2F-regulated genes, it is
possible that such a complex is also recruited to U6 snRN A gene promoters during RB-
mediated repression transcription. By biochemically separating RB repression and HDAC
activities we can now start understanding the mechanistic details of RB repression on

both naked as well as chromatin DNA.

75

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78

CHAPTER 3

THE UNORTHODOX SNAP50 ZINC FINGER DOMAIN
CON TRIBUTES TO CO-OPERATIVE PROMOTER RECOGNITION
BY HUMAN SNAPCI

Abstract

Human small nuclear (sn) RNA gene transcription by RNA polymerases II and III
depends upon promoter recognition by the SNAPC general transcription factor. DNA
binding by SNAPC involves direct DNA contacts by the SNAP190 subunit in cooperation
with SNAP50 and SNAP43.The data presented herein shows that SNAP50 plays an
important role in DNA binding by SNAPC through its zinc finger domain. The SNAP50
zinc finger domain contains fifteen cysteine and histidine residues configured in two
potential zinc coordination arrangements. Individual alanine substitution of each cysteine
and histidine residue demonstrated that eight sites are important for DNA binding by
SNAPC. However, metal-binding studies revealed that SNAPC contains a single zinc
atom indicating that only one coordination site functions as a zinc finger. Of the eight
residues critical for DNA binding, four cysteine residues were also essential for both U1
and U6 transcription by RNA polymerase II and 111, respectively. Surprisingly, the
remaining four residues, while critical for U1 transcription could support partial U6
transcription. DNA binding studies showed that defects in DNA binding by SNAPC alone

could be suppressed through cooperative DNA binding with another member of the RNA

 

1 This work will be published in the following manuscript: Gauri W. Jawdekar, Andrej Hanzlowsky, Stacy
L. Hovde, Blanka Jelencic, Michael F eig, James H. Geiger, and R. William Henry (2006) The unorthodox
SNAP50 zinc finger domain contributes to co-operative promoter recognition by human SNAPC. Journal of
Biological Chemistry. Accepted article in press.

79

polymerase III general transcription machinery, TFIIIB. These results suggest that these
eight cysteine and histidine residues perform different functions during DNA binding
with those residues involved in Zn coordination likely performing a dominant role in
domain stabilization and the others involved in DNA binding. These data further define
the unorthodox SNAP50 zinc finger region as an evolutionarily conserved DNA binding

domain.

Introduction

The human snRNA gene family is unusual in that related member genes are
transcribed by either RNA polymerase II or III depending upon their core promoter
structures, and they thus serve as an interesting model to understand principles of
polymerase selection and activity both during normal and deregulated growth (reviewed
in (8, 12)). Human snRNA genes are defined by the presence of a diagnostic promoter
element called the proximal sequence element (PSE). For both polymerase systems, the
PSE recruits the general transcription factor called SNAPc (27), which is also known as
PTF (25). SNAPC is composed of at least five subunits SNAP190, SNAP50, SNAP45,
SNAP43, and SNAP19 (9-11, 26, 35). SNAP190 can interact with all the other subunits,
and thus provides a central architectural backbone to the complex (22). SNAP190
directly interacts with the transcriptional activator protein Oct-1 during stimulated
transcription of human snRNA genes (5, 24). In the RNA polymerase III pathway,
SNAP190 also interacts with TBP to recruit Brf2-TFIIIB to the TATA box of human U6
snRNA genes (13, 23). Thus, the upstream signal from Oct-1 is conveyed through

SNAP190, stimulating preinitiation complex assembly with other general transcription

80

factors as a prerequisite for RNA polymerase III recruitment. Interestingly, protein kinase
CK2 can phosphorylate SNAP190 to impede DNA binding; however, promoter
recognition by SNAPC can be restored by cooperative binding of TBP to those promoter
sequences containing both a PSE and TATA box, but not sequences lacking the TATA
element (6). This last observation suggests that CK2 can differentially influence RNA
polymerase II and III snRNA transcription by covalent modification of SNAPC to alter its
DNA binding properties even though SNAPC is shared between both polymerase
systems.

The specific binding of SNAPC to the PSE is mediated by a Myb-like DNA
binding domain within the N-terrninal region of SNAP190. Other SNAPC subunits may
additionally function in PSE recognition and provide stabilizing contacts with DNA.
Indeed, UV cross-linking experiments suggest that both SNAP190 (36) and SNAP50 (9)
are in close contact with DNA. In addition, SNAP50 contains an unusual arrangement of
cysteine and histidine residues at the carboxy terminal region of the protein that is
evolutionarily conserved. In other transcription factors, zinc finger motifs function for
nucleic acid binding and/or protein-protein interactions (21). In the current study, we
have focused on the function of the zinc finger region of SNAP50, and we show that this
region is critical for DNA binding by SNAPC. The arrangement of cysteine and histidine
residues within SNAP50 further defines this region as an unorthodox zinc finger domain
that functions in divergent preinitiation complex assembly pathways for RNA polymerase

II and III transcription.

81

Materials and methods
Expression and purification of recombinant proteins

For the EMSA shown in Figure 2-1A, mini-SNAPC was assembled from subunits
individually expressed in E. coli and purified as described before (13). The partial
complex SNAPcy4 was obtained by co-expression in E. coli of SNAP190 (1-505),
SNAP43, SNAP19, and either wild type or mutant SNAP50. The complex was purified
as described (7). TBP was expressed as a GST fusion protein in E. coli and purified as
described (13). The Brf2 and de1 (1-470) proteins were expressed in E. coli and
purified as described before (30). In all cases, GST tags were removed prior to use in

functional assays by thrombin digestion during purification

Electrophoretic mobility shift assay

EMSA was performed in a 20 uL total volume using DNA probes containing a
wild-type or mutant mouse U6 PSE with a mutant human U6 TATA box, or containing
the wild-type human U6 sequence, as described previously (13). DNA binding reactions
using only SNAPc were performed in a buffer containing 60 mM KCl, 20 mM HEPES
pH 7.9, 5 mM MgC12, 0.2 mM EDTA, 10% glycerol, 0.5 ug of poly(dI-dC), and 0.5 ug
of pUC119 plasmid. Reactions were incubated for 20 min at room temperature after
which 5,000 cpm of probe was added, and reactions were incubated an additional 20 min.
Samples were fractionated on a 5% nondenaturing polyacrylarnide gel (39:1) in TGE
running buffer (50 mM Tris, 380 mM glycine, 2 mM EDTA). Reactions containing both
SNAPcy4 and TBP were performed in a buffer containing 100 mM KCl, 20 mM HEPES

pH 7.9, 5 mM MgC12, 0.2 mM EDTA, 10% glycerol, 1 mM dithiothreitol, 0.07% Tween

82

20, 0.2 p. g of poly(dG-dC), and 0.2 ug of pUC119 plasmid. The samples were
fractionated on a 5% nondenaturing polyacrylarnide gel (39:1) in TGEM running buffer
(50 mM Tris, 380 mM glycine, 2 mM EDTA, 5 mM MgClz). Approximately 1 ng of
SNAPcy4, 50 ng human TBP, 240 ng Brf2, and 40 ng of de1 (1-470) was used, as

indicated.

GST-pulldown and immunoprecipitation assays

GST pulldown assays were done as described before (13). For the
immunoprecipitation assays performed in Figure 3-ZB, approximately 20 ng of each
complex containing SNAP50 with the indicated point mutation was used with 1 uL of
anti-SNAP43 antibody, preimmune sera, or buffer alone. Immunoprecipitation reactions
were carried out in HEMGT-ISO buffer (25 mM HEPES, 0.1 mM EDTA, 12.5 mM
Mng, 10% Glycerol, 0.1% Tween-20, 150 mM KCl) containing protease inhibitors and
1 mM DTT at 4°C for 2 hr. The immunoprecipitated material was collected by incubation
with Protein G agarose beads at 4°C for 2 hr. Beads were then collected, washed 5 times
with HEMGT-ISO, and the bound protein was released by boiling in Laemmli buffer.
Recovered proteins were resolved by 15% SDS-PAGE and were transferred to a

nitrocellulose membrane for Western blotting using anti-HA antibodies.

In vitro transcription assays
Endogenous SNAPC was removed from HeLa extracts by anti-SNAP43 antibody
irnmunodepletion as described before (10). In-vitro transcription of human U1 and U6

ysnRN A genes was performed for 1 hr at 30°C using the depleted extract. Approximately

83

5 ng of wild type and mutant SNAPcy4 complexes containing point mutations in

SNAP50 were used for the reconstitution of both U1 and U6 transcription.

Zn binding studies

The amount of Zn associated with recombinant SNAPC was determined by two
methods: 1) Inductively coupled plasma mass spectrometry (ICP-MS), and 2) Flame
Atomic Absorption (FAA). For the ICP-MS studies, SNAPC protein concentration was
determined by Bradford assay using BSA as a standard. The molecular weights of
recombinant SNAPcy4 and SNAPcy3 were calculated as 159,735 and 148,432 g/mol,
respectively. To measure Zn concentration, sample solutions were transferred to a Teflon
vial and brought to dryness on a hot plate. Concentrated nitric acid was added and the
sample was placed on the hot plate and hydrolyzed for 30 min. After cooling, the sample
was diluted with water to bring the acid concentration to 2%, and 2% nitric acid was
added to bring the solution to the desired volume. Ni and Zn standards (Spex Certiprep)
in the concentration range from 0-1000 ppb were prepared in 2% nitric acid. Samples and
standards were each mixed with 20 ppb indium and bismuth standard solution (Spex
Certiprep) as internal standards. Samples were analyzed on an ICP-MS instrument (GV
Instruments) with a flow rate of 0.5 mL/min. Zn66, Zn68 and Ni60 isotopes were
measured and quantified. The responses for zinc and nickel were corrected according to
indium and bismuth response. For the FAA studies, SNAPc protein concentration was
determined by UV absorbance in 6 M urea at 280 nm. The molar extinction coefficient of
176,950 cm"M‘l was used to calculate the molar concentration. SNAPC samples were

transferred to a crucible, dried and ashed at 260 degrees. Concentrated nitric acid was

84

added to the ash and reheated until only white powder remained. The sample was
reconstituted with 5% nitric acid. Zinc standards in the concentration range from 10-
1000 ppb were prepared from zinc metal (Spectrum, 99.9 %) dissolved in concentrated
nitric acid. The samples and standards were analyzed on the flame atomic absorption
instrument (V arian SpectrAA-200) equipped with a zinc hollow cathode lamp operating
at 213.9 nm. Aspiration rate was 1 mL/min.

For the Zinc analysis shown in Figure 2-6, wild type or mutant SNAP50 (315-
411) was expressed as a N-terminal His-SUMO fusion protein from pET28a in E. coli
RIL codonplus. Cells were grown in LB broth containing kanamycin (100 ug/ml) and
chloramphenicol (50 ug/ml) supplemented with 50 uM ZnClz. Approximately 1 L of
culture was used for the expression and purification of each protein. Cells were ruptured
by sonication in buffer A (50 mM Tris pH 8.5, 350 mM NaCl, 10% glycerol, 10 mM
imidazole, 1 mM B-mercaptoethanol) supplemented with 10 uM ZnClz. His-tagged
recombinant proteins were purified by nickel column chromatography followed by
elution with buffer A containing 400 mM imidazole without added zinc. Protein
containing fractions were dialyzed against buffer A containing 1 mM DTT and no added
zinc for 10 hrs at 4°C for subsequent FAA analysis, which was performed as above using
approximately 2 mL of each protein solution (~1 mg/mL) with an aspiration rate of 5
mL/min. Protein concentration was determined in 6 M urea by UV absorbance at 280 nm.
The molar extinction coefficient of 27305 cm"M'l was used to calculate the molar
concentration of the wild type and mutant His-SUMO-SNAPSO (315-411) proteins. Zinc

measurements and protein determination were done in triplicate.

85

Structural Modeling

Two protocols were followed to obtain ab initio predictions of SNAP50 (301-
411). First, the amino acid sequence of SNAP50 was submitted to the Robetta server
(19), and ten models were obtained. Second, lattice-based sampling from extended chains
with MONSSTER (31) and the MMTSB Tool Set (4) were carried out. 2000 structures
were generated in independent runs. The resulting structures were subjected to a short
minimization with CHARMM (1) and evaluated with the scoring function DF IRE (37).
Correlation-based scores were obtained from the original DFIRE scores according to a
recently published method for enhancing scoring functions in protein structure prediction
applications (3 2). The structures were then clustered and average correlation-based scores
were compared between clusters. The structure with the highest correlation-based score
from the cluster with the highest average correlation-based score and more than one
member was then examined and subjected to further refinement through energy
minimization and constrained short molecular dynamics simulations with CHARMM.
The electrostatic potential on the surface of the final structure was calculated from
solutions of the Poisson equation with the PBEQ module (18) in CHARMM. The
program VMD was used for visualization of the final model and electrostatic surface

maps (17).

Results
The C-terminal region of SNAP50 is required for DNA binding by SNAPC.
Promoter recognition by SNAPC is a common early event in both RNA

polymerase II- and III-specific pathways of pre-initiation complex formation at hmnan

86

Figure 3-1. The C-terminal region of SNAP50 is required for DNA binding by
SNAPC.

(A) (Left panel) DNA binding by SNAPC requires cooperation among SNAP190,
SNAP50, and SNAP43. Electrophoretic mobility shift assays (EMSA) were performed
using a U6 probe containing a wild-type PSE and mutated TATA box (AD) or a U6
probe containing a mutated PSE and mutated TATA box (BD). DNA binding reactions
were carried out with SNAP43, SNAP190 (1-505) or SNAP50 individually (100 ng each,
lanes 1-3), or in pair wise combinations of SNAP43 and SNAP190 (1-505), SNAP43 and
GST-SNAP50, and SNAP190 (1-505) and GST-SNAP50 (lanes 4-6). Formation of
SNAPc/DNA complex was observed in reactions containing all three subunits with the
wild type PSE probe (lane 7) but not mutant PSE probe (lane 8). Reactions containing the
wild type and mutant probes with no added proteins are contained in lanes 9 and 10,
respectively. (Right panel) The SNAP50 C-terminal zinc finger domain is required for
DNA binding by SNAPC. EMSA were carried out with SNAP190 (1-505) and SNAP43
with 10, 30, or 100 ng of GST-SNAP50 (1-411), GST-SNAP50 (1-300), or GST, as

indicated.

(B) The C-terrninal SNAP50 zinc finger region is not required for interactions with
SNAP43. Full-length and truncated SNAP50 proteins were labeled with [3°S]methionine
and were used in GST pulldown assays with GST-SNAP43 (lane 3) and GST protein
alone (lane 2). 10% of the total input for each protein is shown in lane 1. A schematic
representation of the various SNAP50 proteins is shown at the left with the positions of
the zinc finger domain and putative LxCxE RB interacting region (black box) indicated.

