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if; LIBRARY

. fl, _ Michigan State
9'" "i ' 7 University
'99 L? to 9 ’2 9

This is to certify that the
thesis entitled

REGULATION OF HUMAN RNA POLYMERASE Ill
TRANSCRIPTION BY RB FAMILY MEMBERS

presented by
XIANZHOU SONG

has been accepted towards fulfillment
of the requirements for the

Master of degree in Cell and Molecular Biology
Science (CMB) program

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Date

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2/05 c:/ClRC/DateDuo.lndd—p.15

 

REGULATION OF HUMAN RNA POLYMERASE III TRANSCRIPTION BY RB
FAMILY MEMBERS
By

Xianzhou Song

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
MASTER OF SCIENCE
Cell and Molecular Biology (CMB) Program

2005

ABSTRACT
REGULATION OF HUMAN RNA POLYMERASE IH TRANSCRIPTION BY RB
FAMILY MEMBERS
By
Xianzhou Song
In humans, the Retinoblastoma tumor suppressor (RB) and its homologues, p107 and
p130, can repress transcription by RNA polymerases (P01) 1, II and HI of genes whose
products are closely linked to cell cycle progression, cell differentiation, cell apoptosis

and cell growth.

For Pol III repression by RB, one mechanism elucidated for tRN A gene regulation is that
RB targets the transcription factor TFIIIB to interrupt Pol III recruitment to the tRN A
promoter. However, RB represses U6 snRNA Pol III transcription by a distinct
mechanism, wherein RB, snRNA activating protein complex (SNAPc), TFHIB, and Pol

HI co-occupy the U6 snRNA promoter during repression.

Herein I demonstrate that p107 and p130 also are able to repress U6 snRNA transcription
in vitro. All RB family members directly interact with multiple subunits of the U6
snRNA general transcription machinery including SNAPc and TFIIIB. Furthermore,
endogenous RB family proteins associate with Pol HI and the A domain of RB is
sufficient for RB binding to P01 1H. Combined with the phenomenon that RB and Pol III
co-occupy the repressed U6 snRN A promoter, a tether and immobilization model is

proposed to explain how RB family members repress U6 snRNA transcription.

To my dear family

iii

ACKNOWLEDGEMENTS

I would like to thank my committee members Dr. R. William Henry, Dr. Susan
Conrad, Dr. Min-Hao Kuo, Dr. Ronald Patterson and Dr. Steven Triezenberg for their
guidance. I especially appreciate my mentor Dr. R. William Henry for his advice.

I am very thankful for the friendships during my graduate training with the
members of the Henry lab, including Dr. Craig Hinkley, Dr. Zakir Ullah, Dr. Heather
Hirsch, Melissa Bosma, Anastasia Gridasova, Gauri Jawdekar, Liping Gu, and
Tharakeswari Selvakumar. In addition, I also thank the Amosti’s lab for kindly providing
materials and equipment for Drosophila related work. I also thank Dean Shooltz for
reading my thesis.

Finally, I strongly want to thank my family for their endless support and for their

faith.

iv

 

TABLE OF CONTENTS

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

LIST OF ABBREVIATIONS .................................................................... viii

CHAPTER 1

LITERATURE REVIEW .......................................................................... l
Retinoblastoma (RB) protein, a tumor suppressor ........................................... 2
The pocket domain family ....................................................................... 4

The molecular mechanisms of transcriptional repression by RB, p107, and p130 ....... 8

Pol III transcription ............................................................................... 13

Regulation of Pol III transcription by RB family members ................................. 24

Summary ........................................................................................... 25

References .......................................................................................... 25
CHAPTER 2

REGULATION OF HUMAN U6 snRNA TRANSCRIPTION BY RETINOBLASTOMA

FAMILY MEMBERS .............................................................................. 38
Abstract .............................................................................................. 39
Introduction ......................................................................................... 40
Materials and Methods ............................................................................ 43

 

Results ............................................................................................... 48

Discussion .......................................................................................... 63
References .......................................................................................... 68
APPENDIX .......................................................................................... 72
Introduction ........................................................................................ 73
Materials and Methods ............................................................................ 74
Results ............................................................................................... 79
References .......................................................................................... 88

vi

LIST OF FIGURES
Fig. 1-1. Schematic drawings of RB, p107 and p130 .......................................... 5

Fig. 1-2. Promoter architectures for different classes of RNA polymerase III transcribed
genes .................................................................................................. 15

Fig. 1-3. Transcription factors required for transcription by RNA polymerase IH. . . . . ....19

Fig. 2-1. All Rb family members are able to repress human U6 snRNA transcription in
vitro .................................................................................................. 49

Fig. 2-2. Endogenous RB family proteins associate with SNAP 43, TBP and P01 IH. . ...54

Fig. 2-3. All RB family proteins interact with multiple components of SNAPc and

TFIIIB ................................................................................................. 58
Fig. 2-4. Pol III and Brgl bind to the RB A domain ........................................... 61

Fig. 2-5. A tether and immobilization model for RB repression of U6 snRN A ............ 66
Fig. A-l. Optimization of Micrococcal Nuclease (MNase) digestion ........................ 80

Fig. A-2. Histone distribution among fi'actions after fractionated fiom sucrose
gradients .............................................................................................. 80

Fig. A-3. Core histones were concentrated by hydroxyapatite column chromatography..82

Fig. A-4. Genomic DNA was depleted from core histones by hydroxyapatite column
chromatography ..................................................................................... 82

Fig. A-S. Chromatin was assembled on pU6/Hae/Ra.2 and the Oct-1 POU domain ...... 85

vii

LIST OF ABBREVIATIONS

Ad ML
de1
Brf

Brgl

DSE
EtBr
HATs
HDACs
HMTS
ICR
PIC

Pol
PSE

PTF

RPBl
RPCl

rRN A

Adenovirus Major late

B double prime

TFHB related protein

Brahma-related gene 1

Chromatin irnmunoprecipitation
Distal sequence element

Ethidium Bromide

Histone acetyltransferases

Histone deacetylases

Histone methyltransferases

Internal control region

Preinitiation complex

RNA polymerase

proximal sequence element

Proximal element transcription factor
Retinoblastoma tumor suppressor protein
RNA polymerase H subunit 1

RNA polymerase III subunit 1

Ribosomal RNA

viii

S-l90

SNAPc

snRNA

TFIIIA

TFIIIB

TFIIIC

TSA

Chromatin assembly S-190 extract

Small nuclear RNA activating protein complex
Small nuclear RNA

TATA binding protein

Transcription factor III A

Transcription factor HI B

Transcription factor III C

Trichostatin A

ix

CHAPTER 1

LITERATURE REVIEW

Retinoblastoma (RB) protein, a tumor suppressor

The concept of tumor suppression has been developing for several decades since first
suggested in cell fusion experiments (43), wherein the tumorigenetic character of cancer
cells was suppressed to some degree after fusion with normal cells. Human
retinoblastoma, a rare pediatric eye tumor, has been extensively researched in correlation
with the concept of “tumor suppression”. After a statistical analysis of retinoblastoma
patients, a “two-hit” hypothesis was proposed to explain the formation of retinoblastoma,
in which two mutations are required for disease initiation (66). This “two-hit” model was
further explained as inactivation of both alleles of a single gene that was responsible for
the suppression of retinoblastoma ( l 9). Soon afier, a retinoblastoma susceptibility gene,
which was abnormal or deleted in some cases of retinoblastoma patients was mapped to
chromosome 13q14 (4, 32, 114). Compelling evidence for the existence of a
retinoblastoma tumor suppressor was provided when the retinoblastoma (RB) gene was
finally cloned and found mutated or deleted in all retinoblastoma tumor samples but not
in samples from normal tissues (33, 35, 76). Subsequently, RB was found to play a role in
other cancers besides retinoblastoma, includes osteosarcomas, soft tissue sarcomas,
leukemias, and many other tumors, where RB is also mutated or inactivated (8).
Additionally during infection by different DNA tumor viruses, RB is inactivated by viral
oncoproteins, including the Simian virus 40 large T antigen, adenovirus ElA protein, and
human papilloma virus E7 protein (21, 25, 127). Together, these observations indicated

that RB is a tumor suppressor that in its wild-type form prevents neoplasm.

Abnormal proliferation is a typical characteristic of cancer. In agreement with its role as a
tumor suppressor, RB was identified as crucial for cell cycle progression control (123).
Overexpression of RB results in the cellular arrest at G1 phase of the cell cycle (40). RB
controls the GI/S transition by repressing the transcription of genes whose products are
required for S phase entry, including cyclinE, Myc, cdc2, PCNA and others (9, 123).
During early G1 phase, RB is hypophosphorylated whereas during late G1 phase, RB is
increasingly hyperphosphorylated by cyclin D/cdk4, cyclin E/cdk2, and cyclin A/cdk2
complexs (1, 50). Consequently during early G1 phase, hypophosphorylated RB binds to
E2Fs and represses E2F dependent transcription, while during the late G1 and S phase,
E2Fs are released from hyperphosphorylated RB and can stimulate target gene

transcription (1, 80, 124).

In addition to its function as a tumor suppressor, RB also plays significant roles in tissue
development, which was first realized in RB knock out mice (12, 59, 60, 73). While RB”
mice are predisposed to pituitary gland tumors (12, 60, 73), which confirmed RB as a
tumor suppressor, RB'l' knock out mice were non-viable and died between days 13 and
15 of gestation with obvious defects in the development of erythroid, nervous system,
lens, and skeletal muscle (12, 59, 60, 73, 134). In RB'I' mice these tissues were not able to
withdraw from the cell cycle during terminal differentiation and underwent extensive
apoptosis (82, 90, 134). RB was shown to serve as co-activator of some tissue specific
transcription factors that are required for terminal differentiation, such as MyoD in the

muscle development, and CBFAl in osteogenic differentiation (41, 117).