87

 

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88

snRNA genes. While the mechanism for PSE binding by SNAPC is not well understood,
this initial event requires extensive cooperation among SNAP43, SNAP50, and
SNAP190, as indicated by the data in Figure 3-1A. EMSA reactions containing all three
subunits supported robust binding by mini-SNAPC to wild-type (lane 7) but not mutant
PSE probes (lane 8), whereas no detectable DNA binding was observed in reactions that
lacked all three complex members (lanes 1-6), consistent with results previously
described (13). Thus, even though SNAP190 contains a bona fide Myb DNA binding
domain, additional components of the complex are required, perhaps serving to directly
recognize the PSE alongside SNAP190.

Whereas the previous experiment was performed with three factors, we postulate
that the recognition of DNA by SNAPC likely involves direct DNA contacts provided by
SNAP50 in addition to the contributions made by SNAP190. Firstly, SNAP50 was UV
cross-linked to the PSE during DNA binding by endogenous SNAPC (9), indicating that
SNAP50 is in close proximity to DNA during promoter recognition. Secondly, SNAP50
contains a putative zinc finger domain within the C-terminal region, and as zinc finger
domains are typically involved in nucleic acid binding and/or protein-protein interactions,
we hypothesized that this region of SNAP50 is involved in DNA binding by SNAPC. To
test this idea, EMSA were performed using mini-SNAPC containing SNAP50 that lacked
the C-terminal cysteine/histidine-rich region. In support of the hypothesis, DNA binding
by mini-SNAPC was completely ablated in reactions performed with SNAP50 (1-300)
lacking the cysteine/histidine-rich region (lanes 15-17), as compared to comparable
amounts of mini-SNAPC containing wild-type SNAP50 (lanes 12-14). In these reactions,

the assembly of SNAP50 (1-300) into a complex along with SNAP43 and SNAP190 (1-

89

505) was as efficient as that observed for wild-type SNAP50 (data not shown),
suggesting that those protein-protein interactions required for complex formation are not
seriously jeopardized by removal of the cysteine/histidine-rich region. Indeed, strong
pairwise interactions between SNAP50 and SNAP43, its major partner in SNAPC, were
maintained for truncated SNAP50 molecules lacking this region (Figure 3-lB). In GST-
pulldown experiments, GST-SNAP43 interacted well with full-length SNAP50 (1-411),
SNAP50 (1-300), and SNAP50 (1-199), but not with SNAP50 (1-124) nor with the
cysteine/histidine- rich region alone (SNAP50 301-411). These data support the idea that
the central region of SNAP50 participates in complex assembly with SNAP43 while the
C-terminal cysteine/histidine-rich region constitutes a DNA binding domain within
SNAP50.

The requirement for the SNAP50 cysteine/histidine rich region for DNA binding
by SNAPC suggests that this region of SNAP50 constitutes a zinc finger domain. To
determine whether zinc is indeed bound by SNAPC, the ratio of zinc associated with a
recombinant SNAPC containing HA epitope tagged SNAP50 along with SNAP190 (1-
505), SNAP43, and SNAP19 (hereafter referred to as SNAPcy4) was determined by
inductively coupled plasma mass spectrometry (ICP-MS). SNAPcy4 was chosen for this
study because this recombinant complex, assembled by coexpression of each subunit in
E. coli, is fully functional for PSE-specific DNA binding and for snRNA gene
transcription by both RNA polymerases II and III (7), and zinc binding under functional
conditions could be examined. As shown in Table 3-1, approximately 1.2 moles of Zn, as
measured for both Zn66 and Zn68, were found associated with each mole of SNAPcy4,

_ while little detectable Ni was associated with SNAPcy4. Similar data was obtained for

90

Table 3-1

This experiment was performed by Andrej Hanzlowsky (Dr. Jim Geiger’s Lab).

91

Table 3-I

Zinc and Nickel analysis of recombinant SNAPc as determined by Inductively-Coupled Plasma
Mass Spectrometry and Flame Atomic Absorption

Method 1: ICP- MS

 

 

 

 

 

Sample [Zn] ppb [Ni] ppb [protein] mg/mL n(Zn)/n(proteln) n(Nl)/n(protein)
SNAPC” (1) 5‘1250.4 - 2.6 1.2 -
”1268.5 - 2.6 1.1 -
- 17.9 2.6 - 0.02
SNAPcy4 (2) a'1415.5 - 2.9 1.2 -
”1443.6 - 2.9 1.2 -
- 35.5 2.9 - 0.03
a = total Zn calculated from Zn66 MVgNW ~ 159.735 glmole
W 2: Atom'c Mpflon b a total Zn 68160181“ from m WWW ~ 148,432 W
Sample .[Zn] ppb [protein] mglmL n(Zn)/n(protein)
SNAPcyd (1) 1956.8 5.3 0.90
SNAPcyG 1642.2 4.1 0.91

 

92

two separate SNAPcy4 preparations. These results are comparable to those obtained by
atomic absorption indicating a molar ratio (nZn/n SNAPcy4) of 0.9 for the four-subunit
SNAPcy4 complex, and for the three-subunit SNAPc73 complex lacking SNAP19. As no
other SNAPC subunits besides SNAP50 contain suitable arrangements of cysteines and/or
histidines for Zn coordination, the parsimonious explanation of these data is that

SNAPcy4 contains a single SNAP50 subunit that binds a single Zn atom.

An evolutionarily conserved zinc-finger domain in human SNAP50 functions for
DNA binding

The amino acid sequence for the human SNAP50 zinc finger domain is shown in
Figure 3-2A, which reveals an unorthodox arrangement of cysteines and histidines within
the C-terrninal 110 amino acids. Typically, zinc finger domains that ftmction for DNA
binding are constituted by multiple repeats of related zinc finger motifs (21). However,
the putative DNA binding domain of human SNAP50 contains nine cysteines and six
histidines that can be grouped into region 1 loosely resembling a TFIIIA-like C2H2 zinc
finger and region 2 resembling a glucocorticoid-like C2C2 zinc finger. While both types
of motifs in other proteins are involved in direct DNA contacts, the discordant
arrangement of zinc finger motifs suggests that DNA binding by SNAP50 is distinct from
mechanisms employed by other zinc finger proteins. In addition, as SNAPcy4 contains
only a single Zn atom, it is likely that only one of the potential Zn fingers actually
coordinates Zn. Interestingly, the results of a BLAST'homology search show that of the
fifteen potential zinc coordination sites in this region, eight are highly conserved among

putative SNAP50 homologues from mammals, insects (Drosophila, Anopheles), fish

93

(Dania), worms (C. elegans), plants (Arabadopsis), slime mold (Dictyostelium), and
parasites from the T rypanosoma, Entamoeba, Plasmodium, and Leishmania genera. This
comparison also shows that the critical zinc coordination sites within region 2 that
constitute the glucocorticoid-like finger motif are well conserved whereas the TFIIIA-like '
C2H2 zinc finger motif within region 1 is not. Nonetheless, two histidines and one
cysteine residue within region 1 are conserved raising the possibility that a function
performed by this region is also maintained across divergent species. The consensus
motif derived from this comparison (Lx4GX6Hx3CxHx2M3YPx11-12Cx2Cx13Px34
Cx2CFx3Hx14G) is distinct from any other family of zinc finger motifs (20), suggesting
that this domain represents a novel zinc finger fold, and is hereafter referred to as the
“SNAP finger” domain.

To examine the function of the cysteine and histidine residues within the C-
terrninal SNAP finger domain of SNAP50, each of these residues was changed to alanine
for functional testing in the context of a partial SNAPC that contains SNAP190 (1-505),
SNAP43, SNAP19, and the various derivatives of full-length HA-SNAPSO. In addition,
an arginine at position 385 was also substituted with alanine. First, to determine whether
the targeted amino acids are critical for complex assembly a two-step affinity purification
of wild type and mutant complexes were performed. Complexes were assembled by co-
expressing four SNAPC subunits in E. coli followed by affinity purification of the
complex via the GST tag contained on the amino terminus of SNAP190 (1-505).
Complexes were liberated from the glutathione agarose beads by thrombin digestion,
which recognizes its cognate site between the GST tag and the SNAP190 coding region.

The soluble complexes were further purified by anti-SNAP43 immunoprecipitation, and

94

Figure 3-2. Single amino acid changes in the SNAP50 subunit abolish DNA binding
by SNAPc.

(A) Sequence alignment of human SNAP50 C-terminal amino acids (301-411) with
corresponding regions from SNAP50 homologues of other species. Putative zinc fingers
similar to TFIIIA (HXsHXloHX3C) and steroid receptors (szszszzC) are indicated as
region 1 and region 2, respectively. The sequence of highly conserved amino acids
derived from this alignment corresponds to Lx4GX6Hx3CxmezgYPx][-lszszum-
4Cx2CFx3Hx14G. This alignment was performed using the Clustal W program. Homo
sapiens (NP003075), Cannis familiaris (XP853813), Bos taurus (AAX08912), Mus
musculus (NP084225), Rattus norvegicus (NP001013230), Drosophila melanogaster
(N P724647), Drosophila pseudoobscura (EAL25490), T rypanosoma brucei (XP827295),
Arabidopsis thaliana (AAO30067), Caenorhabditis elegans-l (NP500819),
Caenorhabditis elegans-Z (NP497807), Plasmodium falciparum (NP702469), Danio
rerio (XP694501), Leishmania major (XP843572), Anopheles gambiae (XP310411),
Dictyostelium discoideum (XP644064), Entamoeba histolytica (XP653151).

(B) Mutations in the SNAP50 zinc finger domain do not disrupt SNAPC assembly.
Approximately 20 ng of each of the SNAPcy4 complex containing substitution mutations
in HASNAPSO were affinity purified first using glutathione agarose to pull down GST-
SNAP190 (1-505) followed by immunoprecipitation with a-SNAP43 antibodies.
Associated wild type or mutant HA-SNAPSO was detected by anti-HA Western analysis
(lanes 6-22). A titration of wild type SNAPcy4 using 8, 4 and 2% of the input material is
shown in lanes 1-3, respectively. Lanes 4 and 5 contain wild type SNAPCy4 recovered
with the protein-G agarose beads alone or with pre-immune serum, respectively. The
bottom panel represents 4% of the input material directly analyzed by anti-HA Western
analysis.

(C) Mutations in the SNAP50 zinc finger domain disrupt DNA binding by SNAPC.
Increasing amounts (3 and 10 ng) of SNAPcy4 with wild type (lanes 2 and 3) or mutant
HA-SNAPSO (lanes 4-35) containing the indicated substitution mutations was tested in an
EMSA for binding to a radiolabeled DNA probe containing a high affinity PSE and
TATA box. Lane 1 shows the probe alone with no added proteins.

95

 

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97

the extent of SNAP50 association, as a measure of complex integrity, was determined by
Western blot analysis against the HA-tag contained at the N-terminus of SNAP50. In this
approach, any SNAP50 detected in this analysis had to be in a complex with both
SNAP43 and SNAP190 (1-505). As shown in Figure 3-2B, the amounts of SNAP50
recovered after the two-step affinity purification were similar for the wild type and
mutant complexes, suggesting that SNAP50 assembly into SNAPC is not markedly
dependent upon any individual cysteine or histidine in this domain.

Next, partially purified wild type and mutant complexes were then tested for PSE
binding function in EMSA to determine whether the cysteine and histidine residues
within the SNAP finger domain are important for DNA binding by SNAPC. The results
revealed three categories of DNA binding effects, as shown in Figure 3-2C. The first
category of mutants exhibited either wild-type DNA binding (H330A, H347A, C377A,
R385) or modest defects (C302A, C312A, H331A, C334A) as compared to the complex
containing wild-type SNAP50. The second category of mutant complexes exhibited
severe defects in DNA binding (H313A, C317A, H319A, H388A), while complexes in
the third category were completely impaired for DNA binding ability (C354A, C357A,
C3 80A, C3 83A). Thus, the cysteine and histidine residues within the latter two categories

are both evolutionarily conserved and critical for DNA binding by SNAPC.

The SNAP50 zinc finger domain is required for RNA polymerase II and III

transcription.
We next tested whether mutations that affected PSE-recognition by SNAPC were

also critical for human U1 snRNA gene transcription by RNA polymerase II and U6

98

snRNA gene transcription by RNA polymerase III. For these experiments, we predicted
that those complexes that exhibited no DNA binding ability would also be defective in
transcription, confirming that DNA binding by SNAPC is an essential aspect of snRNA
gene transcription. Secondly, any previously unappreciated function for SNAP50 in
communication with other components of the general transcription machinery would be
revealed as a defect in transcription for those complexes with wild type DNA binding
activity. As the general transcription machinery for RNA polymerase II and III
transcription are different, it was possible that certain mutations would result in
transcriptional defects in only one polymerase system. However, as shown in Figure 3-
3A, all complexes that were capable of DNA binding also functioned well for RNA
polymerase II and III transcription with the extent of activity essentially parallel for both
systems. Thus, those residues that are not evolutionarily conserved are also not critical
for human snRNA gene transcription by either polymerase. In contrast, those mutant
complexes that were crippled for DNA binding activity (C354A, C357A, C380A,
C3 83A) did not support either RNA polymerase II or III transcription. We consider it
likely that these critical cysteine residues within region 2 of the SNAP finger play a
structural role for SNAP50 function and are likely important for Zn binding. Some
unexpected differences between RNA polymerase II and III transcription were apparent
for those mutant complexes that exhibited reduced, but not ablated, DNA binding. Some
complexes (H313A, C317A, H319A) were moderately active for RNA polymerase III
transcription but did not support RNA polymerase II transcription. Most noticeably, the
complex containing SNAP50 H388A was fully functional for RNA polymerase III

transcription. Thus, the DNA binding defects caused by altering these conserved residues,

99

Figure 3-3. The SNAP50 zinc finger domain is required for human snRNA gene
transcription by RNA polymerases II and III.

(A) HeLa cell nuclear extract was depleted with anti-SNAP43 antibodies to
immunodeplete endogenous SNAPC. In vitro U1 and U6 transcription was then tested in
the absence (lanes 1) or presence of purified SNAPcy4 (5 ng) containing wild type
SNAP50 (lane 3) or mutant SNAP50 with the indicated alanine substitutions (lanes 4-19).
Addition of GST alone did not reconstitute either U1 or U6 transcription as shown in lane
2.

(B) Summary of mutations in the SNAP50 zinc finger domain. Alanine substitution of
cysteines and histidines within the SNAP50 zinc finger domain revealed three classes of
phenotypes, as indicated.

100

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1 Wild type DNA binding, 0302-A, ca12-A, H330-A, H331 -A,
U1 and U6 transcription 0334-A, H347-A. C377-A. Haas-A
II Diminished for DNA binding 0354-A. 635741. 0330A, 6383A
and transcription
11: Diminished for DNA binding 11313-11. 0317-A, H319-A. Haas-A

 

and U1 transcription but active
tor U6 transcription

 

 

 

101

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especially for the H388A substitution are suppressed during RNA polymerase III

transcription.