The pocket domain family

RB, p107 and p130 comprise the “pocket” domain family of proteins. These proteins are
conserved in both sequence and structure (Fig. 1-1). The “pocket” family proteins are
composed of a divergent N-terminal region, a conserved pocket region, and a C-terminal
region. The pocket region consists of two conserved domains, A and B, which are
separated by a variable spacer region (26, 84, 98). The pocket region is more highly
conserved among pocket family members than the N- or C-terminal regions. There is
about 50% amino acid identity between p107 and p130 pocket regions, which is higher
than the 30-35% identity ofp107 and p130 with RB (78, 129, 138). Both p107 and p130
contain a conserved sequence in the spacer region that is not found in RB, and this
sequence is required for strong binding by cyclin A/CDKZ and cyclin E/CDKZ
complexes (42, 77). The pocket domain is critical for most functions of RB family
members and contains multiple binding sites for target proteins. E2F factors bind to the
cleft formed by A and B domain of RB, whereas the LXCXE motif in other proteins,
such as the adenovirus EIA and HPV E7 antigen binds to the B domain. The A domain is
also required for high affinity binding of these proteins to the B domain (74, 131),

suggesting the integrity of pocket domain is important for most RB functions.

RB family members have both common and specialized functions during cell growth. All
RB family members can be inactivated by viral oncoproteins, such as the adenovirus
ElA, SV40 large T antigen and HPV E7 antigen (92). RB, p107 and p130 all serve as
negative regulators of cell proliferation (92), as independent overexpression of each

member in the Saos-2 human osteosarcoma cell line consistently inhibits cell

Fig. 1-1. Schematic drawings of RB, p107 and p130. The amino acid positions of the

pocket domains are indicated.

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proliferation and causes G1 phase arrest (15, 138). However, inhibition of cell
proliferation is cell line specific. Proliferation of C33A human cervical carcinoma cells is
inhibited by over expression of pl 07 and p130, but not by RB (138). In contrast, growth
of T98G human glioblastoma cells is inhibited by pl 30, but not by RB and p107 (69).
Whereas a high frequency of mutations in the RB gene has been observed in tumors,
p130 gene mutations was rarely found. Until now, mutations in the p130 gene have been
detected only in lymphomas, lung cancer and nasopharyngeal carcinoma (1 l, 16, 99). To

date, p107 gene mutations in tumors have not been reported (99).

RB, p107 and p130 all can bind E2F activator proteins and repress E2F-dependent
transcription (99). However, each member of the RB family selectively interacts with
distinct members of the E2F family. RB binds to E2F-1, E2F-2, E2F-3 and E2F-4,
whereas p107 and p130 exclusively bind to E2F-4 and E2F-5 (13). E2F-6 and E2F-7
have not been observed to interact with any RB family members (2). E2F-4 and E2F-5,
but not E2F-(1-3), are rich in quiescent cells and the p130/EZF-4 complex accumulates
in differentiated cells, suggesting that p130 is a co-repressor of E2F-4/-5 in G0 cells (58,
113). Furthermore, the expression pattern of RB family members during the cell cycle is
different. RB expression levels are steady during each phase of the cell cycle (22). In
contrast, p130 exhibits maximal expression during the G0 phase of quiescent or
differentiated cells (72, 113), whereas p107 expression during the G0 phase is hardly
detectable, but its expression levels dramatically increase when cells are stimulated to
enter S-phase (6). This expression pattern correlates somewhat with their binding

activities to E2F family members. RB binds to E2Fs in both quiescent cells and GI phase

 

of cycling cells, p130 forms complexes with E2F-4/-5 in quiescent cells, and p107 binds

to E2Fs mainly in the S-phase of cycling cells (137).

The redundant and specialized functions of RB family members were also displayed in
mice whose RB family members were knocked out either alone or in combination. As
mentioned, RB'l' knock out mice were non-viable coupled with the pronounced defects in
the erythroid, nervous system, lens and skeletal muscle development (12, 59, 60, 73,
134), whereas deleterious effects brought by pl 07 and p130 knock-out are strain
dependent. Mice with C57BU6J genetic backgrounds having either p107'/' or p130"
knock out are viable, fertile, and show no overt phenotype (17, 75). However, in the
BALB/cJ genetic background, p107" ' mice display impaired growth, diathetic
myeloproliferative disorder, and short Gl phase (70). p130”‘ mice show growth arrest
and early embryonic death (71). RB"'/p107'/' and RB” '/p130'/ ' double knock out mice
have similar phenotypes as the RB”' mice with similar genetic backgrounds, but die 2
days earlier during embryogenesis (79). Compared with RBH' mice, both RB+/'/p107"’
and RB+"/pl30"' chimera mice have a broader spectrum of tumors, indicating that RB

family members may cooperate to suppress tumor formation in different tissues (20).

The molecular mechanisms of transcriptional repression by RB, p107, and p130
Among the 100 or more identified RB binding partners, the majority are transcription
factors, including components of the basal transcriptional machinery, chromatin
modifiers, and chromatin remodeling factors (91). The function of these factors for RB

activity remains, in most case, unproven, In contrast, the mechanism of repression by RB

 

family members has been mostly characterized in the E2F dependent RNA polymerase

(Pol) H transcription.

Multiple mechanisms for E2F repression by RB have been described. First, RB binds to
the E2F activation domain and directly blocks its transcriptional activity (30, 44, 49).
Second, RB is recruited by E2F family members and then prevents the formation of
preinitiation complex (PIC) at the core promoter region (105). Third, RB can actively
modify the chromatin structure of the promoter region to repress transcription (14).
Nonetheless, in vivo, the mechanism for repression of E2Fs dependent transcription
remains unclear, and may depend on different promoter contexts. For example, if E2F is
the only transcription activator for a specific gene, it may be enough to negatively block
the E2F activation domain, whereas, if there are some other transcription factors available
besides E2F, blocking E2F may not be enough to repress transcription. Thus, some active
repression mechanism by RB may be required. Each of these mechanisms is discussed in

more detail in the below.

i. RB repression via direct inhibition of the EZF activation domain. The sites within E2F-
1 for RB binding were mapped to the acidic activation region of E2F-l (30). It was
further elucidated by crystal structure analysis that the E2F-1 acidic region binds to the
cleft formed by A and B domains of RB (131). The RB-E2F complex remains able to
bind to E2F binding sites with target promoter (49), Additional evidence suggests that the
interaction between E2F and RB is crucial for RB to repress E2Fs dependent

transcription. The interaction between RB and E2F-1 coincides with the repression of

E2F-1 dependent genes (49). Point mutations in E2F-1 that abrogate binding with RB,

also make E2F-1 dependent transcription unresponsive to RB repression (30).

ii. RB repression via prevention of preinitiation complex (PIC) formation. When RB is
tethered to GAL4, Spl promoted transcription of reporter genes containing upstream
UAS sequences is repressed (105), which suggests RB can also actively repress
transcription via other mechanism than the E2F activation domain occlusion. Thus, the
consequence of RB interference with E2F may be the disruption of pre-initiation complex
(PIC) formation. In a minimal in vitro reconstituted transcription system, RB recruited by
E2F prevents the formation of TFIID-A complex, which is an important step for Pol H
recruitment (105). However, if RB was added after the formation of TFIID-A complex,
E2F dependent transcription becomes resistant to repression (105), suggesting prevention

of pre-initiation complex (PIC) formation is an important step to repress transcription.

iii. RB repression via modification of chromatin structure. The effects on transcription
brought by posttranslational histone modification and chromatin remodeling have been
widely determined (61, 94). The combination of histone H4 K8 acetylation, H3 K14
acetylation, and H3 S10 phosphorylation is often correlated with transcription (61, 101).
Acetylation and phophorylation is presumed to neutralize the positive charge of the
lysines on histone tails and then loosen the tight binding between the histones and DNA.
Loosening DNA from chromatin will ease the recruitment of transcription factors and
RNA polymerase (61, 101). Conversely, the lack of H3 and H4 acetylation and tri-

methylation of H3 K9 is connected to transcriptional repression (61, 101). Levels

10

modified histones are controlled by a set of histone modifiers that are functionally
antagonistic to each other, such as histone acetyltransferases (HATS) and histone
deacetylases (HDACs), and methyltransferases (HMTS) and potential histone
demethylases (61 , 101 , 111). Selective recruitment of those histone modifiers can change

local histone modification status.

RB was found to interact with HDACl-3 of the 7 known HDACs (81, 91). p107 and
p130 are also able to recruit HDACs to repress E2F dependent transcription (28).
HDACl and HDAC 2 have a conserved RB binding motif, LXCXE (91), which binds to
a different site on RB than E2F does. Thus, RB may construct an E2F-RB-HDAC
repressing complex on E2F dependent genes. RB recruited HDACs are able to reduce the
acetylation level of histones at the promoter region on some cell cycle related genes, such
as cyclin B, topoisomerase Ila, and thymidylate synthase. The HDACs activity inhibitor,
trichostatin A (TSA) reverses histone acetylation levels and compromises RB repression
on some genes (112). RB interacts with HDACs in a cell cycle dependent manner.
Phosphorylation of RB by cyclin D/cdk4 abrogates HDACs binding (29, 135). However,
not all genes, such as Cyclin A, are repressed by RB recruited HDACs, (112, 135). Those
genes not influenced by HDACs, may require SWI/SNF or other factors to help
repression (see below). It seems that RB family member mediated deacetylation is a

fundamental step during transcription of some target genes.

Another behavior coupled to RB induced repression is the induced methylation of

nucleosomes on promoters containing E2F binding sites (96, 120). Imunoprecipitated RB

11

 

'F7 in

is able to catalyze histone H3-K9 methylation via 3 RB associated histone methyl-
transferase (HMT) called SUV39H] (96). Overexpression of both RB and SUV39H1
enhances cyclin B repression, whereas SUV39H l/SUV39H2 double knock out mice
show increased cyclin B expression (96). Analysis of the H3-K9 methylation levels by
Chromatin irnmunoprecipitation (Chip) assay revealed detectable methylation of histones
at the cyclin B promoter in RB ”W cells but not in RB" cells (96). RB also interacts with
Heterochromatin protein (HPl), which specifically binds to methylated histone H3-K9.
Correspondingly, HPl on the Cyclin E promoter is only detectable in RB W cells (96).
RB may change histone modifications in a stepwise manner, histone deacetylation
followed by histone methylation. Actively recruited HMTS by RB may be required for
the permanent repression of some genes in differentiated cells (34). p107 and p130 also
physically interact with the SUV39H1, suggesting that a similar mechanism may be used

for gene repression by these other RB-related factors (95).

Chromatin remodeling is another important component of RB-mediated transcription
regulation. ATP-dependent remodeling complexes, such as SWI/SNF complex can
change the nucleosome position on DNA templates. The outcome fi'om changes of
nucleosome position is either the facilitation or prevention of promoter access by
transcription factors and RNA polymerase (94). Yeast having SWIZ/SNFZ knock-out,
changes gene expression both positively and negatively, suggesting that SWI/SNF is
involved in both activation and repression of different target genes (54).

RB, p107 and p130 can associate with histone modifiers and chromatin remodeling

factors, whose recruitment contribute to repression (14). RB induced repression can be

12

contributed by RB recruited Brgl and Brm, the two largest subunits of human SWI/SNF
complexes (24, 118). Both Brgl and Brm associate with RB, and this association is cell
cycle dependent. Brgl and Brm dissociate from hyperphosporylated RB (24, 118). In the
Brgl and Brm deficient SW13 cell line, accumulated hypophosphorylated RB is not
enough to cause cell cycle arrest (135), whereas, co-expression of either Brgl or Brm
with RB enhances repression and cell cycle arrest (135) . Whether Brgl and Brm directly
bind to RB or not have not yet been determined. RB may form either RB-HDAC-
hSWI/SNF or RB-hSWI/SNF complexes on E2F binding sites to execute differential

repression of cell-cycle regulated genes (135).

Generally, RB can repress E2F dependent repression with different mechanisms,

depending on promoter context or cell situation.

Pol III transcription

In eukaryotes, nuclear genes are transcribed by three different but highly related RNA
polymeraseses (Pol), I, II and 111. Each RNA polymerase selectively transcribes distinct
sets of genes (107). P01 I transcribes the large, tandernly repeated, 288, 188 and 5.8S
ribosomal (r) RNA genes, whose products are essential for ribosome structure and
enzymatic activity. Pol H transcribes the protein coding genes (mRNA genes) as well as a
subset of small nuclear (sn) RNA genes. Pol III transcribes a set of genes whose main
common features are that they encode structural or catalytic RNAs such as 5S rRNA,
tRNA, U6 snRNA, and 7SK snRNA (37, 107). Herein, most of the discussion will focus

on transcription performed by Pol III. So far 16 subunits have been characterized in

13

active Pol III that supports in vitro Pol III transcription (57). Some subunits are the
homologues to the subunits of P01 1 and H, such as RFC] (155 KD) and RPC2 (128KD),
the two largest subunits of Pol III, while other subunits are commonly shared between the
Pol I, II and 111, such as RPABCl, 2 and 3 (37, 57). Remaining subunits are dedicated to

Pol III (57).

Promoter structures of Pol III transcribed genes.
Genes transcribed by Pol III fall into four classes (Fig. 1-2) according to promoter

architecture (37, 121).

The promoters of SS rRNA genes (classl) have a conserved A box, C box and an
intermediate elements (IE) (Fig. 1-2) (102, 103). Those elements constitute the internal
control region (ICR) that is required for transcription (103). Xenopus laevis 5S rRNA, the
best-characterized example, has an A box at +50 to +64, an IE at +67 to +72 and a C box
at +80 to +97 DNA sequences (103). Changing the spacing between IE and C box
negatively impacts the transcription efficiency (103). Additional upstream DNA

sequences may also have some effects on 58 rRNA gene transcription efficiency (104).

The promoters of Ad VA] and tRNA (class 2) contain a conserved A box and B box

down stream of the transcriptional start site (Fig. 1-2) (36, 38, 53). Generally, the A box
is proximately located at +8 to +19 bp, while the B box is distally located at +52 to +62
bp (38). The DNA sequences of A and B boxes of tRNA genes partly correspond to the

tRNA D-loop and T-loop and thus the promoter structures are also intimately linked to

14

 

Fig. 1-2. Promoter architectures for different classes of RNA polymerase III
transcribed genes. Class 1(58 rRN A) genes have an internal promoter containing the A
and C boxes plus the IE element. Class 2 (tRNA) promoters have internal A and B boxes.
Class 3 (U6 snRNA) genes have a DSE, PSE, and TATA box for an external promoter.
Class 4 (Vault) genes have combined external and internal promoter elements shared by

other 3 classes of Pol IH transcribed genes.

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the function of the tRNA, and consequently the A and B boxes are highly conserved in all
tRNA genes (3 8). The spacing between the A box and B box is variable and has no
obvious effects on promoter strength. However, in some species, such as yeast and
Drosophila, the upstream DNA sequences up to —50 may somewhat influence

transcription (3 8).

Mammalian U6 and 7 SK snRNA genes have typical class 3 promoters (Fig.1-2) (38, 48,
93). In contrast with other Pol III transcribed genes, all the class 3 promoter elements lie

upstream of transcribed sequences. The core class 3 promoters contain a TATA box and

 

W rum—Ir

proximal sequence element (PSE), which are centered around —27 bp and —56 bp
respectively (38). Besides the core promoter region, an enhancer site named the distal
sequence element (DSE) is present around -239 bp (48). The DSE can be recognized by
several transcription factors including Oct-land Staf (48). So far as known, the DSE and
PSE sites are present at all other U-rich snRNA genes (48). Those U-rich snRNA genes

that do not have TATA box are transcribed by Pol II (48).

Class 4 genes have combined external and internal promoter whose elements are found in
the other three classes (Fig. 1-2). A typical class 4 promoter structure exemplified by
Vault genes has an external PSE, DSE, and TATA box along with internal A and B boxes
(119, 121). As another example, the Epstein-Barr virus small RNA (EBER) gene has

both an external TATA box and internal A and B boxes (55).

17

Basal transcription machinery of RNA polymerase [[1

Depending upon the promoter architecture for each class of Pol HI transcribed genes,
specific basal transcription machinery is required (Fig. 1-3) (48, 100). Class 1 genes
require TFIHA, TFHIB and TFIIIC (100), whereas class 2 genes require only the TFIHB
and TFIHC complexes (100). The basal transcription machinery requirements for class 3
genes are different. Class 3 genes require a variant TFIIIB complex and the snRNA
activating proteins (SNAPc), which is commonly shared by other Pol H transcribed E
snRNA genes (48). So far, the components of basal transcription machinery for class 4

genes are not clear but likely components used for other classes are also utilized for these

 

genes. In the following sections, the components of basal transcription machinery will be _r

discussed in more detail.

TFHIA

5S rRNA gene transcription relies on the gene specific transcription factor TFIHA.
Xenopus laevis TFHIA (39KD) was the first eukaryotic transcription factor to be cloned
and was characterized as zinc finger protein that bound DNA specifically (31, 39, 86).
Xenopus laevis TFHIA possesses 9 zinc fingers that span all ICR regions of SS rRNA
genes (31). The first three N-terminal fingers recognize and strongly bind to the C box.
The last three C-terminal fingers interact with the A box and the middle three fingers (4-
6) loosely contact the intervening promoter region (31). TFHIA binding on the 5S rRNA

gene provides a platform for the productive recruitment of TFHIC.

TFIHC

l8

Fig. 1-3. Transcription factors required for transcription by RNA polymerase 111.
Each identified subunit of transcription factors is indicated. Class 1 (58 rRNA) genes
require Brfl -TFHIB, TFHIA and TFIIIC. Class 2 (tRNA) genes require Brfl -TFIHB and

TFIIIC. Class 3 (U6 snRNA) genes require SNAPc, Brfl-TFIIIB and Oct-l.

l9

Fig.1 -3

 

 

 

 

 

 

Class 1

5S RNA Brf1 -TFl|lB
Class 2

tRNA Brfl-TFIIIB

 

Class3 _" DSE ._.//
U6 snRNA

  
 

SNAPc Brf2-TFlllB

TFIHC is a basal transcription factor that has been widely characterized for its fimction
for SS rRNA (class 1) and tRNA (classZ) transcription (107). The primary function for
TFHIC is to recruit and position TFHIB (107). In S.cerevisiae, TFHIC is composed of six
subunits, 1138, r 131, r 95, r 91, r 60 and r 55 (107). Those 6 subunits form two globular
domains and flexible linker region (109). In S.cerevisiae, TFIHA recruits TFIHC to SS

rRNA gene promoter through direct interactions (5), whereas, TFIHC is recruited to

 

I...
tRNA promoters by directly binding to the promoter A and B block DNA sequences :
(107). In humans, the TFIHC complex can be chromatographically separated as TFHIC]
and TFHIC2 (133). Both TFIHCl and TFHIC2 are required for SS rRNA (class 1) and

tRNA (class 2) transcription (97, 133). The initial promoter recognition of human tRNAs L

is conducted by TFIHC2. TFIHC2 binds to B box of tRNAs promoter then further
recruits TFIHC] and T FHIB (97). TFIHCl may also help U6 and 7SK RNA transcription
(97). TFHIC2 is comprised of 5 Polypeptides of 220, 110, 102, 90 and 63 kD (107).
Besides helping to assemble the pre-initiation complex on Pol HI promters, TFHIC also
display intrinsic histone acetyltransferase activity, which may contribute to full gene
activity. The TFHIC] 10 and TFHIC9O subunits of TFIHC were shown to provide histone

acetyltransferase activity (56, 68).