Suppression of DNA binding defects by cooperation between TFIIIB and SNAPC.
Based on the previously described observations, we hypothesized that the
evolutionarily conserved residues in region 1 (H313, C317, and H319), as well as H388
in region 2 facilitate DNA contacts by SNAPc, either directly or indirectly, and during
RNA polymerase III transcription their activity was restored because TFIIIB stabilized
mutant SNAPC binding to the PSE. To determine whether TFIIIB was capable of
restoring DNA binding activity to SNAPC, it was first necessary to establish an assay that
measured DNA binding by TFIIIB and SNAPC. Human U6 snRNA gene transcription
relies on a variant of TFIIIB called Brf2-TFIIIB, which is composed of TBP, Brf2, and
del (30). In the following experiments, a truncated form of de1 (1-470) was used
because it supports wild-type U6 transcription (ref. (16), data not shown) and can be
expressed and purified more easily than the full-length del (1- 1338). As shown in
Figure 3-4A (left panel), weak binding of Brf2-TFIIIB to U6 promoter probes is observed
when all three components are included in the DNA binding reaction, whereas no binding
was observed in any reactions that did not contain TBP, consistent with the observation
that Brf2-TFIIIB is TATA-box dependent (data not shown). Weak, but nonetheless
cooperative, DNA binding is also observed for TBP plus Brf2 at levels greater than that
observed for either factor alone, as previously described (2, 13, 28). In contrast,
SNAPcy4 bound well and effectively recruited TBP to DNA (Figure 3-4A right panel).

Although Brf2 stimulated DNA binding by TBP, much shorter exposure times were used

102

Figure 3-4. SNAPC recruits Brfl-TFIIIB to U6 promoter DNA in vitro.

(A) SNAPC stimulates DNA binding by Brf2-TFIIIB. Electrophoretic mobility shift
assays were performed using a U6 probe containing a wild-type mouse U6 PSE and a
wild-type TATA-box. DNA binding was carried out in the absence (lanes 1-8) or
presence (lanes 9-12) of wild type SNAPcy4. Reactions containing individual TBP, Brf2,
and de1 (1-470) subunits are shown in lanes 2-4. Reactions containing pair wise
combinations of TBP with Brf2, TBP with de1 (1-470), and Brf2 with de1 (1-470) are
shown in lanes 5, 6, and 8. DNA binding by the complete Brf2-TFIIIB complex in the
absence of SNAPC is shown in lane 7. Additional reactions were performed with SNAPC
alone (lane 9) or in combination with Ber-TFIIIB subunits (lanes 10-12), as indicated.
Lane 1 shows migration of the probe alone. The relative positions of the various
SNAPcy4/Brf2-TFIIIB complexes are shown on the right.

(B) Coordinated DNA binding by SNAPC and TBP facilitates higher order complex
assembly with Brf2 and de1. DNA binding reactions were performed with the indicated
combinations of SNAPcy4 and Brf2-TFIIIB subunits. These results suggest that
preinitiation complex assembly follows the order SNAPC>TBP>Brf2>deL

103

 

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Probe

12345 6789101112131415

104

to visualize DNA binding for reactions containing SNAPC (right panel), and for
exposures that exhibited prominent DNA complexes containing SNAPc, the DNA
binding by Brf2-TFIIIB alone was essentially undetectable (not shown, and Figure 3-4B).
Thus, at least under these conditions, SNAPC plays a dominant role for TBP recruitment
to U6 promoter DNA. Interestingly, serial addition of Brf2 then de1 resulted in
complexes that migrated incrementally more slowly, consistent with the idea that
increasingly larger complexes are being assembled on the DNA. The amount of the
SNAPcy4-DNA complex was not further affected by Brf2 and de1. In contrast, the
amount of the SNAPcy4/TBP-DNA complex was diminished by Brf2 addition, and the
SNAPcy4/TBP/Brf2-DNA complex was diminished by de1 addition. These last
observations suggest a substrate-product relationship during complex assembly, and are
consistent with the idea that Brf2 and de1 both exhibit a binding preference for the
higher order complexes containing TBP than that complex containing only SNAPC.

To further refine the pathway for assembly of SNAPC and Brf2-TFIIIB on U6
promoter DNA, combinations of each factor were tested for DNA binding ability in
EMSA. As shown in Figure 3-4B, in the absence of SNAPC, none of the Brf2-TFIIIB
components bound to DNA when tested singly or in combination under conditions that
support robust DNA binding by SNAPcy4. Interestingly, SNAPcy4 was capable of
recruiting TBP, but not Brf2 or de1, when tested in pair wise combination, whereas
Brf2 could be incorporated into the complex only in reactions containing SNAPcy4 and
TBP. This observation further supports the premise that Brf2 preferentially recognizes the
SNAPc/TBP promoter-bound complex. Similarly, deI was not recruited to the

' SNAPc/TBP complex, but again it required the presence of Brf2 for DNA association.

105

Based on these results, we propose a sequential assembly pathway with the initial
promoter recognition performed by SNAPC and TBP followed by Brf2 and del. The
eventual recruitment of del is predicted to enable RNA polymerase III recruitment
under transcription conditions.

To test the hypothesis that Brf2-TFIIIB can suppress DNA binding defects in
SNAPC, recombinant SNAPC containing wild type or mutant SNAP50 were tested for
DNA binding in the absence or presence of each component of Brf2-TFIIIB (Figure 3-5).
For this assay, examples from each category of mutant SNAP50 were tested including
H388A and H313A that were fully or partially active in U6 transcription, respectively, as
well as C383A that was devoid of measurable activity for either DNA binding or
transcription. In these assays, the same U6 promoter sequence was used for DNA binding
as that contained on the reporter plasmids that were previously used for in vitro U6
transcription. As expected, wild-type recombinant SNAPc bound DNA well and
supported robust recruitment of Brf2-TFIIIB. In contrast, mutant SNAPc containing
SNAP50 (H388A), SNAP50 (C383A), or SNAP50 (H313A) were each inactive when
tested alone for DNA binding. Thus, both wild-type and mutant SNAPc bound with
reduced affinity to the U6 promoter sequences tested in this assay relative to those
experiments done with the artificial high affinity PSE promoter probes as shown in
Figures 3-2 and 3-4. Importantly, DNA binding by the SNAP50 (H388A)-containing
complex was restored by TBP, although not to levels seen with the wild type SNAP50
complex, and higher order complex formation with Brf2 and de1 occurred at nearly
wild type levels. Thus, Brf2-TFIIIB was capable of restoring DNA binding activity to

mutant SNAPC. This result stands in contrast to the SNAP50 (C3 83A)-containing

106

Figure 3-5. TFIIIB suppresses DNA binding defects in SNAPC.

DNA binding reactions were carried out as previously described for the artificial AC
probe instead using the natural U6 promoter containing DSE, PSE, and TATA-box
elements. Reactions were performed with 8 ng of SNAPcy4 containing either wild type
SNAP50 (lanes 2-8) or mutant SNAP50 with the substitutions H388A (lanes 9-12),
C383A (lanes 13-16), and H313A (lanes 17-20). Reactions additionally containing TBP,
TBP and Brf2, or TBP, Brf2 and de1 (1-470) were performed as indicated. DNA
binding by mutant SNAPcy4 harboring the H388A mutation was restored by TBP alone
whereas TBP plus Brf2 were required to restore DNA binding by SNAPcy4 harboring the
SNAP50 (H313A) mutation. No DNA binding under any condition was observed for
SNAPcy4 containing the SNAP50 (C3 83A) substitution.

107

Wt H388-A CS83-A H313-A

 

‘______—___fi
mSNAPcy4 - +++++++++++++++++++
de1(1-470)- ---+— ++---+—--+---+
Brl‘2 ---+++-+--++--++--++
TBP --+++----+++-+++-+++
mSNAPc/TBP/BrlZ/deix
mS NAPclTBP/Brfz _
mSNAPchB
mSNAP

Free
Probe

 

1 2 3 4 5 6 7 8 91011121314151617181920

108

complex that was unable to bind DNA under any conditions. Interestingly, TBP did not
restore DNA binding by the SNAP50 (H313A)-containing complex but Brf2 plus TBP
did, suggesting that the H313A substitution presents a more dramatic defect to DNA
binding by SNAPC. Nonetheless, deI could be recruited at reduced levels by this
SNAP(:(H3 13A)/T BP/Brf2 complex, consistent with the markedly reduced transcription

supported by this complex for in vitro U6 transcription.

The C-terminal SNAP50 zinc finger binds zinc.

As the previous analyses revealed that certain amino acids within the SNAP
finger domain are differentially required for DNA binding and snRNA gene transcription,
we postulated that those amino acids that are absolutely critical for both functions are
also important for zinc binding. To test this hypothesis, Zn binding studies of wild type
and mutant SNAP50 were undertaken. While our initial analysis of zinc content was
performed for the four-member complex (SNAPcy4), suitable amounts of mutant
complexes for zinc analysis were not obtained in this context. Nor were we able to obtain
sufficient amounts of full-length SNAP50 (1- 411) or truncated SNAP50 (301-411) for
these studies when expressed individually in E. coli. However, suitable amounts of
truncated SNAP50 (315-411) were obtained when expressed as fusion protein with a His-
SUMO N-terminal tag, and zinc binding studies of this protein were therefore pursued.
As shown in Figure 3-6, analysis of SNAP50 (315-411) by flame atomic absorption
showed that this region of SNAP50 bound substantial amounts of zinc (~0.7 mole
zinc/mole protein). This level of zinc binding by the isolated SNAP50 zinc finger domain

' is comparable to that seen for full-length SNAP50 (1 -41 1) in the context of the four-

109

Figure 3-6. The SNAP50 zinc finger domain binds zinc.
Wild type and mutant SNAP50 (315-411) containing the indicated mutations were tested

for Zn content by flame atomic absorption, and the calculated molar ratios of zinc to
protein are shown. Error bars indicate the relative standard deviation.

110

Ratio Zn/Protein

1.00

 

0.80

0.70
0.60 '
0.50-
0.40

0.30

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0.10...-

 

 

 

 

 

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Protein ID. 0 o

111

protein complex SNAPcy4 (~0.9 mole zinc/mole protein), indicating that the SNAP
finger domain is likely responsible for zinc binding by SNAPC. Zinc binding by SNAP50
(315- 411) was markedly reduced by individual alanine substitution at positions C354,
C357, C380, and C383, whereas alanine substitutions of C377 and H388 did not
substantially affect zinc binding. Overall, these data indicate that those cysteines within
the C354x2C357x22C3 80x2C3 83 motif are important for zinc binding by SNAPC, whereas
the adjacent C377 and H388 residues are not. Moreover, SNAP50 (315-411) harboring
either the C354A:C380A or C354A:C383A double alanine substitutions was further
incapacitated as compared to SNAP50 C354A or C383A, but zinc levels were
comparable to the reduced levels observed for SNAP50 harboring the single C380A

substitution. Thus, C380 plays a more critical role in zinc binding than C354 and C383.

Discussion

DNA binding by SNAPc is a cooperative event wherein SNAP190, SNAP50, and
SNAP43 are all required for promoter recognition (ref. (13), and Figure 3-1). Our data
demonstrate that an unorthodox zinc finger domain in SNAP50 plays a critical role in this
process. A comparison of this region with SNAP50 homologues from other species
revealed that the arrangement of many cysteine and histidine residues within the SNAP50
C-terrninal region is remarkably well conserved (Figure 3-2A), and a mutational analysis
of all histidine and cysteine amino acids throughout this region showed that these highly
conserved residues are critical for DNA binding by SNAPC.

The high degree of sequence conservation within the SNAP finger domain

' throughout evolution suggests that SNAP50 function is conserved in other species.

112

Indeed, Drosophila PBPSO (Proximal element binding protein 50 kDa), a homologue of
human SNAP50, makes direct DNA contacts within the U6 and U1 promoters although
the promoter sequences recognized by human SNAPC and Drosophila PBP are different
(34). Besides SNAP50, Drosophila also maintains homologues of SNAP43 and
SNAP190, but not of SNAP45 or SNAP19. The identification of SNAPSO- and SNAP43-
related proteins in Trypanosoma and Leishmania suggests an ancient fimction for SNAPC
in non-translated RNA production, in this case RNA polymerase II transcription of
spliced leader (SL) RNA. Trypanosoma SNAPc also contains a Myb domain-containing
protein (3, 29) reminiscent of human SNAP190, which contains an unusual Myb DNA
binding domain (35). The conservation of these three subunits throughout evolution
remarkably parallels the experimental definition of a minimal human SNAPC composed
of SNAP190 (1-505), SNAP50, and SNAP43 that retains full activity in RNA polymerase
II and III transcription (7, 24).

Interestingly, the overall spacing of potential zinc coordination sites within
SNAP50 does not resemble other known zinc finger motifs, although the arrangement for
a subset of these cysteine residues resembles that of hormone receptor DNA binding
domains (20), consistent with a role in DNA binding for this region in SNAP50.
Nonetheless, the unusual arrangement of zinc coordination sites combined with
secondary structure predictions suggest that human SNAP50 is the founding member for
a novel class of zinc finger domains that we refer to as the SNAP finger domain.

To date, an experimental structure of SNAP50 has not been determined.
Therefore, computational methods were employed to predict model structures to assist

our understanding of the mechanism for DNA binding by SNAP50. Comparative

113

modeling, which is often successful in other cases, was not possible because no sequence
homologs of SNAP50 with known structures are available. However, as the SNAP finger
domain is sufficiently short (~100 residues) ab initio modeling based only on the amino
acid sequence and the predicted secondary structure was performed. Although ab initio
structure prediction methods in general cannot accurately predict protein structures at the
level of experimental structures, it is often possible to obtain approximate models of
relatively small domains (<100 residues) with an overall root mean square deviation of S-
10 A from the correct, native structure.

The first round of ab initio prediction with the Robetta server resulted in ten
models for the SNAP finger domain. The resulting models were substantially different;
however, the conserved cysteines 354, 357, 380, and 383 were in sufficiently close
proximity to serve as zinc coordination sites in 5 out of the 10 models. Such a result is
non-trivial given that the pair C354/C357 is separated by 22 residues from C380/C383,
which lends support to the hypothesis that these four cysteines coordinate zinc in a novel
zinc finger fold topology. In order to examine a wider range of possible structures and
arrive at a model for the entire SNAP finger domain, additional ab initio sampling was
carried out under the constraint that the two pairs of residues, C354/C380 and
C357/C3 83, are each in close proximity. The best-scoring model consists mainly of 8-
sheets and a small a-helical segment according to the predicted secondary structure
(Figure 3-7A), and the predicted structure is shown in Figure 3-7B. Submission of the
model shown to the DALI server (14, 15) resulted in two known structures with remotely
similar topologyzdomain II from calpain, a cysteine protease (PDB ID: IKXR) and a

beta-propeller domain of sialidase (PDB ID: lEUT). However, the structural similarity is

114

Figure 3-7. Model for the SN AP50 zinc finger domain.
(A) Predicted secondary structure of SNAP50 (301-411) from SABLE (39).

(B) Stereo image of the predicted model structure for the C-terminal domain of SNAP50
(residues 305-411). Conserved cysteines C354, C357, C3 80, C383 (yellow) are shown in
a zinc coordination geometry (zinc is shown in orange). The conserved residues H313,
C317, H319, and H388 are shown in green, and other conserved residues are shown in
blue.

(C) Electrostatic potential projected onto the molecular surface of the SNAP50 model in
the same orientation as in figure 78 (left) and rotated to show the back (right).

Modeling of SNAP50 zinc finger domain was done by Dr. Michael Feig.

115

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117

sufficiently low to suggest that the predicted SNAP finger fold has a novel architecture.
The current model for the SNAP finger domain highlights the zinc coordination by C354,
C3 57, C380, and C383, and is independently supported by experimental data indicating
that these four cysteine residues are involved in zinc binding. The proposed model is
vaguely reminiscent of the GATA-l zinc finger motif wherein an or-helix plays a critical
role in DNA recognition (33), but is substantially different because of a much longer
inserted sequence between the two pairs of cysteines. In the SNAP50 model, other highly
conserved residues, G315, P341, and P376, are located at critical turn regions for
stabilization of the proposed structure.

Interestingly, this SNAP50 model also brings the conserved residues H313, C317,
C319 in proximity with H388, which could provide an alternate metal binding site. We
note, though, that the binding of an additional metal atom to SNAPC is not supported by
the experimental data, and the exact function of these residues remains unclear. These
residues may stabilize structures involved in DNA binding by SNAP50, or instead
participate directly in DNA contacts. Of note, these residues are located adjacently to the
a-helix, which in other zinc finger proteins is frequently used to make specific base
contacts within the major groove during DNA binding (20). However, only the C-
terminus of the corresponding a-helix within the SNAP finger domain model is fully
exposed, which would likely limit major groove contacts. Thus, stable DNA binding
through this region may require additional contacts by flanking residues. Interestingly,
the electrostatic surface potential for the SNAP finger domain (Figure 3-7C) shows that
the presented model clearly distinguishes between positively and negatively charged

faces. A large well-defined positively charged surface patch surrounding the a-helix

118

suggests the potential for DNA binding, although a specific mode of interaction between
SNAP50 and DNA cannot yet be predicted because of the uncertainty associated with
this working model. Nonetheless, the current study provides insight into the DNA
binding properties of SNAPC, and identifies the evolutionarily conserved zinc finger
domain of SNAP50 as critical for promoter recognition and human snRNA gene

transcription.

Acknowledgements

We thank Craig Hinkley, Heather Hirsch, Liping Gu, Dorothy Tappenden, and Justin
Bammer for technical assistance, and Tharakeswari Selvakumar for critical reading of the
manuscript. We thank David Szymanski for assistance with the ICP-MS. This research

was supported by the NIH Grants R01-GM59805 and R01-GM063894 to R.W.H. and

J .H.G., respectively.

119

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123

CHAPTER 4

THE HEXIMl REPRESSOR OF P-TEFB STIMULATES
TRANSCRIPTION OF ITS 7SK snRNA COREPRESSOR PARTNER
BY RNA POLYMERASE III

Abstract

The HEXIM1/7SK snRNA ribonucleoprotein complex inhibits RNA polymerase II
transcription of HIV-1 and cellular genes through interactions with the elongation factor
P-TEFb. The 7SK promoter is a type 3 RNA polymerase III promoter, with a core
consisting of a TATA box recognized by TBP and a proximal sequence element (PSE)
recognized by SNAPC. Here we show that HEXIMI co-purifies and is associated with
SNAPC. HEXIMl occupies an endogenous 7SK snRNA gene promoter and stimulates
7SK snRNA gene transcription in vivo. Consistent with a role as an activator of RNA
polymerase III transcription, HEXIMl stimulates the binding of SNAPc and TBP to
DNA containing their cognate promoter elements. The cyclinTl and cdk9 components of
P-TEFb are dispensable for 7SK transcription by RNA polymerase 111, but are required
for U1 snRNA gene transcription by RNA polymerase 11. These findings reveal a novel
role for HEXIMI as an activator of expression of its 7SK snRNA co-repressor partner
and implicate this regulatory circuit as a potential attenuator of global RNA polymerase

II transcription.

124

Introduction
Small nuclear (sn) RNA genes are among the most actively transcribed genes in human
cells with initiation events occurring approximately every 2-4 seconds during periods of
active grth (6). While initially believed to be constitutively transcribed and relatively
immune to regulatory influence, the transcription of human snRNA genes is regulated
both during the cell cycle (19) and in response to DNA damage (10). Many factors that
have been extensively investigated because of their regulatory roles during human
neoplasm initiation and progression also regulate human snRNA gene transcription.
These include the p53 and Retinoblastoma tumor suppressor proteins, as well as the
protein kinase CK2 and c-Myc oncoproteins (3, 4, 9, 10, 17, 20). That these critical
tumor-related factors control human snRN A gene transcription portends a global role for
snRNAs in controlling cellular growth.

The human snRN A gene products play central roles in global gene expression,
most commonly through their function in RNA processing (26). Examples include the U-
rich snRNAs U1, U2, U4, U5, and U6 that are required for messenger RNA splicing. In
addition to their classical roles in RNA metabolism, snRNA complexes can also stimulate
RNA polymerase II transcription possibly by coupling transcription initiation or
elongation to downstream splicing events (8). More recently, U1 snRNA was shown to
associate with TFIIH and to stimulate RNA polymerase II transcription (24), perhaps by
stimulating TFIIH activity during transcriptional initiation. The cyclin H/cdk7
components of TFIIH phosphorylate the carboxy terminal domain (CTD) of the RNA
polymerase 11 largest subunit to stimulate early initiation events (1, 7), but whether U1

snRN A modulates that process is not known. Additional CTD phosphorylation by the

125

cyclin T1/cdk9 kinase component of P-TEFb stimulates RNA polymerase II elongation
(29). The P-TEFb transcriptional elongation factor, originally characterized as a RNA
polymerase II elongation factor in Drosophila, was subsequently linked to HIV-1
transcription and replication initiated from the HIV long terminal repeat (LTR) (28, 46).
Interestingly, P-TEF b kinase activity and consequently RNA polymerase II elongation
from the HIV-1 LTR are inhibited by a ribonucleoprotein complex composed of
HEXIMI and 7SK snRNA (34, 42). Thus, U1 and 7SK snRNAs associate with the
cyclin/cdk complexes, TFIIH and P-TEFb, respectively, but with opposite effects on gene
transcription. These observations suggest that the relative levels of U1 and 7SK snRNA
in the cell may control cellular transcription potential and furthermore, the regulated
transcription of snRN A genes may be important for maintaining this balance.

Human snRN A genes can be grouped according to their promoter structures that
dictate polymerase specificity for transcription. Some genes such as U1 and U2 are
transcribed by RNA polymerase II, and others such as U6 and 7SK are transcribed by
RNA polymerase III (11, 15). The use of different polymerases and transcription
machinery may allow the cell to discretely control the transcription of distinct classes of
snRNA genes. Regardless of polymerase choice, human snRNA genes contain a distal
sequence element (DSE) that functions as an enhancer of transcription and contains
binding sites for the Oct-1 (32) and STAF (33, 36) activator proteins. Each gene also
contains a proximal sequence element (PSE) that is located within the core promoter
region that is recognized by the general transcription factor called the snRNA activating
protein complex (SNAPC) (35), or the PSE-binding transcription factor (PTF) (32). A

TATA box element located at a fixed distance from the PSE determines the initial

126

pathway of preinitiation complex assembly for transcription by RNA polymerase III (25).
In this context, the TATA-box is recognized by the TATA-box binding protein (TBP) as
part of the snRNA-specific version of the TFIIIB complex (38, 41). The assembly of
SNAPC along with TFIIIB pemrits RNA polymerase III recruitment, whereas the absence
of the TATA box by default directs PSE- and SNAPc-dependent preinitiation complex
assembly with TFIIA, TFIIB, TFIIE, and TFIIF for transcription by RNA polymerase II
(23).

To identify factors that associate with SNAPC and are potentially involved in
human snRN A gene transcription, a biochemical fractionation of SNAPc from HeLa cell
extracts was performed. We report that endogenous HEXIMl associates with SNAPC
during chromatographic purification and furthermore, HEXIMl associates with human
snRNA genes in vivo as detected by chromatin immunoprecipitation (ChIP). In vitro,
recombinant HEXIMl stimulates promoter recognition by SNAPC and by TBP to their
cognate cis elements within a 7SK-like probe, and in vivo HEXIMl activates 7SK
snRNA, thus revealing a novel positive function for HEXIMl in regulating RNA
polymerase III transcription. Other components of P-TEFb including cyclin T1 and cdk9
are dispensable for U6 and 7SK snRNA gene transcription by RNA polymerase III in
vitro, but are required for U1 transcription by RNA polymerase 11, suggesting that the
function of HEXIMl for RNA polymerase III transcription is separate from its role in

regulating P-TEFb kinase activity.

127

Methods and Materials

UV cross-linking.

The conditions for UV cross linking are essentially as described (35), except that
reactions were performed with highly purified SNAPc fractions. The purification of

SNAPC was described previously (14).

Electrophoretic mobility shift assays.

EMSA were performed essentially as described (16) using recombinant HEXIMI
expressed in E. coli as a N-terrninal GST-fusion protein (GST-HEXIMI). After protein
expression, GST-HEXIMI was affinity-purified by binding to glutathione agarose beads
followed by thrombin digestion to release the untagged full-length HEXIMI protein. As
indicated, reactions also included recombinant full-length human TBP or mini-SNAPC
containing SNAP190 (1-505), SNAP43, and SNAP50. The amounts of each protein are
indicated in the figure legends. The radiolabeled DNA probes used contained a high
affinity wild-type mouse U6 PSE and human U6 TATA box. Additional reactions were
performed with probes that contain debilitating mutations in each element. Protein-DNA
complexes were separated on a 5% polyacrylamide gel in 0.5X Tris-borate-EDTA (TBE)
running buffer at 150 V. The HEXIMl-TBP complexes were resolved in running buffer

containing Tris-glycine-EDTA-Mg2+ (TGEM).
Chromatin immunoprecipitation assay.
Normal human mammary epithelial cells (184B5) were maintained inDulbecco’s

minimum essential media (Gibco) containing 5% fetal bovine serum (Gibco), and

128

penicillin-streptomycin at 37°C with 5% C02. ChIP assays were performed as described
previously (18) using 184B5 cells that were grown to ~75% density. Cells were treated
with 1% formaldehyde for 30 min at room temperature. After cell lysis, the DNA was
fragmented into 500-800 bp fragments by sonication. Immunoprecipitation reactions
were performed overnight at 4°C using chromatin from approximately 107 cells per
reaction and approximately lug of each antibody. The anti-HEXIMI antibody (C8162)
was generated in rabbits using a peptide (CSH419) corresponding to the C-terminal 18
amino acids of HEXIMI. The anti-SNAP43 (C848, (14)) and anti-TBP (SL2, (27))
antibodies were described previously. The anti-RNA polymerase III antibody (MIl'70)
was raised in rabbits against the C-terminal 18 amino acids of hRPClSS as previously
described (40). The RNA polymerase 11 antibody (8WG16) was purchased from Covance
Research Products. The anti-cdk9 (SC-484) and anti-cych (SC-8127) antibodies were
purchased from Santa Cruz Biotechnology. Immunoprecipitated DNA was analyzed by
PCR using primers specific to the genes indicated. The primers used for amplification of

each gene are as follows:

U6 forward: 5’-AAGACGCGCAGGCAAAACG-3’

U6 reverse: 5’-CGGTGTTTCGTCCTTTC-3’

7SK forward: 5’-T'ITI‘GGGAATAAATGATATTTG-3 ’
7SK reverse: 5’- GAGGTACCCAGGCGGCGCACAAG-3’
U1 forward: 5’- CACGAAGGAGTI‘CCCGTG-3’

U1 reverse: 5’-CCCTGCCAGGTAAGTATG-3’

U1 upstream forward: S’-GAACTTACTGGGATCTGG-3’

129

U1 upstream reverse: 5’-GAGACAACTGAGCCACTTG-3’

GAPDH forward: 5’-AGGTCATCCCTGAGCTGAAC-3’

GAPDH reverse: 5’- GCAATGCCAGCCCCAGCGTC-3’

PCR products were separated by 1.5 % agarose gel electrophoresis in 0.5X TBE buffer,

stained with ethidium bromide, and visualized with Kodak imaging software.

In vitro transcription.

Antibody irnmunodepletion of HeLa cell nuclear extracts were performed as described
(13) and the depleted extracts were used for in vitro transcription assays for 1hr at 30°C.
In vitro transcription of human U6 snRNA, U1 snRNA, AdML, and AdVAI genes were
performed as described (25, 35). 7SK transcription was analyzed by a riboprobe
protection assay using the plasmid pBS-7 SK, containing the 7SK promoter from —245 to
+1 fused to an inverted B-globin sequence, and conditions identical to those used for U6
in vitro transcription. Transcripts were separated by denaturing PAGE and visualized by

autoradiography.

Transient transfection assay.

For the HEXIMl knockdown experiment, control siRNA and the HEXIMl-specific
annealed siRNA (Si-RNA ID# 17952) were purchased from Ambion Biotechnology (5’-
GGAUCCGAGCCGAGAUGUU-3’). HeLa cells were grown to 50-60% confluence in a
six-well plate and transiently transfected with 0.2 nmol of the siRNA using
Lipofectarnine 2000 (Invitrogen). After 8 h, the medium containing the transfection

reagent was supplemented with Dulbecco’s modified Eagle’s medium with 5% fetal

130

bovine serum and antibiotics. Cells were also co-transfected with 300 ng of the pBS-7SK
reporter plasmid containing an inverted B-globin sequence driven by the 7SK snRNA
core promoter (-245 to +1). The pBS-Ul reporter plasmid was constructed by replacing
the 7SK promoter region with that of U1. The pU6/I-Iae/Ra.2 has already been described
(25). Cells were harvested 30 hr post transfection. For HEXIMl overexpression, HeLa
cells were transiently transfected with 10 ng pCGN-HEXIMl plasmid or pCGN empty
vector and 300 ng of the pBS-7SK reporter plasmid. Total RNA was extracted using
TRIZOL reagent (Invitrogen). Equal amounts of total RNA were further used in an
RNase protection assay with a radiolabeled probe specific to the inverted B-globin

sequence.

Results
Endogenous HEXIMl co-purifies with SNAPC,

Previous UV cross linking experiments performed with partially-purified SNAPC
/PTF, hereafter referred to as SNAPC, revealed that two subunits, SNAP190/PTF01 and
SNAP50/PTFB, were in close proximity to the PSE dtu‘ing DNA binding by SNAPC (12,
45). However, a third unidentified factor of 70 kDa apparent molecular weight was also
covalently attached to the DNA in a PSE-specific manner (35). As no subunits within
SNAPC correspond to this size, this observation suggested that an additional unknown
factor could function in human snRN A gene transcription through interactions with the
general transcription factor SNAPc and with DNA. Therefore, an extensive biochemical
fractionation of SNAPC was employed to identity this associated factor. The purification

scheme for endogenous SNAPC (Figure 4-1A) is the same as that previously described

131

(14). Typically, the SNAPC present in the last Mono-S step of fractionation has been
purified approximately 104-fold to the point that individual proteins can be isolated and
identified (data not shown).