TFHIB

TFHIB is commonly required for all class 1, 2 and 3 gene transcription. TFHIB recruits
Pol [H by direct contact (122). In S.cerevisiae, TFIHB was identified as a complex of
TBP, B’ (Brfl, 70KD) and B”(de1, 90KB) (63). TBP is required for most Pol 1H

transcription. Brf shares sequence and structure similarity with TFHB (18), a factor in the

21

Pol II basal transcription machinery, and is thus named as TFHB-related factor (1 8). In
contrast with the single TFIHB in S.cerevisiae (62), there are different hsTFIIIB variants
required for class 1, 2 and 3 gene transcription in humans (107). There are several forms
of human Brf, including Brfl , Ber, and Brf-V2 (a splice form of Brfl ), as well as human
de1, including de1 and the de1 alternative splice forms, del-V2 and del-V3
(128). TFHIB containing TBP, Brfl and de1 supports tRNA and 5S rRNA transcription It
(67, 87). However, class 3 gene transcriptions do not require Brfl but instead require I

Brf2 (108). Ber-v2 may be also required for class 3 genes during in vivo transcription

x—J I

(85). Recombinant TBP, Brf2 and de1 can reconstitute class 3 gene transcription using

 

a minimal in vitro system (57). The roles played by the del-V2 and del-V3 splice l.
forms remain to be investigated. TFIHB is recruited on Pol III promoters through

different ways. For class 1 and 2 TATA box less genes, TFHIB is recruited by direct

interaction with TFHIC (100). For class 3 genes, TFHIB can directly bind to the TATA

box (100). After DNA binding of T F IIIB promotes the DNA melting for formation of the

open complex (65).

SNAPc

The transcription of human U6 and 78k snRNA genes (class 3) specifically requires the
small nuclear RNA activating protein complex (SNAPc). SNAPc is also required for
other U-rich snRN A gene transcription by RNA polymerase H. The requirement of
SNAPc is due to the external PSE elements on snRN A promoters. SNAPc, also named
proximal element transcription factor (PTF), is composed of at least 5 identified subunits:

SNAP190 (130), SNAPSO/PTFB (3, 45), SNAP45/PTF5 (106, 132), SNAP43/PTF7 (47,

22

132) and SNAP19 (46). Immunodepletion of SNAPc from cell extract blocks U1 and U6
snRN A transcription (46). SNAPc specifically binds to PSE DNA sequences; and its
binding activity on DNA is increased with cooperation fiom TBP (88). In an in vitro
transcription system, mini-SNAPc comprised of recombinant SNAP190 (1-505),
SNAPSO and SNAP43 supports U6 (89) and U1 (L. Gu, personal communication)

snRN A transcription. SNAPc cooperates with TFHIB to recruit Pol III on U6 promoters
(88). On a chromatinized template, the direct interaction between Oct-1 on the DSE and

SNAP190 on the PSE helps SNAPc to settle on the PSE (136).

The P0111] transcription cycle

Once the preinitiation complex is formed, it takes about 5 minutes at 22 °C for yeast
tRNA to complete initiation process, including Pol HI recruitment, DNA helix melting
and initial RNA synthesis (23). P01 HI with the help of TFIHB melts DNA around start
site more than l4bp length, but it is sensitive to temperature (64). After initiation, Pol III
starts elongation without an obvious pause and elongates RNA at an average rate of ~20
nt/s at 20 °C in yeast (83). During elongation, the preinitiation complex does not
dissociate from the promoter region immediately, and is used to re-initiate several rounds
of transcription (23). This reinitiation process, as well as Pol III recycling has been well
established in yeast tRNA and U6 snRNA transcription (27). Transcriptional reinitiation
strongly enhances the transcription rate by cutting out the relatively long time spent on
initiation (23). The termination of Pol III transcription relies on a cluster of T residues

(four or more) next to the transcribed region (7).

23

Regulation of Pol III transcription by RB family members

Abnormal hyperactivity of Pol III was observed in mice with myelomas about three
decades ago (110). Those findings prompted investigation into the mechanism for normal
Pol III activity in normal cells. Although the connection between the abnormal Pol [II
activity and cancer is yet to be investigated, the mechanisms for Pol III regulation have
advanced significantly since the 19908 (126), and recently RB, p107 and p130 have been
closely linked to the regulation of Pol 1H activity (1 16, 126). Global Pol HI repression by
RB, p107 and p130 was observed both in vivo and in vitro (51, 52, 116, 126). Transient

overexpression of either p107 or p130 represses Pol IH transcription in vivo (116), and in

 

lfli ' I'm—‘1.om _

vitro, recombinant p107 and p130 repress Ad VAl , tRNA and 5S rRNA transcription

(1 16). In RB'I’ mice primary fibroblasts, Pol III activity, as detected by nuclear run on
assay was elevated, whereas general Pol II activity was not affected (126). As with p107
and p130, recombinant RB represses Pol III in vitro transcription (51, 52, 126), whereas a
natural occuring mutant RB found in cancer does not repress Pol III transcription (126).
Pol III activity varies during cell cycle and has maximal activity at the S and G2 phases
with minimal activity at GO, early G1, and M phases (125). This variation in Pol III
activity is consistent with the activity of RB. RB loses its repression activity starting

during middle G1 phase (123).

For Pol HI transcription, different molecular mechanisms for RB repression have been
proposed, according to the different features of each class of Pol III transcribed gene. RB
was found to interact with both TFIIIB and TFHIC2 (10), two factors required for class 1

and 2 gene transcription. The interaction between RB and TFHIB disrupt the contacts

24

between TFIIIB and other components of basal transcription machinery as well as Pol III,
and further prevents the recruitment of Pol III (115). As for U6 snRNA (class 3) gene

repression, RB was found to interact with SNAPc, however, RB did not abrogate the Pol
III occupation on U6 promoter (51, 52). Those findings suggest RB may repress different

class of Pol 1H genes by different mechanisms.

Summary

_ I u ‘. VD'Y‘IJ'.I.1
1.

RB family members can repress P01 1, Pol II, and Pol HI transcription of genes that play

essential roles in cell cycle control, cell growth, differentiation and apoptosis. Pol III

 

Ir

activity is presumed to regulate cell growth potential, and it is both academically and
practically significant to know the molecular mechanism for how RB family members
repress Pol III transcription. The following chapter will focus on discussing the

repression mechanism of U6 snRNA transcription by RB, p107, and p130.

25

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37

CHAPTER 2

REGULATION OF HUMAN U6 snRNA TRANSCRIPTION BY

RETINOBLASTOMA FAMILY MEMBERS

38

Abstract

The Retinoblastoma tumor suppressor (RB) represses U6 snRNA gene transcription by
RNA polymerase HI (Pol HI). Whether the other two RB related family members p107
and p130 regulate U6 snRN A transcription is unknown. My research shows that p107 and
p130 repress U6 snRNA transcription at levels comparable to RB. All RB family

members directly interact with multiple components of two U6 snRN A transcription

 

”-
complexes, SNAPc and TFIIIB, which are required for U6 snRNA transcription. The U6
snRNA specific TFHIB is composed of three proteins, TBP, Brf-2 and de1.
Interestingly, del selectively interacts with RB only. An additional association between
RB family members and P01 IH was observed and the A domain of RB is sufficient for (L

RB binding to P01 HI, which suggests the mechanism for U6 snRN A repression by RB
family members could involve direct interactions with the polymerase. Combined with
the previous finding that RB and P01 IH co-occupy the U6 snRNA promoter during
repression, a tether and immobilization model is proposed to explain the mechanism for

U6 snRNA transcriptional repression by RB family members.

39

Introduction

RB family members (RB, p107 and p130) play essential roles in controlling cell growth,
proliferation, cell-cycle progression, differentiation and apoptosis (5, 16, 17, 22). Cell
cycle control by RB family members is closely connected to their role in regulating the
transcription of genes containing E2F binding sites, whose products control the cell cycle
transition (1, 31). There are multiple mechanisms adopted by RB to repress E2F
dependent Pol II transcription. First, RB blocks E2F transcriptional activity through
direct binding to its activation domain (8, 10, 12). Second, RB prevents the formation of
the preinitiation complex (PIC) at target gene promoters (21). Third, RB represses Pol H

transcription by modifying the chromatin structure (6).

In addition to the regulation of E2F-dependent Pol H transcription, RB family members
also regulate the transcription by Pol I and P01 1H (3, 4, 13, 32). All RB family members
repress Pol HI activity both in vivo and in vitro (2, 13, 35). P01 IH activity as detected by
nuclear run on assay is elevated in RB'/' mice primary fibrolasts (35). Furthermore, Pol
HI activity reflected by 5S RNA, tRN A, and Ad VAl transcription is also down-
regulated in cells transiently expressing p107 or p130 (27). Recombinant RB represses in
vitro transcription of all classes of Pol 111 genes (13, 19), whereas recombinant p107 and
p130 were shown to repress the in vitro transcription of Pol IH transcribed SS RNA,
tRNA and Ad VAl gene (27). That p107 and p130 also regulate U6 snRNA transcription
has not been demonstrated. The significance of Pol I and Pol IH repression by RB is that
the P01 1 and P01 1H products including rRNA, tRNA and U6 snRNA are closely related

to protein synthesis and cell growth (33).

4O

One major goal of our laboratory is to understand the mechanism of how RB family
members repress Pol HI transcription using U6 snRNA transcription as a model. The
mechanisms elucidated on Pol H repression by RB are not able to explain Pol 1H
repression by RB. One reason is that Pol HI transcription requires different transcription

factors than E2F (9).

Based on promoter architecture, genes transcribed by RNA Pol [H can be divided into
four classes, class 1, 2, 3 and 4 (Fig. 1-2), represented by the SS RNA, tRNA, U6 snRNA
and Vault genes, respectively (9, 11, 30). Except for class 4 genes, the transcription
factors required for the other 3 classes genes are well identified (Fig. l-3). The only
member of class 1, the SS rRNA gene, requires TFHIA, TFIHB and TFIHC. Class 2
genes (tRNA and Ad VAl) require TFHIB and TFIHC complexes, whereas class 3 genes
(U 6 snRNA) require SNAPc and TFHIB (20). The TFHIB required by class 1 and 2
genes contains TBP, del and Brfl, whereas the TFIIIB required by class 3 genes
contains TBP, del and Brf2 (23). Consistent with their differing transcription factor
requirements, different mechanisms for RB repression of Pol IH transcription have been
observed. For class 1 and 2 gene repression, RB targets TFIHB by directly binding to
Brfl or TBP to disrupt the interaction between TFIHB and TFIHC (26). For U6 snRNA
(class 3) gene repression, RB targets SNAPc via SNAP43 and SNAP50, and TFHIB via
TBP and del (13). Interestingly, RB and RNA polymerase IH co-occupy a repressed U6

snRNA promoter, suggesting that RB is not necessary to block preinitiation complex

41

formation at the U6 snRN A gene promoter (14). Instead, the interactions among RB,

SNAPc, and TFIIIB may be important for correctly targeting RB to the promoter.

The phenomenon of RB and RNA polymerase III co-occupying a repressing U6 snRNA
promoter (14) raises the question, by what mechanism does RB inactivate Pol HI on the
U6 promoter. Other questions that will be addressed are whether p107 and p130 also
repress U6 snRNA transcription and whether all RB family members share common

mechanisms and functions for U6 snRNA repression.

RB, p107 and p130 share a conserved pocket A/B domain (Fig. l-l), which is responsible
for most RB family member functions (28). In addition to overlapping fimctions in cell
proliferation control, RB, p107 and p130 maintain some unique features (6). For
example, RB binds to B2Fl-E2F4, but p107 and p130 bind to E2F4 and E2FS only (6).
The expression pattern of each RB family member during the cell cycle is also different.
Protein p130 is highly expressed in quiescent cells, and its expression level decreases
rapidly when G0 stage cells are stimulated to enter the cell cycle. In contrast, p107
expression level is very low during the G0 stage of the cell cycle, and its level rises
quickly when cells enter into S phase. RB expression levels are moderate and quite stable
in each phase of the cell cycle (6). p107 and p130 were also found to repress class 1 (5S
RNA) and class 2 (tRNA) gene Pol IH transcription by targeting TFIHB (27). Initial
chromatin irnmunoprecipitation (Chip) assays performed in our lab (unpublished data)
showed that p107 and p130 also occupy the U6 snRN A promoter, indicating that p107

and p130 may repress U6 snRNA transcription.

42

 

15F

The data presented here show that RB, p107 and p130 all repress U6 snRNA Pol HI
transcription in vitro. RB, p107 and p130 can interact with multiple subunits of SNAPc
and TFHIB including the SNAP190, SNAPSO and SNAP43 subunits of SNAPc, and the
Brfl and TBP subunits of TFHIB. Interestingly, deI was found to interact with RB
only, the significance of this difference for RB, p107 and p130 repression remains to be
investigated. Additionally, endogenous RB, p107 and p130 associate with Pol 1H and the
A domain of RB is responsible for the P01 IH association. Combined with the previous
finding that SNAPc and TFHIB bind to RB in a region other than the A domain (14), a
tether and immobilization model is proposed to explain how RB inactivates the P01 IH on

the U6 snRNA promoter during repression.

Materials and Methods

Subcloning of GST-p107 and GST-p130

Partial p107 and p130 sequences were subcloned correspondingly from pCMV-p107 and
pDEF3-pl30 (36), which contain full length cDNAs encoding p107 and p130. The PCR
primers used for p107 subcloning are 5’-
GACTAGTCTAGAATTGCTGTACTGTGTGAACTGC-3’ and S’-
GACGCGGATCCTTAATGATTTGCTCTTTCACTGAC-3’, which correspond to
amino acids fiom 249 to 1068 of p107. The PCR primers used for p130 subcloning are

5 ’-GACTAGTCTAGAGGTI‘CAGGAACAGAGACTGCTG-3 ’and S ’-
GACTAGCTAGCCCGTCGGGAGGTGACCAGTCG-3’, which correspond to amino

acids fi'om 372 to 1139 of p130. The PCR products p107 and p130 were individually

43

inserted into a pETl lc-based expression vectors with an in-frarne GST tag fused to the
N-ternrinus of p107 and p130 to make pGST-p107 (249-1068) and pGST-p130 (372-
1139). The DNA sequence of pGST-p107 (249-1068) and pGST-pl30 (372-1139) was

checked by sequencing.

Preparations of recombinant proteins

Recombinant GST-p107 (249-1068), GST-RB (379-928), and GST were expressed in
E.coli BL21 DE3. GST-p130 (372-1139) was expressed in E.coli BL21 DE3+ cultured in
the media with 37ug/mL of chloromphenical. Protein expression was induced by IPTG
(1mg/mL) for 18 hours at 16 °C. Bacteria were homogenized with Wheaton Dounce
Homogenizer (15 mL volume, 25 —30 strokes with B pestle) in HEMGT-lSO buffer (25
mM Hepes pH 7.9, 0.5 mM EDTA, 12.5 mM MgC12, 10% glycerol, 0.1% Tween-20, 150
mM KCl ) plus protease inhibitors (1 mM sodium bisulfate, 1 mM benzamidine, 1 uM
pepstatin A, 0.5 mM PMSF) and 1 mM DTT. Protein extracts were incubated with
glutathione sepharose 4B beads (Amersham Biosciences Inc.) for 4 hours at 4 0C. Beads
were washed with HEMGT-lSO buffer for 3 times and the bound GST-fusion proteins
were eluted with HEMGT-l 50 buffer containing 50 mM reduced glutathione. The eluted
GST fusion proteins were dialyzed against Dignam buffer D (20 mM HEPES, pH 7.9,
0.2 mM EDTA, 5 mM MgC12, 10% glycerol, 0.1% Tween-20, 100 mM KCl) using a 28-
Well Microdialysis System apparatus (GIBCOBRL Inc.). The GST fusion proteins were
concentrated to more than 100 ng/ul through YM-30 cenuicon devices (Millipore Inc. 1
Cat# 4208). The concentration, molecular weight, and purity of GST fusion proteins were

checked by SDS-PAGE and Coomassie blue staining.

In vitro transcription assay

To assay repression by RB family proteins, GST-RB family fusion proteins were added
to the following in vitro transcription systems of U6 snRNA, Adenovirus (Ad) VAl and
Ad major late (ML) as previously described (13, 14). For in vitro U6 snRNA
transcription, 250 ng of pU6/Hae/Ra.2 and 2 ul HeLa cell nuclear extract were used in a
20 ul reaction. For Ad VA] in vitro transcription, 250 ng of pBSM13+VA1 and 2 pl
HeLa cell nuclear extract were used in a 20 ul reaction. For Ad ML in vitro transcription,
250 ng Ml3-Ad ML and 4 ul HeLa cell nuclear extract were used in a 25 ul reaction. A11
in vitro transcription reactions were carried out at 30°C for 1 hour. The transcription
levels of Ad VAI and Ad ML were assessed by the amount of incorporated a32P-CT P,
and the U6 snRN A transcription levels were assessed by the amount of u32P-CTP
radiolabeled RNA probe protected by pU6/Hae/Ra.2 transcripts after being subjected to a
T1 RNAase protection assay. Transcripts were separated by denaturing 6% PAGE and

visualized by autoradiography.

Co-immunoprecipiation and Western blot assay

Co-irnmunoprecipitation assays were performed by incubating 600 pl of N2 HeLa cell
nuclear extract with 3 ug of goat IgG, anti-p107 (Santa Cruz, sc-318), anti-p130 (Santa
Cruz, sc-317), or anti-RB (Santa Cruz, M-lS) antibody at 4°C for overnight. Then, for
each sample, 1 mL of HEMGT-l 50 containing protease inhibitors and 1 mM DTT was
added together with 25 ul of Protein G agarose beads (Upstate Inc. Cat# 16-266) for

another 4 hour rotation at 4°C. The beads were washed with 1 mL of HEMGT-l 50 3

45

times, and the proteins bound to beads were eluted by boiling in Laemmli buffer. The
eluted proteins were separated by 7.5% SDS-PAGE followed by transfer to nitrocellulose
membrane using a serni-dry transfer method. Proteins on nitrocellulose membrane were
detected by Western blot analysis (14) using either anti-Pol 111 (largest subunit, RPCl)
(MI-170), anti-Pol II (8WG16), anti-SNAP43 (CS48), or anti-actin antibodies. In the
reciprocal co-immunoprecipitation assay, 600 pl of N2 HeLa cell nuclear extract was
incubated with 3 pg of rabbit preirnmune serum or rabbit Pol HI (largest subunit, RPCl)
(MI-170) anti-serum. Co-immunoprecipitated proteins were then detected by Western

analysis, using antibodies specific to RB (M-lS), TBP (SL2) and actin.

Co—mmuoprecipitation assay with the treatment of Ethidium Bromide

The normal structure of DNA in HeLa cell nuclear extract was disrupted by treating with
ethidium bromide (EtBr) (18). EtBr was added to HeLa cell nuclear extract to 100 pg/mL
final concentration before being used for co-irnmunoprecipitation assay. The HeLa cell
nuclear extract with EtBr was clarified by centrifugation at 13000 rpm (16,000 g) at 4°C
for 5 minutes. 600 p1 of clarified HeLa cell nuclear extract was incubated with 3 pg of
goat IgG, anti-p107, anti-p130, or anti-RB antibodies at 4°C overnight. Other conditions
were same as described above, except 100 pg/mL of EtBr was added to the HEMGT-ISO

buffer for the wash steps.

GST pull-down assay from HeLa cell nuclear extract

GST fusion proteins used in GST pull-down assays were prepared as described before

(14). GST pull-down assays were performed by incubating 300 pl of HeLa cell nuclear

46

extract with 1 pg of either recombinant GST, GST-RB (379-928), GST-RB containing
the A, B and C domains (379-870), GST-RB containing the A and B domains (379-772),
GST-RB containing the A domain (3 79-577), GST-RB containing the B and C domains
(645-870), GST-RB containing the B domain (645-772), GST-RB mutant with Aexon 22
(379-928), or GST-RB (379-928) with C706F mutation at 4°C for overnight.
Recombinant GST or GST fusion proteins together with associated proteins were
precipitated through binding to 25 pl of glutathione sepharose 4B beads (Amersham
Biosciences Inc.) by incubation at 4°C for 4 hours. After washing the beads 3 times (lmL
per wash) with HEMGT-lSO buffer containing protease inhibitors and 1 mM DTT, the
bound proteins were eluted by boiling in Laemmli buffer. The eluted proteins were
separated by 7.5% SDS-PAGE and then transferred to nitrocellulose membrane. The
recovered proteins on nitrocellulose membrane were detected by Western blot using
either anti-Pol HI (largest subunit, RPCl), anti-Brgl (H-88), anti-actin or anti-GST
antibody. The result of Western blot assay using anti-GST antibody indicated relative

amount of GST and GST fusion proteins recovered in the GST pull-down process.