To determine whether a similar pattern of protein cross-linking to PSE probes was
possible with highly purified SNAPc as was previously described for crude fractions, UV
cross linking experiments were performed with fractions obtained after the Mono-S step
of fractionation. DNA binding reactions were performed with radioactive wild type (wt)
or mutant (mu) PSE probes containing bromodeoxyuridine. Reactions were also
performed in the presence of excess unlabeled wt or mu PSE competitor DNA. After
DNA binding, reactions were cross linked with UV light, digested with micrococcal
nuclease, and the molecular weight of proteins cross linked to DNA were estimated by
SDS-PAGE and autoradiography. As shown in Figure 4-1B, three proteins of
approximately 70, 54, and 50 kDa were detected in reactions performed with the wt PSE
probe either alone (lane 2) or with mu PSE competitor DNA (lane 5). No cross linking
was observed in reactions using the mu PSE probe (lane 3) or with wt PSE probe plus wt
PSE competitor DNA (lane 4). Thus, all three proteins are specifically bound to DNA in
a PSE-dependent fashion. Cross linking of the 70 and 50 kDa proteins in this experiment
is similar to that previously described using less purified SNAPc-enriched fractions (3 5).

To determine the identity of the cross-linked proteins, the Mono-S fractions were
separated by SDS-PAGE and proteins were detected by Coomassie blue staining.
Subsequently, those proteins that were of similar molecular weight as the radiolabeled
species observed in the UV cross-linking experiment were excised for lysyl

endopeptidase digestion and N-terminal peptide sequencing. All peptides that were

132

Figure 4-1. Endogenous HEXIMl associates with SNAPC.
(A) Schematic representation of the chromatographic steps used to purify SNAPc.

(B) A polypeptide of ~ 70 kDa is cross-linked specifically to the PSE during DNA
binding by SNAPC. DNA binding reactions were performed using Mono-S fractions
enriched in SNAPC and homogeneously labeled probes that were substituted with
bromodeoxyuridine and contained either a wild-type (wt) or mutated (mu) mouse U6 PSE
(12). Reactions were performed with the wt or mu probes alone (lanes 2 and 3), or wt
probe in the presence of excess wt (lane 4) or mu (lane 5) unlabeled competitor DNA.
Proteins were cross-linked to DNA with UV light, digested with DNase I and
micrococcal nuclease, and size fractionated by 12.5% SDS-PAGE. Proteins covalently
linked to the remnants of the radiolabeled probe DNA were visualized by
autoradiography. Lane 1 contains protein size markers. N-terrninal sequencing of the 50,
54, and 70 kDa proteins from untreated Mono-S fractions identified the proteins to be
SNAP50 and HEXIMl, as indicated.

(C) A minor population of cellular HEXlMl co-purifies with SNAPC. The
phosphocellulose P-ll fractions were characterized for the presence of HEXIMI and
SNAP43 by Western blot analysis. Increasing amounts of P1 l-A (lanes 1 to 3), P1 l-B
(lanes 4 to 6), P11- C (lanes 7 to 9), and P1 l-D (lanes 10 to 12) were separated by
SDSPAGE electrophoresis, and the presence of SNAPC and HEXIMl were detected by
Western analysis.

(D) HEXIMI co-fractionates extensively with SNAPC. Twenty mL of highly purified
SNAPC fractions from the Mono-S stage of purification were analyzed by

Western analysis using antibodies against HEXIMl (top panel) or SNAP43 (middle
panel). Five mL of the same fractions were also analyzed by EMSA for PSE-binding
activity (bottom panel) using radioactive probes containing a high-affinity mouse U6 PSE
and TATA box. The positions of unbound probe (free probe) and SNAPC bound to DNA
are indicated. The peak of SNAPC DNA binding activity is contained in fractions 61 to
66, whereas the peak of HEXIMI is found from fractions 58 to 69.

This experiment was performed by R. William Henry.

133

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obtained from the 70 and 54 kDa proteins correspond to HEXIM1 and peptides from the
50 kDa protein were found to belong to SNAP50, previously identified as contacting the
PSE during DNA binding by SNAPC (12). For all three cases, no peptides from other
proteins were obtained, including the recently identified HEXIM2 protein that shares
extensive similarity with HEXIM1 (2, 44). We speculate that the smaller HEXIM1
protein (labeled HEXIM1*) is a proteolytic product of larger full length HEXIM1. Thus,
HEXIM1 present in the SNAPc-enriched fractions is in close proximity to the PSE during
DNA binding by SNAPC.

Next, antibodies were generated against HEXIM1 to follow the fi‘actionation of
HEXIM1 during SNAPc purification. As shown in Figure 4-1C, most SNAPc is detected
in the P1 l-C fraction (lanes 7-9), as expected, whereas most HEXIM1 is present in the
P1 1- B (lanes 4-6) and P1 l-D (lanes 10-12) fractions, with a similar but lesser amount
detected in the P1 l-C fraction containing SNAPC. Neither SNAPC nor HEXIM1 were
detected in the P1 l-A fraction (lanes 1-3). Therefore, a substantial but minor proportion
of HEXIM1 co-fractionates with SNAPC during phosphocellulose chromatography. The
population of HEXIM1 that does co-purify with SNAPc was more extensively analyzed
as shown in Figure 1D. Fractions obtained from the Mono-S step of fractionation
revealed an extensive co-purification of HEXIM1 (top panel) and SNAP43 (middle
panel). The fractionation pattern for SNAP43 closely resembled the pattern of SNAPC
DNA binding activity in electrophoretic mobility shift assay (EMSA; bottom panel).
However, significant amounts of HEXIM1 were also detected in fractions that were
devoid of SNAP43 and SNAPC DNA binding activity, indicating that even at this late

stage of purification, HEXIM1 can be chromatographically separated from SNAPc. Only

136

modest amounts of HEXIM1 associated with SNAPc during co-irnmunoprecipitation
from HeLa nuclear extracts or SNAPc-enriched fractions, and the SNAPc/DNA complex
was not obviously super shifted using anti-HEXIM1 antibodies (data not shown),
suggesting that HEXIM1 is not a stable component of endogenous SNAPc. Together,

these data indicate that HEXIM1 and SNAPc may only loosely associate.

HEXIM1 cooperates with SNAPC and TBP for snRNA gene promoter recognition
The observation that HEXIM1 was cross linked to DNA in a PSE-specific fashion
indicated that HEXIM1 could target these promoters through cooperative DNA binding
with SNAPC or TBP, which also function for both RNA polymerase II and III
transcription of snRN A genes. Therefore, EMSAs were performed to investigate whether
HEXIM1 could cooperate with SNAPC and TBP for promoter recognition. In the
following experiments, low levels of each factor were purposefully chosen to minimize
DNA binding by each factor alone. As shown in Figure 4-2A, neither HEXIM1 nor
recombinant mini-SNAPC alone bound strongly to thSE/thATA probe DNA (lanes 2
and 3, respectively), but inclusion of both factors resulted in cooperative complex
formation (lane 4) that was not observed in reactions containing SNAPC plus comparable
amounts of GST (lane 5). A similar pattern of cooperation between HEXIM1 and SNAPc
continued with the thSE/muTATA box probe (lanes 11-15), but complex formation was
not observed in reactions performed with probes containing mutations in the PSE,
regardless of the TATA box status (lanes 6-10 and 16-20). The HEXIM1-SNAPC
complex was super shifted by anti-HEXIM1 and anti-SNAP190 antibodies, but not by

IgG (data not shown), indicating that both factors are components of this complex.

137

Figure 4-2. HEXIM1 cooperates with SNAPC and TBP for DNA binding.

(A) HEXIM1 cooperates with SNAPC for DNA binding in a PSE-specific manner.
Approximately 30 ng of mini-SNAPC (mSNAPc) containing SNAP190 (1-505),
SNAP43, and SNAP50, was used for DNA binding either alone or with 100 ng of
HEXIM1 or GST. Reactions containing only HEXIM1 were also performed as indicated.
Reactions were performed with dsDNA probes containing combinations of wt and mu
PSE with wt and mu TATA as shown schematically. Reactions containing only the
dsDNA probes are shown in lanes 1, 6, 11, and 16.

(B) HEXIM1 cooperates with TBP for DNA binding in a TATA box specific manner.
Approximately 100 ng of TBP was tested for DNA binding either alone, or with 100ng of
HEXIM1 or GST, as indicated. Reactions containing HEXIM1 alone were also
performed, as indicated. The identity of each probe is shown schematically above the
figure.

(C) HEXIM1 stimulates preinitiation complex assembly on snRNA promoters. HEXIM1
stimulates SNAPC and TBP binding. Similar amounts of SNAPC, HEXIM1 and TBP as
above were used and tested for DNA binding on probes containing combnations of wt or
mu PSE with wt or mu TATA box. Reactions containing SNAPC and TBP are shown in
lane 2. The presumptive identities of the various protein-DNA complexes are shown on
the left.

138

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Therefore, HEXIM1 and mini-SNAPC can cooperate for DNA binding in a PSE-
dependent fashion. This result can also explain the PSE dependent cross linking of
HEXIM1 to DNA previously observed in the UV cross linking experiments with highly
purified SNAPc fractions (Figure 4-1).

We next tested whether HEXIM1 can influence DNA binding by TBP (Figure 4-
28). Neither HEXIM1 nor TBP alone bound to the thSE/thATA box (lanes 2 and 3,
respectively), but 000perative DNA binding was observed in reactions containing both
factors (lane 4), and in this experiment, GST did not influence TBP binding (lane 5). In
contrast to the PSE-dependent cooperation between HEXIM1 and SNAPc, the
cooperative DNA binding by HEXIM1 and TBP was TATA box dependent, as PSE
mutation did not affect complex formation (lanes 6-10), whereas no cooperative complex
formation was observed in reactions where the probes contained mutations in the TATA
box (lanes 11-20). Thus, HEXIM1 can cooperate with either SNAPC or TBP, and the
ability of HEXIM1 to form complexes on DNA is dictated by the DNA binding
specificity of its cooperating partner.

TBP recruitment by SNAPc is thought to be part of the preinitiation complex
assembly pathway. We therefore tested whether HEXIM1 can influence TBP recruitment
by SNAPC. As shown in Figure 4-2C, inclusion of HEXIM1 in reactions that contain
SNAPc and TBP caused a noticeable retardation in mobility of the SNAPc-TBP-DNA
complex, consistent with the idea that higher order complexes are being formed (compare
lanes 3 and 4 with lane 2). Interestingly, this effect was not observed on probes that
contained muPSE/thATA, thSE/muTATA or muPSE/muTATA sequences. Only the

HEXIM1-TBP complex was seen with the probe containing a muPSE/thATA box,

141

(lanes 6 and 7) while the HEXIM1-SNAPC complex was seen with the probe containing
thSE/muTATA box (lane 9 and 10). It should be noted that SNAPc and TBP were both
detected in the HEXIM1-SNAPc-TBP-DNA complex, but not HEXIM1, using antibody
supershilt assays (data not shown). It is possible that HEXIM1 only transiently associates
with the DNA and therefore was not detected in the HEXIM1-SNAPc-TBP-DNA
complex. Nonetheless, together these observations suggest that HEXIM1 stimulates DNA

binding by SNAPC and TBP.

Differential snRNA promoter occupancy in vivo by P-TEFb subunits.

The association between HEXIM1 and SNAPc observed during biochemical purification
of SNAPC suggests that HEXIM1 may play a role in human snRNA gene transcription.
Therefore, chromatin immunoprecipitation experiments were performed to determine
whether endogenous HEXIM1 associates with various snRN A gene promoters, including
the 7SK and U6 snRNA genes that are transcribed by RNA polymerase III, and the U1
snRNA gene that is transcribed by RNA polymerase II. In addition to HEXIM1,
immunoprecipitation reactions were performed using antibodies directed against SNAPC,
TBP, and RNA polymerases II and 111. As HEXIM1 is a component of P-TEFb, snRNA
gene promoter association by the cyclinTl and cdk9 subunits of P-TEFb subunits were
examined (Figure 4-3). As expected, the SNAPC and TBP immunoprecipitated samples
were enriched for all snRNA gene promoters examined (lanes 6 and 7), relative to the
IgG control (lane 5), whereas RNA polymerase III was present only at the U6 and 7SK
genes (lane12), and RNA polymerase 11 only at the U1 snRNA gene promoter (lane 11).

Interestingly, 7SK promoter DNA was enriched in all P-TEF b specific

142

Figure 4-3. HEXIM1 occupies endogenous snRNA promoters.

Chromatin was harvested from human mammary epithelial cells (18435) as described
previously (18) and immunoprecipitation reactions were performed using IgG (lane 5) or
anti-SNAP43 (lane 6), anti-TBP (lane 7), anti-HEXIM1 (lane 8), anti-Cdk9 (lane 9), anti-
Cyclin T1 (lane 10), anti -RNAP 11 (lane 11), and anti-RNAP III (lane 12) antibodies.
Immunoprecipitated DNA was analyzed by PCR for enrichment of 7SK snRNA, U6
snRNA, and U1 snRNA core promoters using primers specific for each gene. Enrichment
of GAPDH exon 2 and U1 upstream DNA were examined as negative controls.
Amplification for a lO-fold serial dilution (10% to 0.01%) of input chromatin is shown in
lanes 1-4.

143

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immunoprecipitations using HEXIM1 (lane 8), cdk9 (lane 9), and cyclin T1 (lane 10)
antibodies, whereas U6 promoter DNA was only enriched in the HEXIM1-specific
reactions. U1 promoter recovery by P-TEFb antibodies resembled the U6 pattern, with
strong recovery by HEXIM1 antibodies. The modest U1 promoter enrichment by cyclin
T1 immunoprecipitation suggests that the cyclin/cdk component of P-TEFb could
regulate U1 transcription by RNA polymerase II. In all immunoprecipitations, enrichment
of the U1 upstream region and GAPDH exon 1 was not observed. These results indicate
that multiple components of endogenous P-TEF b associate with the 7SK gene promoter
in vivo and thus P-TEF b might regulate 7SK transcription by RNA polymerase 111.
Furthermore, 7SK, U6, and U1 transcription may be differentially regulated by P-TEFb
even though the 7SK and U6 promoters are highly similar and all genes share a

requirement for the PSE, and thus SNAPC, for efficient transcription.

The cyclin/cdk component of P-TEFb is not required for RNA polymerase III
transcription.

Whereas RNA polymerase II elongation is regulated by P-TEFb, which
phosphorylates the CTD of the RNA polymerase 11 largest subunit, a direct role for the
cyclin/cdk sub-complex of PTEF b in snRNA gene transcription by RNA polymerase II or
III has not yet been established. RNA polymerase 111 does not contain a similar proven
target for P-TEFb phosphorylation and thus it might be immune to P-TEFb action.
Nonetheless, P-TEFb could participate in regulation of snRNA gene transcription by

RNA polymerase 111 through an unanticipated mechanism.