GST pull-down assay with in vitro translated proteins

Each subunit of SNAPc, TFHIB and Oct-1 was individually transcribed and translated in
TNT T7 coupled reticulocyte lysate system (Promega Cat. # L4610). During in vitro
translation, 35S-methione was incorporated into proteins for labeling. Between 3 to 20 pl
of crude 3SS-labelled protein (depending on the protein concentration) were incubated
with 1 pg of GST, GST-p107, GST-p130 or GST-RB in HEMGT-ISO buffer at 4°C for 2

hours. Then 1 mL of HEMGT-l 50 buffer containing protease inhibitors and 1 mM DTT

47

was added together with 20 pl of glutathione sepharose 4B beads to each sample for
another 2 hours. The beads were washed with 1 mL HEMGT-ISO buffer for 3 times.
Bound proteins were eluted fi'om beads by boiling in Laemmli buffer. Eluted proteins
were separated by 12.5% SDS-PAGE. The recovered GST fusion proteins in each sample
were monitored through Coomassie blue staining, and the 3’SS-labelled proteins in SDS-

PAGE were detected by autoradiography.

Results

All RB family members repress U6 snRN A transcription in vitro

To test whether p107 and p130 are able to repress U6 snRNA (class 3) transcription, in
vitro repression assays were performed with recombinant GST fusion RB family proteins.
The GST-RB (3 79-928) used here contains the NB pocket domain and the C-terminal
region, which is competent for repression on all three classes of Pol HI transcribed genes
(14). GST-p107 (249-1068) and GST-p130 (372-1139) used here both contain the A/B
pocket domain and C-terminal region (Fig. 2-1 A), which are active for in vitro
repression on SS RNA (class 1) and Ad VAl (class 2) transcription (27). First, the size
and purity of GST fusion proteins were checked by SDS-PAGE and Coomassie blue
staining. As shown in Fig. 2-1 B, the size of each recombinant protein, compared to
protein size standards, matched the expected molecular weight. Smaller molecular weight
proteins may be truncated forms of GST-p107 (249-1068) and GST-p130 (372-1139), or
other miscellaneous proteins from E.coli. The GST-p107 (249-1068) and GST-p130

(3 72-1 139) were not previously expressed in our laboratory, and therefore, the expression

of the full-length proteins was further confirmed by Western blot analysis. As shown in

48

Fig. 2-1. All RB family members are able to repress human U6 snRNA transcription
in vitro. (A) Schematic representations of GST-p107 and GST-p130. (B) Analysis of
recombinant GST-p107 (249-1068) (lane 2), GST-p130 (372-1139) (lane 3), GST-RB

(3 79-928) (lane 4) and GST (lane 5) proteins by SDS-PAGE and Coomassie blue
staining. Protein molecular weight marker is shown in lane 1. (C) GST- p107 (249-1068)
and GST-p130 (372-1139) are specifically recognized by p107 and p130 antibodies.
Western blot analysis was performed using p107 (top gel) and p130 antibodies (bottom
gel). Different amounts of recombinant GST-p107 (249-1068) or GST-p130 (372-1139)
were loaded (lanes 2-5). GST-RB (379-928) (lanel) was used as a negative control. (D)
GST-RB (379-928), GST-p107 (249-1068) and GST-p130 (372-1139) repress the P01 HI
transcription of Ad VA] and U6 snRNA genes, but not Pol II transcription of Ad ML
gene. Approximately, 0, 200, 400, 800 ng of GST-RB (379-928) (lanes 1 to 4), GST-
p107 (249-1068) (lanes 5 to 8), or GST (lanes 13 to 16) were added to in vitro
transcription assays using HeLa cell nuclear extracts. Lanes 9-12 contain 0, 100, 200, 400

ng of GST-pl 30 (372-1139). Transcripts were processed as described previously (l3, 14).

49

Fig. 2-1
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50

Fig. 2-1 C, GST-p107 (249-1068) and GST-p130 (372-1139) were recognized by anti-
p107 and anti-p130 antibody, respectively, but GST-RB was not recognized by any one
of those antibodies, indicating GST-p107 (249-1068) and GST-p130 (372-1139) were
successfully cloned and expressed. Antibodies against p107 and p130 were designed

upon the non-conserved C-tenninal region, and do not to cross-react with each other.

Next, these proteins were tested for their ability to repress Ad VAl , U6 snRN A, and Ad
ML transcription. The in vitro repression assay of Ad VAl transcription served as a
positive control for repression activity of the GST fusion proteins, whereas the repression
assay on Ad ML transcription served as negative control to indicate that the repression by
the GST-fusion proteins was gene specific. As shown in Fig. 2-1 D, both Ad VAI and U6
snRNA gene transcription decreased as increasing amounts of GST-RB (lanes 1 to 4),
GST-p107 (lanes 5 to 8) and GST-p130 (lanes 9 to 12) were added in each repression
assay. Ad VAl and U6 snRNA gene transcription were barely detectable when 800 ng of
GST-RB (lane 4) or 400 ng of GST-pl 30 (lane 12) were included in the reactions,
although transcription was still detectable when 800 ng of GST-p107 (lane 8) was present
in the reaction. As controls, the transcription levels of Ad VAl (top gel, lanes 13-16) and
U6 snRN A (middle gel, lanes 13 to 16) were not affected by addition of an equivalent
mass of GST to the reactions. This analysis suggests that GST-p107, GST-p130 and
GST-RB can repress Ad VAl and U6 snRN A gene transcription, and the repression
activity is from RB family proteins. As a control of gene specific repression, the Ad ML
transcription levels (bottom gel, lanes 1 to 4, S to 8, 9 to 12) were not influenced by

increasing amounts of GST-RB (lanes 1 to 4), GST-p107 (lanes 5 to 8), GST-p130 (lanes

51

9 to 12), or GST (lanes 13 to 16), suggesting the repression by GST fusion proteins tested
here was also gene specific. The in vitro repressions of Ad VAl by RB family members
and the repression of U6 snRN A by RB found here are consistent with previous findings
(13, 14). Furthermore, this is the first example of U6 snRNA gene repression by p107 and

p130 in vitro.

Endogenous RB family members associate with multiple subunits of RNA
Polymerase III, SNAPc and TFIIIB, and associations are not DNA dependent

One typical character for RB family protein mediated repression is the stable and direct
binding to some RNA polymerase accessory factors, such as E2F (31). This direct and
stable binding character for RB family proteins also were found in RB family protein
induced Pol III repression (26, 27). Previous findings showed that endogenous RB family
proteins associate with TFIHB (26, 27), and endogenous RB was also found to associate
with SNAPc (13). However whether endogenous p107 and p130 associate with SNAPc
was not investigated. Another interesting phenomenon is that RB and P01 IH co-occupy
the repressed U6 snRN A promoter (14), which suggests that Pol HI may be retained at

the U6 snRNA promoter through some RB mediated mechanism.

To test whether endogenous RB family proteins could bind to Pol III, and whether
endogenous p107 and p130 also associate with SNAPc and TFHIB, a co-
irnmunoprecipitation assay was performed followed by Western blot analysis. HeLa cell
nuclear extract was incubated with either goat IgG, anti-p107, anti-p130, or anti-RB

antibodies for co-immunoprecipitation followed by Western blot analysis using either

52

anti-Pol HI (largest subunit, RPCl), anti-Pol H (largest subunit, RPBl), anti-SNAP43, or
anti-actin antibodies. As shown in Fig. 2-2 A, significant levels of Pol HI(RPC1) and
SNAP43 were detected in the samples irnmunoprecipitated with the anti-p107 (lane 4),
anti-p130 (lane 5) and anti-RB (lane 6) antibodies but not with IgG (lane 3). However,
Pol II was not detectable under the conditions used here for irnmunoprecipitation and
Western blot analysis. As a non-specific co-irnmunoprecipitatin control, actin was not
detected in any of those co-irnmunoprecipitation samples. Thus, all endogenous RB

family members specifically associate with Pol IH (RPCl) and SNAP43.

To further confirm the association between RB and P01 HI (RPCl), a reciprocal co-
immunoprecipitation assay and Western blot analysis was performed. HeLa cell nuclear
extract was incubated with either preirnmune or anti-Pol HI (RPCl) serum for co-
irnmunoprecipitation, followed by Western blot analysis using either anti-RB, anti-TBP
or anti-actin antibodies. Detection of TBP was performed to determine whether TFHIB
mediates the association between RB and P01 HI. As shown in Fig. 2-2 B, a significant
level of endogenous RB was detected in the sample irnmunoprecipitated with anti-Pol HI
serum (lane 4) but not with the preirnmune serum (lane 3). Neither TBP nor actin was
detected in a significant level in any of the irnmunoprecipitated samples (lanes 3 and 4).
The results shown by Fig. 2-2 B, not only confirm that endogenous RB associates with
Pol IH (RPCl), but also suggest that the association between RB family members and P01
[H were unlikely mediated by TFHIB complex, even though RB was found to interact

with TFHIB complex in previous research (26). p107 and p130 seemed also co-

53

Fig. 2-2. Endogenous RB family proteins associate with SNAP 43, TBP and Pol III.
(A) Endogenous Pol III, SNAP43, but not Pol II and actin were co-immunoprecipitated
with p107, p130 and RB. 600 pl of HeLa cell nuclear extract was incubated with 3 pg of
p107 (lane 4), p130 (lane 5) or RB (lane 6) antibodies as well as with IgG antibody (lane
3). Protein-antibody complexes were precipitated by affinity purification using the
protein G agarose beads (Upstate Inc.). The bound proteins were resolved by 7.5% SDS-
PAGE, and the association of Pol HI, Pol H, SNAP43 and actin with RB family proteins
was detected by Western blot analysis. Lanes 1 and 2 show the relative amount of Pol 1H,
Pol II, SNAP43 and actin present in 12 and 3 pl of nuclear extract. (B) Endogenous RB
but not TBP or actin co-immunoprecipitated with Pol H1. 600 pl of HeLa cell nuclear
extract was incubated with rabbit preimmune (lane 3) and Po] HI (largest subunit, RPCl)
anti-serum (lane 4) antibodies. Subsequently, RB, TBP and actin antibodies were used for
Western blotting analysis. Lanes 1 and 2 show the relative amount of RB, TBP and Actin
present in the 30 and 12p] of HeLa cell nuclear extract. (C) The association of RB family
proteins with Pol HI(RPC1), SNAP43 and TBP is not DNA dependent. 600 pl of HeLa
cell nuclear extract was immunoprecipitated with p107 (lanes 3 and 7), pl 30 (lanes 4 and
8) and RB (lanes 5 and 9) antibodies as well as goat IgG antibody (lanes 2 and 6)
respectively. The reactions were carried out with (lanes 6-9) or without (lanes 2- 5) 100
p g/mL EtBr. Precipitated proteins were resolved by 7.5% SDS-PAGE, and the
association of Pol III, SNAP43, TBP and actin with RB family proteins was assessed by
Western blot analysis using the appropriate antibodies as indicated. Lane 1 shows the
relative amount of Pol HI (RPCl), SNAP43, TBP and actin present in 12 pl of HeLa cell

nuclear extract.

54

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irnmunoprecipitated with Pol HI (RPCl) (data not shown), further suggesting that all RB

family proteins associate with Pol IH (RPC 1).

In order to ensure that the associations of RB family proteins and P01 IH/SNAP43/TBP
were not mediated by DNA, EtBr was added to the co-immunoprecipitation experiments
to disrupt the normal DNA structure that is required for protein binding (18, 25). Two
sets of co-irnmunoprecipitation assays were performed in parallel for comparison, one
that was treated with EtBr and the other set that was not treated. Except for the EtBr
treatment, all other operations of those two sets of co-immunoprecipitation assay were
the same. Irnmunoprecipitated proteins were analyzed by Western blot, using anti-Pol HI,
anti-SNAP43, anti-TBP, and anti-actin antibodies. As shown in Fig. 2-2 C, Pol IH

(RPC 1), SNAP43 and TBP were present in the irnmunoprecipitates of RB family proteins
(lanes 3,4 and 5), suggesting that RB farme proteins associate with Pol IH(RPC1),
SNAP43 and TBP. Furthermore, the co-immunoprecipitation levels of Pol 1H, SNAP43,
TBP are comparable between the samples treated with (lanes 6,7,8 and 9) or without
(lanes 2,3,4 and 5) EtBr, which indicates that the associations between RB family
proteins and Pol III (RPCl), SNAP43 and TBP are not likely affected by the DNA
present in the HeLa cell nuclear extract, at least in maintaining the association, though the

DNA disruption by EtBr need to be tested in further.

RB family members can interact directly with multiple components of SNAPc and

TFIIIB complexes

56

The results of previous co-irnmunoprecipitation assay showed that endogenous RB, p107
and pl 30 associate with SNAP43 and TBP, and the RB can interact directly with the
subunits SNAPSO and SNAP43 of SNAPc, TBP, Brfl and de1 subunits of TFIHB (13,
14). It is possible that p107 and p130 may associate with SNAPc or TFHIB by interacting
directly with subunit(s) belonging to SNAPc or TFHIB. To test the possibility that p107
and p130 can interact directly with SNAPc and TFHIB subunit(s), a GST-pull down
assay with purified recombinant GST-p107 (249-1068) and GST-p130 (372-1139) was
performed. The parallel GST pull-down assay with GST-RB (379-928) and GST were
used as a reference and negative control, respectively. Each subunit of SNAPc, TFHIB
and Oct-l was individually expressed in rabbit reticulocyte lysate system and labeled
with 3SS-methione. Approximately, 1 pg of GST-p107 (249-1068), GST-p130 (372-
1139), GST-RB (379-928), and GST was incubated with equivalent amount of each in
vitro translated 35 S labeled protein. The levels of 3SS-labeled proteins recovered by GST
fusion proteins were detected by autoradiography. As shown in Fig. 2-3 A, a significant
amount of SNAP190 (1-505), SNAPSO and SNAP43 but neither SNAP45 nor SNAP19
of SNAPc were detected in samples treated with GST-p107 (249-1068) (lane 3), GST-
p130 (372-1139) (lane 4) and GST-RB (379-928) (lane 5). For the TFIHB complex, Brfl
and TBP were detected in all pull-downs with the GST tagged RB family proteins (lanes
3 to 5). Interestingly, another subunit of TFHIB, del , was only present in the GST-RB
(379-928) pull-down (lane 5). Neither Brf2, exists only in the TFHIB used by U6 snRNA
transcription, nor the U6 snRNA DSE binding protein, Oct-l, were detected in any pull
down reactions (lanes 3 to 5). No association was detected with GST by any 35 S labeled

proteins, suggesting that those subunits detected in the pull-down with the GST tagged

57

Fig. 2-3. All RB family proteins interact with multiple components of SNAPc and
TFIIIB. (A) GST pull-down experiments were performed to test the interaction between
RB family proteins and individual subunits of SNAPc and TFHIB. Each SNAPc and
TFIIIB subunit was expressed in vitro using rabbit reticulocyte lysates, and proteins were
labeled with [3 5 S] methionine. The identities of SNAPc and TFHIB are indicated. Lane 1
contains 10% of 35S-labeled proteins used as an input. Interactions for [3 5 S] methionine
labeled proteins were tested with GST (lane 2), GST-p107 (249-1068) (lane 3), GST-
p130 (372-1139) (lane 4) and GST-RB (379-928) (lane 5). Proteins were separated by
SDS-PAGE and visualized by autoradiography. (B) Equivalent mass of GST proteins was
recovered by glutathione sepharose beads. 1 pg each of GST (lane 2), GST-p107 (249-
1068) (lane 3), GST-p130 (372-1139) (lane 4) as well as GST-RB (379-928) (lane 5)
were prebound to glutathione sepharose beads. The proteins on beads were resolved by
SDS-PAGE and visualized by Coomassie blue staining. Lane 1 contains protein

molecular weight marker.

58

Fig. 2-3

 

 

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' SNAP19

 

 

Bd1

 

Bdm Brf1-TFIIIB

 

TBP

 

Brf2-TFIIIB

 

m Brr2

 

 

 

 

m Oct-1

59

RB family proteins specifically interact with the RB family proteins. The results of Fig.
2-3 B shows that equal amounts of GST, GST-p107, GST-p130 and GST-RB were

recovered during GST pull down processing. In summary, all RB family members may
associate with SNAPc and TFIHB through direct interacts with subunits within SNAPc

and TFHIB.

RNA polymerase III associates with the A domain of RB

RB has been shown to associate with SNAPc, TFHIB and P01 HI (RPCl). To understand
the mechanism of how RB represses U6 snRNA transcription, it is important to identify
the domain(s) of RB responsible for its association with those proteins. Previous work
already identified that repression of U6 snRN A by RB requires the RB pocket A/B
domain and C domain (14). Correspondingly, GST-RB containing the NB and C
domains (379-870) maintains the strong binding with SNAP43, SNAP50, TBP, Brfl and
del (14). In contrast, GST-RB containing the pocket A/B domain (379-772) merely 1
maintains strong interaction with SNAP50, and the GST-RB containing the A domain
(379-577) does not bind to any one of those target proteins (14). To determine the regions
of RB required for binding to Pol III, a series of truncated or mutated GST-RB fusion
proteins were used in GST pull down assays. GST tagged proteins were incubated with
HeLa cell nuclear extract. Proteins co-precipitated by GST tagged proteins were detected
by Western blot analysis using anti-Pol IH (largest subunit, RPCl), anti-Brgl, anti-actin,
and anti-GST antibodies. Brgl analyzed here served as a positive control, which was
already known to associate with RB (7). The schematic representation of the various

GST-RB proteins is shown in Fig. 2-4 A, and the GST pull-down results are shown in

60

Fig. 2-4. Pol III and Brgl bind to the RB A domain. (A) Schematic representation of
the truncated or mutated RB proteins used in the GST pull-down assay. (B)
Characterization of the RB domains required for its associations with the RNA
polymerase 1H and Brgl. GST pull down analysis was performed by incubating 300 p1 of
HeLa cell nuclear extract with 500 ng of GST (lane 2), GST-RB (379-928) (lane 3),
GST-RB A/B/C domains (379-870) (lane 4), GST-RB A/B domain (379-772) (lane 5),
GST-RB A domain (379-577) (lane 6), GST-RB B/C domains (645-870) (lane 7), GST-
RB B domain (645-772) (lane 8), GST-RB Aexon22 (379-928) (lane 9) and GST-RB
C706F (379-928) (lane 10). Precipitated proteins were resolved by 7.5% SDS-PAGE.
The association of Pol HI, Brgl and actin with RB protein was assessed by Western blot
analysis using Pol HI (subunit RPCl) antiserum, Brgl and actin antibodies. Lane 1 shows
the relative amount of Pol III (RPCl), Brgl and actin present in 15 pl of HeLa cell
nuclear extract. Western blot analysis using GST antibody indicated the relative amount

of GST tagged RB proteins recovered after processing.

61

 

 

 

 

 

 

 

 

 

 

 

 

 

 

GST-RB (379-928)
GST-RB (379-870)
GST-RB (379-772)
GST-RB (379-577)
GST-RB (645-870)

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GST-RB (379-928) A exon 22

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62

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GST

Fig. 2-4 B. Both Pol 1H and Brgl were efficiently precipitated by GST-RB (379-928)
(lane 3), GST-RB A/B/C domains (379-870) (lane 4), GST-RB A/B domain (379-772)
(lane 5), GST-RB A domain (379—577) (lane 6), and GST-RB C706F (379-928) (lane
10), but not by GST-RB B/C domains (645-870) (lane 7), GST-RB B domain (645-772)
(lane 8), and GST-RB Aexon 22 (379-928) (lane 9). Western blot analysis against GST
indicated the relative amount of GST fused RB proteins recovered after GST-pull down
processing. Conclusively, the A domain of RB is responsible for binding to P01 HI

(RPCl) as well as Brgl.