145

To test whether P-TEFb is required for snRNA gene expression, P-TEFb was
removed from HeLa nuclear extracts by irnmunodepletion using antibodies directed
against individual P-TEFb subunits, and the effect on in vitro transcription was
determined. As shown in Figure 4-4A, Western blot analysis revealed that treatment of
extracts with antibodies against cdk9 (lanes 4-6) and cyclin T1 (lanes 7-9) resulted in an
approximate 90% reduction of both cdk9 (top panel) and cyclin T1 (middle panel)
relative to the IgG treated extracts (lanes 10-12) and untreated nuclear extracts (lanes 1-
3). Endogenous HEXIM1 levels were reduced by approximately 50% in the P-TEFb-
depleted samples. Thus, multiple components of P-TEFb are effectively removed by
either treatment. This result is also consistent with the idea that not all HEXIM1 is
associated with the catalytic P-TEFb subcomplex. As shown in Figure 4-4B, U1
transcription by RNA polymerase II was markedly inhibited for both the cdk9 (lane 2)
and cyclin T1 (lane 3) depleted extracts relative to transcription levels supported by the
IgG depleted (lane 4) or mock treated extracts (lane 5). Under these conditions
adenovirus major late (AdML) transcription was modestly affected only in the cdk9
immunodepleted extracts (data not shown). Thus, P-TEFb is required for U1 in vitro
transcription by RNA polymerase 11, possibly for CTD phosphorylation, and is consistent
with a previously postulated role for P-TEFb in 3’ end formation of U2 snRNA (21, 30).
In contrast with RNA polymerase II snRNA gene transcription, neither 7SK nor U6
snRN A gene transcription was substantially affected by P-TEFb depletion, indicating that
P-TEFb is not essential for in vitro snRNA gene transcription by RNA polymerase 111.
Even though cdk9 and cyclin T1 associate with the 7SK snRNA gene promoter in vivo,

the apparent bystander status for cyclin T1 and cdk9 during RNA polymerase III

146

 

Figure 4-4. P-TEFb is differentially used for snRNA gene transcription by RNA
polymerases II and III in vitro.

(A) Multiple P-TEFb subunits are removed from extracts by antibody irnmunodepletion.
HeLa cell nuclear extracts were subjected to immunodepletion using anti-Cdk9 (lanes 4-
6), anti-Cyclin T1 (lanes 7-9), and IgG (lanes 10-12) antibodies that were covalently
cross-linked to protein G agarose beads. Lanes 1-3 contain a titration of the untreated
extract. A portion of the extracts was separated by 12.5% SDS-PAGE, and analyzed by
Western blot analysis using antibodies directed against cdk9, cyclin T1, and HEXIM1, as
indicated.

(B) P-TEFb is required for U1 transcription by RNA polymerase II, but not for 7SK and
U6 snRNA transcription by RNA polymerase III. ln-vitro transcription assays for U1,
U6, and 7SK snRNA genes were performed with HeLa nuclear extract (lane 1) or extract
that had been immunodepleted for P-TEFb components (lanes 2 and 3). Lanes 4 and 5
show transcription from extract that was mock depleted with IgG or beads, respectively.

147

 

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148

transcription suggests that the 7SK gene might be a site for assembling the inactive P-

TEFb ribonucleoprotein complex, perhaps as the 7SK snRNA is being transcribed.

HEXIM1 activates 7SK transcription in vivo

Next the effect of HEXIM1 over-expression on 7SK reporter gene expression was
tested (Figure 4-SA). Over-expression of HEXIM1 resulted in a modest, but reproducible,
increase in 7SK transcription (lane 4) relative to that seen in cells transfected with the
7SK reporter plasmid either alone (lane 2) or with an empty expression vector (lane 3). In
this experiment, over-expression of HEXIM1 did not affect endogenous actin or rRNA
levels. We note that HEXIM1 over-expression did not significantly affect 7SK
transcription during nuclear run-on assays in HeLa cells (data not shown), suggesting that
in vivo HEXIM1 levels are not limiting for 7SK transcription unless the 78K gene copy
number is increased such as during transient transfection. We were unable to achieve a
substantial reduction in endogenous HEXIM1 levels by anti-HEXIM1 irnmunodepletion
(data not shown), and thus could not determine whether HEXIM1 contributes to snRN A
gene transcription in vitro. Nonetheless, the observation that HEXIM1 is present at
snRNA promoters in vivo suggests that this protein could directly regulate transcription.
To examine the possibility that HEXIM1 regulates RNA polymerase III transcription of
snRNA genes, the steady state levels of HEXIM1 were reduced by using siRNA specific
for HEXIM1 and the effect on 7SK reporter gene expression was tested. As 7SK snRNA
binds directly to HEXIM1, any change in 7SK snRNA levels may confound the
assessment of HEXIM1 function, and therefore the 7SK snRNA encoding sequence in

this reporter gene was replaced with an inverted B-globin sequence. As shown in Figure

149

 

Figure 4-5. HEIMl positively regulates 7SK snRNA transcription in vivo.

(A) Over-expression of HEXIM1 increases 7SK snRNA transcription. HeLa cells were
cotransfected with pBS-7SK reporter gene alone (lane 2) or with either pCGN (lane 3) or
pCGN-HA-HEXIMI (lane 4). Cells were harvested 24 hr after transfection and processed
for whole cells extract preparation and total RNA collection. Endogenous HEXIM1 and
actin levels were measured by Western blot analysis. Endogenous 18S and 28S rRNA
levels were monitored by agarose gel electrophoresis and ethidium bromide staining. 7SK
snRNA reporter gene transcription was measured by RNase T1 protection assay and
phosphoimager analysis. The experiment was performed five times and the average 7SK
reporter gene activity is shown in the graph at the bottom. Statistical significance was
estimated using a Student’s T-test and error bars are the standard deviation.

(B) siRNA mediated reduction in HEXIM1 does not affect 7SK transcription. Hela cells
were

cotransfected with an inverted B-globin reporter construct driven by a human 7SK
snRNA promoter (pBS-7SK) alone (lane 1) or along with either the control siRNA (lane
2) or siHisl (lane 3). 30 hr after transfection cells were harvested and processed for
whole cell extract preparation and total RNA collection as in (A). This experiment was
performed three times.

150

 

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4-SB, transient transfection of the si-HEXIMlcaused 90% reduction of endogenous
HEXIM1 (lane 4) relative to levels observed during transfection of the control siRN A (si-
ctrl lane 3). Actin levels were similar for each treatment as were the steady state 188 and
28S rRNA levels. Interestingly, 7SK reporter gene transcription by RNA polymerase III
was not affected with HEXIM1 knock down, suggesting that endogenous HEXIM1 is not
essential for transcription. Interestingly, a lower migrating form of HEXIM1 (labeled as
HEXIM1 *) was not affected in this knockdown. The sequence of this lower migrating
form of HEXIM1 is not known and whether it plays a role in snRNA transcription is yet
to be determined. Nonetheless, the over-expression and knockdown data suggests that
HEXIM1 stimulates 7SK snRNA transcription although HEXIM1 is not essential for this

process.

Discussion

As the products of human snRNA genes play critical roles in numerous steps of
productive global gene expression, the rate of cellular grth and the ability to proliferate
may be sensitive to steady state snRNA levels. In particular, global RNA polymerase II
transcription may be sensitive to the cellular levels of U1 and 7SK snRNA that associate
with the cyclin/cdk complexes, TFIIH and P-TEFb, respectively (24, 34, 42). Both of
these complexes directly regulate RNA polymerase II transcription. Interestingly,
HEXIM1 forms a cyclin/cdk inhibitor complex with 7SK snRNA to down regulate P-
TEFb activity (31, 43). In this context, HEXIM1 is thought to play a negative role in

RNA polymerase II transcription. The data presented herein reveal an unexpected

153

positive role for HEXIM1 in transcription of its 7SK snRNA co-repressor partner by
RNA polymerase III.

The role of HEXIM1 in snRNA gene transcription was first suggested because
HEXIM1 associated with the general transcription factor SNAPc during chromatographic
fractionation of HeLa cell extracts. As SNAPC is required for transcription of human
snRNA genes including U1, U6, and 7SK, a role for HEXIM1 in transcription of all these
genes was postulated. Whereas our results suggest a positive role for HEXIM1 in 7SK
snRNA gene regulation, however, HEXIM1 function does not seem to be essential for
snRN A gene transcription, even though endogenous HEXIM1 was resident at these gene
promoters in vivo. While efficient HEXIM1 knockdown was observed, a shorter form of
HEXIM1 (HEXIM1‘) was not affected. Interestingly, HEXIMI" and not HEXIM1
associates with the DNA-binding domain within SNAP50 (22), when this region of GST-
SNAPSO was used as a bait to pull out interacting proteins from HeLa cell nuclear extract
(data not shown). The sequence of HEXIM1 * is not known, but it seems likely that it
may have internally deleted sequences, as both N- and C-terminal specific antibodies
were able to recognize HEXIM1 * in a Western analysis (data not show). Thus it is likely
that an unidentified splice variant of HEXIM1 exists in the cell and may play a role in the
regulation of snRN A gene transcription.

U6 transcription has been reconstituted in vitro with recombinant factors and
highly purified RNAP III, and in this system no requirement for HEXIM1 was observed
(5). Perhaps traces of HEXIM1 co-purify with RNAP III or with SNAPC purified from
insect cells. It seems more likely; however, that the role of HEXIM1 can be bypassed in

the in vitro transcription system, For example, if HEXIM1 serves to facilitate

154

preinitiation complex assembly, this function might be dispensable in vitro where the
template is naked DNA and the transcription initiation factors are in excess (20), and thus
HEXIM1 activity for U6 regulation may be restricted to a context that is not yet
appreciated.

Our data further indicate that only a minor proportion of HEXIM1 co-fractionates
with SNAPC, suggesting that HEXIM1 partitions into multiple complexes consistent with
the idea that HEXIM1 plays multiple independent roles in the cell (43). Indeed, the
function of HEXIM1 for RNA polymerase III transcription is independent of P-TEFb
kinase activity, as 7SK in vitro transcription is not sensitive to cyclinTl and cdk9 levels.
Thus, it is interesting that cyclinTl and cdk9 were both found associated with the
endogenous 7SK snRNA gene promoter. One possibility is that P-TEF b kinase activity is
required for RNA polymerase III transcription in the cell, but this requirement is not
revealed by in vitro assays. However, the catalytic subunits of P-TEFb were not detected
at the highly related U6 snRN A gene promoter, and although the role of HEXIM1 for U6
transcription by RNA polymerase III is not known, this observation suggests that RNA
polymerase III phosphorylation by P-TEFb is not essential for RNA polymerase III
transcription. An alternative explanation is that the 78K snRN A gene serves as a site for
P-TEFb assembly of the cyclinT/cdk9 kinase complex with the regulatory HEXIM1/7 SK
snRNA complex, as the 7SK snRN A is being transcribed.

As endogenous HEXIM1 associates with PSE-containing promoter sequences
within the cell an important question remains as to how HEXIM1 is targeted to these
genes. We observed that at protein concentrations where little DNA binding is detected

by either SNAPC or HEXIM1 alone, HEXIM1 cooperates with SNAPC for PSE-

155

dependent promoter recognition. Furthermore, HEXIM1 also cooperated with TBP for
TATA-box dependent binding, suggesting that HEXIM1 plays a more general role to
assert its influence on 7SK snRNA gene transcription by facilitating cooperative
preinitiation complex assembly at snRN A gene promoters.

Human 7SK snRNA, along with other snRNAs, are expressed at high levels in the
cell. In part, these levels are maintained by exceptional transcription efficiencies dictated
by the typical promoter structure of snRNA genes (3 7). In fact, the relatively compact
and powerful promoters of some snRN A genes have lent themselves to widespread use in
biotechnology and medical applications to drive high-level expression of effector RNA
molecules. However, snRN A gene promoters are sensitive to complex regulatory control
that may have downstream effects on global RNA production and cellular proliferation. It
is especially intriguing that 7SK snRNA gene transcription is regulated by its functional
partner HEXIM1. We speculate that P-TEFb stimulates HEXIM1 production by RNA
polymerase II, and in turn, HEXIM1 stimulates 7SK snRNA gene transcription in a
process that is expected to down regulate P-TEFb activity as increased levels of the
HEXIM1/7SK snRNA complex are formed. Thus, one possibility is that HEXIM1 levels
may function as a barometer for cellular gene expression levels via feedback regulation
of its co-repressor partner. It is further possible that HEXIM1 contributes to cell growth
control in specialized contexts. For example, an intriguing idea is that HEXIM1
antagonizes HIV-1 transcription by directly maintaining P-TEFb in an inactive state and
ensuring that the levels of its 7SK snRNA co-repressor partner are adequate for this
function. Indeed, a recent model was proposed wherein the HIV-1 Tat protein competes

with HEXIM1 for binding to the cyclin T1 component of P-TEFb to increase levels of

156

active P-TEF b and HIV-1 gene transcription (39). It will be important to determine the

context, timing, and cell-type specificity for HEXIM1 regulation of 7SK transcription.

157

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162

CHAPTER 5

SUMMARY

Non-translated RNAs synthesized by RNA polymerase III contribute to the
biosynthetic growth capacity of a cell (6). Deregulated transcription of these RNAs may
cause unrestricted grth and therefore it is important to regulate RNA polymerase III
transcription. Interestingly, the RB and p53 tumor suppressor proteins (2, 9) as well as
the CK2 and Myc oncoproteins (1, 3) that are important for regulating cell proliferation,
also regulate transcription of these genes. This observation indicates that transcriptional
regulation of these RNA polymerase III transcribed genes may play an important role in
controlling cellular proliferation.

Genes transcribed by RNA polymerases 111 include those encoding SS rRNA,
tRN A and U6 snRNA that are molecular components of the cellular machinery governing
multiple steps in the flow of genetic information in cells. Of these genes, the human U6
snRN A gene is interesting because subtle changes in the core promoter architecture can
switch transcription from RNA polymerase III to II (5). Thus the U6 snRNA gene and
related family members provide a good model system to study the molecular mechanism
of polymerase preference and activity during both normal and deregulated growth.

All human snRN A genes contain a proximal sequence element (PSE) located in
the core promoter region that is recognized by the general transcription factor called
SNAPC. SNAPC plays a pivotal role in snRNA gene transcription by providing core

promoter recognition and coordinating TBP activity as part of nucleating the preinitiation

163

complex assembly for both RNA polymerase II and III. In contrast to other promoter
recognition complexes such as SL1 and TFIIIC that specialize in transcription by a single
RNA polymerase, SNAPC is more functionally versatile due to its role in both RNA
polymerase II and III transcription. SNAPC is composed of at least five subunits namely,
SNAP19, SNAP43, SNAP45, SNAP50, and SNAP190. Although SNAP190 has been
shown to contain a Myb domain in its sequence that contributes to DNA binding by
SNAPC, it is not sufficient for this process (10). Indeed DNA binding by SNAPc is a
cooperative event wherein SNAP190, SNAP50, and SNAP43 are all required ((7) and
Figure 3-1A). These observations suggested that additional contacts are necessary for
DNA binding. A role for SNAP50 in this process was suggested because cross-linking
experiments showed that SNAP50 is in close proximity to the DNA (4). My mutagenesis
studies revealed that SNAP50 does indeed play a role in SNAPC DNA binding through a
highly conserved zinc finger domain in its C-terminus, that functions for preinitiation
complex assembly for both RNA polymerase II and III transcription.

As SNAPC binding to the PSE is a crucial early event in the preinitiation complex
assembly at snRNA promoters, it is a target for regulatory factors. Indeed the
Retinoblastoma (RB) tumor suppressor protein, which was shown to repress of U6
snRNA gene transcription, does interact with the SNAP50 subunit of SNAPC (8), This
observation suggested that the RB-SNAPSO interaction could disable DNA binding by
SNAPC thus explaining the mechanism for RB repression. However, subsequent studies
have shown that RB does not affect DNA binding by SNAPC, indicating that RB may use
some other mechanism. For example, it may recruit co-factors to repress U6

transcription. For other RB target genes such as the RNA polymerase II transcribed E2F-

164

regulated genes required for progression through the cell cycle, RB has been shown to
recruit histone deacetylases (HDAC3) and components of the SWI/SNF ATP-remodeling
complexes (12). Therefore, I examined the role of these RB co-factors during repression
of U6 transcription. My studies revealed a role for HDACs and SWI/SNF in RB
repression of U6 snRN A transcription. My data further indicates that endogenous
HDACs and SWI/SNF proteins associate with the U6 promoters in 18435 cells that
retain RB function but not in HeLa cells in which the RB firnction is compromised. These
observations suggest that recruitment of co-repressor to the U6 snRN A promoter may be
dependent upon RB ftmction. As a first step to address whether RB recruits co-repressor
proteins to the U6 promoter in vivo I have successfully established RB overexpression in
HeLa cells using transient transfection assays. Interestingly, my preliminary data suggests
that one other RB co-factor called DNA methyl transferase 1 (DNMTl) associates with a
U6 promoter in vivo only in the RB-transfected cells but not in a control empty vector-
transfected or mock-treated cells as assayed by transient transfection assay followed by
ChIP experiments. These results suggest that HDAC, SWI/SNF, DNMTl could be
involved in RB-mediated repression of U6 transcription.

My results further indicate that RB repression and HDAC activity are
biochemically separable. Using this system, it will be possible to determine whether RB
co-factors are recruited sequentially to the U6 promoter and whether chromatin affects
this process. For example, RB can form different repressor complexes that contain
HDACs or HDACs plus SWI/SNF. Indeed a RB-HDAC-SWI/SNF complex and the RB-
SWI/SNF complex have been shown to repress cyclin E and cyclin A, respectively,

whose genes products are required for cell cycle progression The sequential disassembly

16S

of the RB-SWI/SNF/HDAC complex to one lacking HDAC activity is proposed as one
mechanism governing RB repression of different genes at distinct phases of the cell cycle
(11). It is possible that RB may utilize a similar mechanism wherein it associates with
multiple corepressor complexes. My studies indicate that RB stimulates U6 promoter
association by HDAC2 and the Brgl component of SW1/SNF complex (Chapter 2), Thus,
an important future avenue of research will be to determine if and how RB coordinates
the activity of multiple co-repressor proteins for repression of U6 transcription.

RB repression of U6 snRNA transcription is a good model system to understand
the mechanistic details of RB repression, as we have established in vitro repression
assays using naked- as well as chromatin-DNA templates, and in vivo assays to study
promoter association by RB and its cofactors. A link between the tumor suppressor
function of RB and U6 repression has been suggested due to the observation that the

region encompassing the AB pocket and the C-terminus of RB are the same regions
required for tumor suppression and U6 repression by RB (9). These studies will help

clarify the mechanism of RB repression as an important tool to understand RB activity

during tumor suppression.

166

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Hinkley, C. S., H. A. Hirsch, L. Gu, 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.

Hirsch, H. A., L. Gu, and R. W. Henry. 2000. The retinoblastoma tumor
suppressor protein targets distinct general transcription factors to regulate RNA
polymerase III gene expression. Mol Cell Biol 20:9182-91.

Hirsch, H. A., G. W. Jawdekar, K. A. Lee, L. Gu, and R. W. Henry. 2004.
Distinct mechanisms for repression of RNA polymerase III transcription by the
retinoblastoma tumor suppressor protein. Mol Cell Biol 24:5989-99.

Wong, M. W., R. W. Henry, B. Ma, R. Kobayashi, N. Klages, P. Matthias, M.
Strubin, and N. Hernandez. 1998. The large subunit of basal transcription factor
SNAPc is a Myb domain protein that interacts with Oct-1. Mol Cell Biol 18:368-
77.

Zhang, H. S., M. Gavin, A. Dahiya, A. A. Postigo, D. Ma, R. X. Luo, J. W.
Harbour, and D. C. Dean. 2000. Exit from G1 and S phase of the cell cycle is

167

l2.

regulated by repressor complexes containing HDAC-Rb-hSWI/SNF and Rb-
hSWI/SNF. Cell 101:79-89.

Zhu, L. 2005. Tumour suppressor retinoblastoma protein Rb: a transcriptional
regulator. Eur J Cancer 41:2415-27.

168

APPENDIX A

MULTIPLE SUBUNITS OF SNAPC CO-EXPRESSED IN E. COLI
ARE ACTIVE FOR TRANSCRIPTION BY HUMAN RNA
POLYMERASE 11 AND 1111

DNA binding by SNAPC is a crucial early event during preinitiation complex
assembly for both RNA polymerase II and III transcribed human snRNA genes. Although
mini-SNAPC (mSNAPc) that assembled by mixing the individual subunits namely
SNAP190 (1-505), SNAP50, and SNAP43 is able to function in DNA binding, it is
crippled for in vitro transcription (1). One possible explanation is that a post-translational
modification of SNAPC is crucial for its activity and that this event is not successfully
recapitulated in E. coli cells. This might also explain the observation that SNAPC
expressed in a Baculaovirus expression system functions for transcription (2). Another
possibility is that SNAPC does not efficiently obtain its fully active, native conformation
when its subunits are expressed separately and reassembled. To test this hypothesis we
devised a co-expression system wherein the individual subunits of mSNAPc were
simultaneously expressed in the same E. coli cell. The co-expressed SNAPC is referred to

as mSNAPcy4 hereafter.

 

lFigures A-lB and A-lC used in this document were published in the following manuscript: Andrej
Hanzlowsky, Blanka Jelencic, Gauri W. Jawdekar, Craig S. Hinkley, James H. Geiger, and R. William
Henry (2006) Co-expression of multiple subunits enables recombinant SNAPC assembly and function for
transcription by human RNA polymerase 11 and 111. Protein Expression and Purification Expression
Vol.48; pp.215-223

169

mSNAPcy4 is functional for DNA binding and TBP recruitment to a U6 snRNA
promoter

We wanted to determine whether mSNAPcy4 was functional. The composition of
mSNAPcy4 obtained by using a co-expression system is shown in Figure A-1A. The
ability of mSNAPcy4 to bind DNA in an electrophoretic mobility shift assay was tested
as shown in Figure A-lB. Increasing amounts of mSNAPcy4 was able to bind
thSE/thATA probe DNA (lanes 2 and 3) and thSE/muTATA probe DNA (lanes 10
and 11), however failed to bind to a muPSE/thATA DNA probe (lanes 6 and 7) and
muPSE/muTATA DNA probe (lanes 14 and 15). The mSNAPcy4 was functional for TBP
recruitment to a probe containing thSE/thATA DNA (lane 4) but not to a
thSE/muTATA DNA (lane 12). These results show that the mSNAPcy4 obtained using
the co-expression system does indeed behave similarly to mSNAPc for DNA binding and

TBP recruitment.

mSNAPcy4 is able to reconstitute human U1 and U6 snRNA transcription in vitro
We next wanted to test whether mSNAPcy4 was able to reconstitute in vitro
transcription initiated from a plasmid containing the human U1 snRNA promoter as
shown in Figure A-2A. HeLa cell nuclear extract was either mock depleted with pre-
immune rabbit sera or depleted with anti-SNAP43 to remove endogenous SNAPC. In the
absence of endogenous SNAPC, U1 transcription is reduced considerably but not in the
mock depleted extract (compare lanes 1 and 2 with lane 3). When increasing amounts of
mSNAPcy4 were added to the transcription reaction U1 transcription was restored (lanes

4 to 10). Similarly, as shown in Figure A-2B, U6 snRNA transcription was also restored

170

by increasing amounts of mSNAPcy4 (lanes 3 to 9), but not by a non-specific protein like
GST (lane 10). These results show that mSNAPcy4 is indeed functional in an in vitro
transcription assay. Thus we have been able to establish a system for co-purification of
functional mSNAPcy4 that contains SNAP190 (1-505), SNAP10, SNAP43, and SNAP19.
This system is overall far superior as it yields nearly a pure and homogenous complex,
and the quantity of protein obtained is suitable for further structure-function

characterization of this multi-protein transcription factor.

171

Figure A-l. mSNAPcy4 is competent for DNA binding and TBP promoter
recruitment

(A) Purified recombinant mSNAPcy4 was separated by 15% SDS-PAGE and visualized
by staining with Coomassie blue (lane 2). Lane 1 contains a protein size markers.

(B) Increasing amounts of mSNAPcy4 (3 ng and 10 ng) were added to EMSA reactions
containing dsDNA probes harboring a wt PSE and wt TATA, mu PSE and wt TATA, wt
PSE and mu TATA, or mu PSE and mu TATA box, as indicated. Lanes 4, 8, 12, and 16
contain approximately 50 ng of recombinant human TBP in addition to IOng of
mSNAPcy4. Reactions containing only the DNA probe are shown in lanes 1, 5, 9, and 13.

172

 

 

 

 

 

A - g.
s 5
<0 (I)
2 E
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— an-
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75 —— . MW
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50 — C m —SNAP50
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25 - o
20 — a
15 __ ~ —SNAP19
10 — u '
1 2
Coomassie Blue staining
B

PROBE M— W W

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probe

12 3 4 5 6 7 8 910111213141516
EMSA

 

173

Figure A-2. mSNAPcy4 supports human snRNA gene transcription in vitro by both
RNA polymerase II and III

(A) The HeLa cell nuclear extract used for human U1 in vitro transcription assay was
either mock depleted with a preimmune rabbit sera or anti-SNAP43 antisera to
immunodeplete endogenous SNAPC. Reduction of the U1 signal upon removal of
endogenous SNAPC is shown in lane 3. Increasing amounts of mSNAPcy4 (0.3 ng, 1 ng,
3 ng, 10 ng, 30 ng, 100 ng, or 300 ng) was able to reconstitute correctly initiated
transcription from a human U1 promoter as shown in lanes 4 to 10. Lanes 1 and 2 show
the U1 signal obtained from either untreated or mock-depleted reactions. RT represents a
read-through transcript.

(B) In vitro transcription of human U6 snRNA was carried out using HeLa cell nuclear
extract that was treated as before in. Increasing amounts of mSNAPcy4 (0.3 ng, 1 ng, 3
ng, 10 ng, 30 ng, 100 ng, or 300 ng) was able to reconstitute correctly initiated
transcription from a human U6 promoter as shown in lanes 3 to 9. Lane 2 shows the
reduced U6 signal upon removal of endogenous SNAPC. Approximately 300ng of GST
was added to the transcription reaction instead of mSNAPCy4 as shown in lane 10.

174

 

 

a-SNAPc depleted
l

‘ 11"» ' .V ij'“ " 7:3 Anny», ..~-._.\, ‘~ . '. In .. 't . .1
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l

. ., 1 -- ~1:»' ,, . .
‘
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v t
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175

References

Hanzlowsky, A., B. Jelencic, G. Jawdekar, C. S. Hinkley, J. H. Geiger, and R.
W. Henry. 2006. Co-expression of multiple subunits enables recombinant

SNAP(C) assembly and function for transcription by human RNA polymerases II
and 111. Protein Expr Purif 48:215-23.

Henry, R. W., V. Mittal, B. Ma, R. Kobayashi, and N. Hernandez. 1998.
SNAP19 mediates the assembly of a functional core promoter complex (SNAPc)
shared by RNA polymerases II and III. Genes Dev 12:2664-72.

176

APPENDIX B

DIFFERENTIAL U6 snRNA PROMOTER ASSOCIATION IN VIVO
BY RB FAMILY MEMBERS

Pocket proteins associate with a U6 sNRNA promoter in vivo.

The Retinoblastoma (RB) tumor suppressor protein is thought to contribute to growth
control through its regulation of RNA polymerase III transcription Therefore to test
whether RB function at U6 snRNA promoters is linked to growth, chromatin
immunoprecipitations assay was performed from normal mammary epithelial cells
(1 84B5) harvested at increasing cell densities. Populations of cells were harvested at low
(~25%), intermediate (~75%), and high (~100%) cell densities. As shown in Figure B-
1A, both U6 (top panel) and U2 (middle panel) promoter DNA were significantly
enriched in chromatin immunoprecipitations performed with anti-SNAP43 antibodies
(lane 6) at all cell densities as compared the GAPDH exon2 negative control (bottom
panel). Interestingly, measurable levels of RB were detected at the U6 snRNA promoter
only in those cells harvested at intermediate cell density, but not at the low or high
densities (lane 7). The pattern for RB enrichment in these experiments suggests that RB
may regulate these genes when cells are actively growing but not when the cells have
exited the cell cycle. Therefore, to characterize the relative percentage of cells in each
phase of the cell cycle, cells grown at the different densities were analyzed for DNA
content by flow cytometry. Indeed, the relative number if cells in GO/Gl increases as the

cell density increases, indicating that when grown to 100% confluence most cells have

177

Figure B-l. U6 snRNA promoter occupancy by RB and RB family members is cell
density dependent.

(A.) U6 promoter occupancy by RB is sensitive to the cell density. Chromatin was
harvested form normal human mammary epithelial cells (184B5) grown to approximately
25%, 75%, and 100% cell density. Immunoprecipitation reactions from each chromatin
sample were performed using rabbit pre-immune sera (lane 5), anti-SNAP43 (lane 6), or
anti-RB antibodies (lane 7). Immunoprecipitated material was analyzed for U6 snRNA
and U2 snRN A promoter DNA (upper and middle panels, respectively) or GAPDH exon
2 DNA (bottom panel) by PCR using primers specific to each gene. Lanes 14 represent
10-fold serial dilution (10% to 0.001%) of input chromatin. Cells grown to similar
density were processed for propidium iodide staining and the relative percentages of cells
in 60/01, S, or 62 phase of the cell cycle were determined using FACS analysis.

(B) Cell density influences distinct RB family member association with the human U6
promoter. Chromatin was collected from human 18485 cells that were grown to either
intermediate or high densities. Immunoprecipitation reactions were performed using IgG
(lane 2), anti-RB (lane 3), anti-p130 (lane 4), or anti-p107 (lane 5) antibodies.
Immunoprecipitated DNA was analyzed for U6 snRNA (upper panel) and Cyclin A
(lower panel) promoter DNA by PCR. Lane 1 indicates the input DNA.

178

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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E E
< 8 "
E O
%lnput égg 25%;:
74:: ‘ o. a as E 2’ 8 is is
1,: "l— -- ~— -- 125% F u. .. _. intermediate
4--- U6 snRNA
t-..- us snRNA L- -— i.-. .. .. 75% g, __ huh
25:. f. .. .._.. .._.. .._. 110070 :
."1. b, . 'I' - intermediate
9-75: ”EL; an. m - 12570 CYCIII'IA .. ..- - high
”‘ 02 snRNA may... .._.... 75% ' ‘1 "2 '3‘ “"2" "5
b 25%
._ ]75%
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5»...- 1 2 3 4 5 6 7
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25% 75% 100%
GO/G1‘67 71 E
s 25 2o 12
62 8 9 3

179

likely exited the cell cycle. However, the difference in cell cycle profile for cells grown at
low and intermediate densities is subtle, and thus other contributing factors may govern
whether RB regulates U6 transcription. One possible explanation could be that at lower
cell-density most of the RB protein is in the hyperphosphorylated form and as the cell
density increases there is a shift to the hyphosphorylated form of RB, which is believed to
be transcriptionally active. Indeed there has been a report showing that as cell density
increased from 13% to 43% there was an increase in hyperphosphorylated-RB as
observed by Western blot analysis. However, as the cell density reached 75% and 100%
there was more hypophosphorylated-RB (1).

Prior studies have suggested that all three members of the RB family can regulate
RNA polymerase III transcription (3, 5). Therefore, I wanted to determine whether p107
and p130 also associate with the U6 snRNA promoter. Chromatin immunoprecipitation
assays were performed from cells grown to intermediate or high cell density and using
antibodies for each RB family member. Enrichment of the cyclin A promoter was
examined as a positive control for the p107 and p130 immunoprecipitations (6). As
shown in Figure B-1B, the U6 snRNA promoter was enriched in the a-RB and a-p107
immunoprecipitations at the intermediate cell density conditions, but not when cells were
grown at high densities. RB was not present at the cyclin A promoter during either
growth condition whereas the cyclin A promoter was enriched in the p107
immunoprecipitation from cells grown at both intermediate and high density. In contrast,
p130 associates with both promoters only at high cell densities. None of the RB pocket
proteins was detected at the U6 promoter at low cell density nor was enrichment of the

GAPDH exon 2 DNA observed (data not shown). Together these results suggest that all

180

three RB family members may participate in regulation of U6 snRNA expression but
under different grth conditions. Data from our lab indeed shows that p107 and p130
also repress U6 snRNA transcription in vitro by RNA polymerase III (Xianzhou Song,
unpublished data). Thus my data has revealed that both RB and p107 associate with U6
snRNA promoter DNA when cells are actively dividing. When cells have exited the cell
cycle due to contact inhibition, neither RB nor p107 occupy a U6 snRNA promoter, but
instead p130 now associates with the U6 promoter. Previously, p130 was shown to be
mostly active in the GO/Gl phase while p107 is active in the S-phase (reviewed in (4).
These observations are consistent with previous reports that both p107 and p130 can
regulate RNA polymerase III transcription (5) My data indicates that RB and p107 may
regulate RNA polymerase III activity during active growth and transition through the cell
cycle whereas p130 may be active during growth arrest, and suggest that pocket protein
family members regulate hmnan U6 snRNA gene transcription at distinct phases of cell

growth.

Methods and Materials

Human mammary epithelial 184BS cells were used for the cell density experiments. The
cells were seeded as follows: 0.15x107 cells into 40 plates (25%), 0.6x107 cell into 10
plates (75%), and 1.2x107 cells into 5 plates (100%). After 48 hrs cells were harvested
form each density pool and processed for collecting chromatin as described before (2). A
portion of the chromatin corresponding to 1x107 cells was then used for each
immunoprecipitation reaction using anti-SNAP43 (C848), anti-RB (SC-1538) antibodies,

or an irrelevant preimmune serum. For the ChIP experiment shown in Figure B-lB, anti-

181

p107 (SC-318), anti-p130 (SC-317) antibodies were additionally used.
Immunoprecipitated DNA was examined for enrichment of U6 snRNA, U2 snRN A, and
Cyclin A promoter DNA or GAPDH exon2 DNA using PCR amplification. For the
FACS analysis done Figure B-lA, a portion of the harvested cells were washed and fixed
with ice cold 70% ethanol. Subsequently, cells were stained with Propidium Iodide and

the DNA content was analyzed using a FACS Vantage flow cytometer.

182

References

1. Hannan, K. M., B. K. Kennedy, A. H. Cavanaugh, R. D. Hannan, I. J.
Hirschler-Laszkiewicz, L. S. Jefferson, and L. I. Rothblum. 2000. RNA
polymerase I transcription in confluent cells: Rb downregulates rDNA
transcription during confluence-induced cell cycle arrest. Oncogene.

2. Hirsch, H. A., G. W. Jawdekar, K. A. Lee, L. Gu, and R. W. Henry. 2004.
Distinct mechanisms for repression of RNA polymerase III transcription by the
retinoblastoma tumor suppressor protein. Mol Cell Biol 24:5989-99.

3. 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.

4. Mulligan, G., and T. Jacks. 1998. The retinoblastoma gene family: cousins with
overlapping interests. Trends Genet 14:223-9.

5. Sutcliffe, J. E., C. A. Cairns, A. McLees, S. J. Allison, K. Tosh, and R. J.
White. 1999. RNA polymerase III transcription factor IIIB is a target for
repression by pocket proteins p107 and p130. Mol Cell Biol 19:4255-61.

6. Takahashi, Y., J. B. Rayman, and B. D. Dynlacht. 2000. Analysis of promoter
binding by the E2F and pRB families in vivo: distinct E2F proteins mediate
activation and repression. Genes Dev 14:804-16.

183

APPENDIX C

RB INTERACTS WITH MULTIPLE COMPONENTS OF THE U6
snRNA-SPECIFIC GENERAL TRANSCRIPTION MACHINERY
FOR PROMOTER RECRUITMENTl

RB interacts with the U6 snRNA-specific general transcription machinery.

For RB to enact repression of U6 snRNA transcription RB might be recruited to
the promoter either directly by binding to specific DNA control elements or through
interactions with the general transcription factors. As RB does not have a DNA-binding
domain, RB most likely is recruited to the DNA via interactions with SNAPC and TFIIIB.
Therefore, GST pulldown experiments were performed to determine the region(s) of
SNAP50 that is required for interaction with RB. The truncated SNAP50 mutant proteins
used are represented in Figure C-lA. GST-RB (379-928) and the 35S-labeled SNAP50
proteins were expressed as before (4). GST-RB (379-928) interacted with the full-length
SNAP50 (1-411) protein and this interaction was specific, as no interaction was seen with
GST (lanes 2 and 3). SNAP50 (1-300) and SNAP50 (301-411) proteins interacted with
GST-RB (379-928) suggesting that there might be two regions in SNAP50 involved in
RB interaction. A bigger C-terminal deletion of SNAP50 containing amino acids 1-199
also interacted with RB, however the region in SNAP50 containing amino acids 1-124
and 123-199 did not interact with RB suggesting that the region in SNAP50 around

amino acids 123/124 could be important for RB interaction. Interestingly, a LxCxE motif

 

' Figure C-2 used in this document was published in the following manuscript: Heather A. Hirsch, Gauri
W. Jawdekar, Kang-Ae Lee, LiPing Gu, and R. William Henry (2004) Distinct mechanisms for repression
of RNA polymerase III transcription by the Retinoblastoma tumor suppressor protein. Molecular and
Cellular Biology Vol.24; pp.5989-5999

184

is present within this region, amino acid residues 109-113. The LxCxE sequence, which
is a known motif for RB binding (3), may be important for SNAP50 interaction with RB.
However, a truncation of SNAP50 containing amino acids 123-300, which lacks the
LxCxE motif, also interacts strongly with RB, suggesting that a region outside the LxCxE
motif may provide a region for interaction with RB. Whether the LxCxE motif can
contribute to interactions with RB is not known. Other amino-terminal truncations tested,
including SNAP50 (301-411), SNAP50 (123-411), SNAP50 (200-411) lacking the
LxCxE motif interacted weakly with RB. Together these data provide evidence that the
region of SNAP50 between amino acids 1-199, 123-300, and 301-411 may be important
for RB interaction and provide a basis for finther mutational analysis.

The region(s) in deI that are important for RB interaction were mapped in a
GST pulldown assay. 0 and N- terminal deletion mutants of del were cloned and
expressed as 35S-labeled proteins. The de1 protein contains a SAN T domain in the N-
terminal amino acids. Though the exact function of the SANT domain is not known, in
other proteins it has been implicated in DNA binding (1). de1 contains a series of
repeats in the C-terminal two-thirds of the protein with potential phosphorylation sites.
As shown in Figure C-1B, full length del (1-1338) protein, de1 (471-1338) protein,
and de1 (823-1338) protein interacted strongly with GST-RB (379-928) (lane 2) but not
with GST or beads alone (lanes 3 and 4). However, the N-terminal region containing
amino acids 1-470 did not interact with GST-RB (379—928), suggesting that the C-
terminal two-thirds of del is important for RB interaction and the amino acids between
823-1338 are sufficient for this interaction. RB could potentially be recruited to the U6

promoter via these interactions.

185

Figure C-l. Characterization of the regions in SNAP50 and de1 that are required
for RB interaction.

(A) The RB pocket domain interacts with at least three regions in SNAP50. Schematic
representation of SNAP50 proteins containing the indicated deletions is shown in the left
panel. GST pulldown analysis was performed to map the regions in SNAP50 that interact
with RB. SNAP50 proteins containing deletions were expressed In vitro using rabbit
reticulocyte lysate and labeled with 35 S methionine. Equal amounts of the SNAP50
proteins were incubated with approximately 1 ug GST- RB (379- 928) (lane 3) and 1 pg
GST (lane 2). Complexes were collected with glutathione agarose beads for 2 hrs at 4°C.
Bound proteins were washed and eluted in Laemmlie buffer. Proteins were separated by
SDS-PAGE and visualized by autoradiography. Lane 1 shows 10% of the input.

(B) The C- terminal region of del is important for RB interaction. Schematic
representation of the de1 proteins harboring the indicated deletions are shown In the lefi
panel. GST pulldown assays were carried out with3 5S-methionine-labeled de1 proteins
and GST-RB (379- 928) as in (A).

186

 

 

 

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N
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[x
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[ SNAP50 (1-411)

 

 

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' SNAP50(301-411)

 

 

 

 

1 199
~ SNAP50 (1.199)
1 124 L
:1] SNAP50 (1-124)
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SNAP50 (123.199)
123 300

SNAP50 (123-300)

 

SNAP50 (123-411)

 

 

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l “

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187

10 % INPUT

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6:
5.
m
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(5

GST
15.15: Beads

de1 (1-1338)

 

l>>>>>>>>>>>§l

 

 

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" "-‘ —. j]: : h

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sill de1 (873-1338)

»>>>>>>>>»>>>>:| ;' 2731;; ~

  

 

   

 

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188

To further understand the mechanism of RB repression of U6 transcription, the RB
domains that are required for repression of U6 transcription were characterized.
Therefore, RB proteins containing the A domain but lacking additional regions of the
carboxy terminus were analyzed. Previous observations show that RB interacts with
SNAPC (4) and TFIIIB (2, 6). That RB does not occupy a human U1 snRNA gene
promoter even though SNAPC is required for transcription of U1 by RNA polymerase II,
suggests that RB interaction with SNAPC and TFIIIB represents a mechanism by which
RB is specifically recruited to the U6 snRNA promoter to repress transcription. GST pull
down experiments were performed to identify the regions of RB necessary for interaction
with components of SNAPC and TFIIIB. Each component of the basal transcription
machinery was individually expressed and labeled with 35S-methionine in rabbit
reticulocyte lysate. Expression of these labeled proteins is shown in Figure C-2B (lane 1).
Equivalent amounts of GST-RB (3 79-928) or GST-RB proteins containing the indicated
truncations were incubated with the labeled proteins. Strong interactions between GST-
RB (379-928) and two components of SNAPC, SNAP43 and SNAP50 were observed.
Specific interactions of GST-RB (379-928) with the TBP, Brfl, and de1 components of
TFIIIB were also observed, although the interactions with TFIIIB appear to be weaker as
compared to those with SNAPC components. These interactions are specific because
neither GST nor beads alone bound to any of the SNAPC or TFIIIB proteins (lanes 6 and
7). In contrast GST-RB (379-928) did not interact with Brf2 or Oct-l in these assays
(lane 2). More interestingly, GST-RB (379-870), which repressed U6 transcription,
maintained the ability to interact with components of SNAPC and TFIIIB (lane 3), but

GST-RB (3 79-772), which failed to repress U6 transcription, showed

189

Figure C-2. The A/B pocket domain and the C-terminal region of RB are required
for interactions with RNA polymerase III-general transcription machinery.

(A) Schematic representation of the GST-RB proteins containing the indicated deletions.

(B) Characterization of the RB regions required for interactions with RNA polymerase HI
basal transcription machinery. GST-pull down analysis was performed to determine the
region of RB that can interact with each component of RNA polymerase III basal
machinery. SNAP43, SNAP50, TBP, Brfl, Br12, del, and Oct-1 were expressed in
vitro and labeled with 35S-methionine. Lane 1 shows 10% of each protein that was added
to the reaction. The various GST-RB proteins containing deletions were incubated with
each 35S-methionine labeled protein (lanes 2-5). GST or beads alone were used as
controls (lanes 6 and 7). The 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.

190

 

re re:-

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IO.--’J.

 

 

 

 

 

 

, , . l TBP

 

 

 

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' . 7": ,3.
n,
.

 

 

 

 

 

 

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GST-RB (379—870)
GST-RB (1379-772)
GST-RB (379-577)

 

GST-RB
Protein ID

U6 snRNA
repression

SNAP43

SNAP50

TBP

de1

Ber

 

 

GST-RB (379-928)
GST-RB (379-870)
GST-RB (379-772)
GST-RB (379-577)

 

 

 

 

 

 

 

 

191

 

reduced interactions with SNAP43, TBP, del, and Brfl (lane 4). Thus these interactions
might be critical for RB repression of U6 snRN A transcription (summarized in the table).
Although GST-RB (379-772) interacted strongly with SNAP50, this interaction alone is
not sufficient to maintain RB repression. Finally, GST-RB (379-577), which contains
only the A domain, was not able to interact with any of the SNAPC or TFIIIB proteins
(lane 5) consistent with its inability to repress transcription. These observations show that
the NB pocket domain and the C-terminal region of RB are important for interactions
with the U6 snRNA-specific general transcription factors. It is possible that RB is
recruited to the promoter DNA via multiple protein-protein interactions with SNAPc and
TFIIIB. Sequential ChIP experiments indeed show that RB co-occupies the same U6
snRNA gene promoter with SNAPC and TFIIIB in vivo (5). Interestingly, proteins
harboring progressively increasing C-terminal deletions lose their ability to repress
transcription, indicating that the NB pocket domain and the C region are necessary for

repression of RNA polymerase III transcription (5).

192

J“ W. us. “A:

 

References

1. Aasland, R., A. F. Stewart, and T. Gibson. 1996. The SANT domain: a putative
DNA-binding domain in the SWI-SNF and ADA complexes, the transcriptional
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