Discussion

The significance of RB family proteins in tumor suppression, cell-cycle progression
control, cell differentiation and cell apoptosis is well established (5, 16, 17, 22). RB
family proteins may also control cell growth by regulating the transcription of non-
translated genes, such as ribosomal RNA, tRNA, and U6 snRNA that determine the
protein synthesis rate (33). In vitro repression assays of Ad VAl and U6 snRNA genes
with recombinant RB family proteins (Fig. 2-1) supports the idea that all RB family
members can repress Pol III transcription (27). Whether p107 and p130 are able to
repress U6 snRNA transcription in vivo is yet to be investigated, but it is likely that p107
and p130 do repress U6 snRNA transcription in viva, because endogenous p107 and p130
were found to occupy an endogenous U6 snRN A promoter (unpublished data by G.

J awdekar).

63

Pol IH activity varies during the cell cycle progression, and has the highest activity
during S and G2 phases but lowest activity during G1 and M phases (15, 34). RB family
proteins may repress Pol IH activity in a cell cycle dependent manner. RB and p130
target TFIHB during G0 and G1 phases of cell cycle. RB is hypophosphorylated during
G0 and G1 phase, and the hypophosphorylated RB binds to TFIHB component, Brfl
(24). Whether RB family proteins target SNAPc in a cell cycle dependent manner is not
known. All RB, p107 and p130 repress U6 snRNA transcription in vitro (Fig. 2-1), but
whether they have overlapping or unique functions on U6 snRNA repression during cell
cycle is not clear. RB, p107 and p130 may have some unique functions on U6 snRNA
repression during cell cycle. Another possible distinction among RB family members on
U6 snRNA repression is that different members of RB family may function in different
tissues because of the discrete expression levels of RB family members among different

tissues (29).

Except for the delsubunit of TFHIB, all subunits of SNAPc and TFHIB that interact
with RB also interact with p107 and p130 (Fig. 2-3). In addition, Pol HI (RPCl) was
found to associate with all RB, p107 and p130 (Fig. 2-2), and thus, it is likely that all RB
family members share some common mechanism in repressing Pol IH transcription. de1
was detected to interact with RB only, and TBP was found to bind to p107 much stronger
than RB and p130 (Fig. 2-3), however, the significance of these differences for RB, p107

and p130 to regulate the P01 HI transcription is not known.

64

Previous findings in our lab demonstrated that the mechanism for RB to repress U6
snRNA transcription is distinct from the mechanism for tRNA repression (14). RB
represses tRNA transcription by disrupting the preinitiation complex (PIC) formation at
tRN A promoter (26). However RB does not disrupt the preinitiation complex (PIC)
formation at the U6 snRN A promoter. The evidence for this idea is that RB, SNAPc,
TFIHB, and P01 HI co-occupy the U6 snRN A promoter during repression (14). In the
present work, RB was found to associate with multiple subunits of SNAPc, TFHIB and
P01 HI. Although the integrity of SNAPc, TFHIB and P01 HI complexes is yet to be
investigated, RB may function through a novel mechanism to repress the U6 snRN A. The
idea is that RB tethers Pol HI to SNAPc or TFIIIB and further prevents the Pol 111 from
translocation that is required for transcription (Fig. 2-5). One other evidence for this
model is that RB uses different regions to bind to SNAPc and TFHIB than to Pol IH. As
identified here, the RB A domain is sufficient to associate with Pol HI (Fig. 2-4). Though
GST-RB Aexon 22 (379-928) has an intact A domain; it does not precipitate Pol HI
(RPCl) and Brgl (lane 9 of Fig. 2-4). One possible reason is that internally truncated B
domain influences the regular conformation of RB A domain. Previous findings indicate
that RB binds subunits within SNAPc and TFHIB using a region other than A domain
(14), and the domain(s) in RB responsible for its binding to SNAPc and TFIHB is
discrete from the one binding to P01 IH (RPC l ). Whether RB interacts with Pol HI
directly or this interaction is mediated by other factor(s) is not known. Whether RB and
P01 1H association contributes to U6 snRNA repression is yet to be investigated.
Additional data from our lab (unpublished data by T. Selvakumar) demonstrated that RB

enriches the open complex on the U6 promoter, indicating RB tethers Pol HI but does not

65

Fig. 2-5. A tether and immobilization model for RB repression of U6 snRN A
transcription. RB simultaneously binds to Pol III and Pol IH accessory factors including
SNAPc and TFIIIB using discrete binding sites. One proposed consequence of RB
binding is that RB prevents Pol 111 from translocation required for Pol IH transcription.

RB may bind to P01 IH directly or indirectly through other unknown factor(s).

66

 

m-m .9”.

67

cause Pol HI to lose integrity. Since p107 and p130 were also demonstrated to associate
with Pol IH and multiple components of SNAPc and TFIIIB, it is possible that p107 and

p130 also use a tether and immobilization model to repress U6 snRN A transcription.

68

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71

APPENDIX

72

Introduction

In eukaryotes genes are packaged into chromatin. Thus, histone modifications and
changes in chromatin structure play a significant role in both positive and negative
regulation of gene transcription. For example, histone acetylation levels which are
controlled by histone acetyltransferases (HATS) and histone deacetylases (HDACs),
significantly influence gene transcription (4). Another example for a role of histone
modification in transcriptional regulation is histone methylation, which is controlled by
methyl transferases (HMTS) and putative histone demethylase (4, 5). Chromatin structure
can also be remodeled by the SWI/SNF complex and its effects on gene transcription can
be either negative or positive (2). It is possible that histone modification and chromatin
remodeling affect U6 snRN A transcription. As described by Zao et a1. (2001), there is a
nucleosome positioned between DSE and PSE sites of the U6 snRNA promoter, which
are separated by approximately 150 bp. The positioned nucleosome mediates the

coordinative binding between Oct-1 POU domain on the DSE and SNAPc on the PSE

(6).

RB interacts extensively with HDACs, HMTS and the SWI/SNF subunits Brg-l/Brm (1),
which are linked to histone modification and chromatin remodeling. RB induced histone
modification or chromatin remodeling has been demonstrated to facilitate gene repression
(1). Whether RB can repress U6 snRNA transcription by changing the structure of a
nucleosome at the U6 snRN A promoter is unknown. To explore the role of chromatin
structure in U6 snRN A transcription, a chromatinized U6 snRN A template was generated

in vitro.

73

Materials and Methods

Preparation of Drosophila S-190 chromatin assembly extract

The preparation of Drosophila S-190 chromatin assembly extract followed the protocol
created in Kadonaga laboratory (3). Approximately 80 g of Drosophila melanogaster
embryos staged from 0 to 6 hours were collected. After extensive washing with water,
embryos were dechorionated in 50% bleach for 90 seconds. Dechorionated embryos were
cleaned by rinsing with Embryo Wash buffer (0.7% NaCl, 0.04% Triton X-100) and

ddeO. Roughly cleaned embryos were moved to a glass beaker and further washed 2

 

times with cold (4°C) Embryo Wash buffer to remove chorion particles. Trace Embryo
Wash buffer left in the cleaned embryos was washed out with Saline Wash (0.7% NaCl),
and then trace Saline Wash buffer was further washed once with Buffer R (1.5 mM
MgC12, 1 mM DTT, 10 mM KCl, 10 mM B-glycerophosphate, 0.5 mM EGTA, 10%
glycerol, 10 mM HEPES, pH 7.5, 0.2 mM PMSF). Buffer R was then added to double
volume of settled embryos. Embryos in Buffer R were homogenized in a 40 mL Wheaton
Dounce Homogenizer until no intact embryos were left. Embryo homogenates were then
centrifuged at 7,650 X g for 5 min at 4°C. After the centrifugation, the golden brown
supernatant between the top foamy layer and bottom pellet was transferred to a new tube
using a syringe with 18-gauge needle. Mng was added to give a final concentration of 7
mM, and then the supematants was clarified through centrifirgation in an SW41 rotor
(Beckman Co.) at 40,000 rpm (198,000 X g) for 135 min at 4°C. Afier centrifuge
clarification, a middle liquid layer with yellow brown color, which is the S-190

chromatin assembly extract, was collected and frozen quickly in liquid nitrogen and

74

placed in -80°C storage. Pre-clarified S-l90 was clarified one more time through
centrifugation in a SW50.1 rotor at 45,600 rpm (192,000 X g) for 135 min at 4°C before

S-l90 was used to assemble chromatin.

Preparation of Drosophila histones

a) Preparation of embryo nuclei

150 g of Drosophila melanogaster embryos with age from 0 to 12 hours were collected
and dechorionated with 50% bleach for 90 seconds. After dechorionation, embryos were
extensively rinsed with Embryo Wash buffer and ddH20. The dechorionated embryos
were weighed and suspended in 3 mL of Buffer B (0.5 mM EGTA, 2 mM EDTA,
prepared in Buffer A with 15 mM Tris, pH 7.5, 0.1% (v/v) 2-mercaptoethanol, 0.34 M
sucrose, 0.15 mM spermine, 15 mM NaCl, 0.5 mM spermidine, 60 mM KCl, 0.25 mM
PMSF) per gram of embryos. Embryos in Buffer B were homogenized using a Yamato
homogenizer. The homogenate was filtered through miraclotlr (Calbiochern) into Sorvall
GSA rotor bottles to remove debris. Homogenate was subjected to centrifugation at 8,000
rpm (10,400 X g) for 20 min at 4°C to precipitate nuclei. After centrifugation, the loose
nuclei pellet in the tubes was transferred to another centrifuge tube and suspended in 200
mL of Buffer A. Homogenate in Buffer A was centrifuged again at 8,000 rpm (10,400 X
g) for 10 min at 4°C. The nuclei pellet in the tube was collected for second wash. Finally,
nuclei pellet was suspended in 30 mL of Buffer A to obtain a DNA and protein

concentration of 100 Abszw units/mL.

b) Test micrococcal nuclease (MNase) digestion

75

1 mL of embryo nuclei suspended in Buffer A was transferred to 1.5 mL microcentrifuge
tubes at 37°C and added with CaClz to a final concentration of lmM. The genomic DNA
was digested with 0.4 units of MNase. Digestion continued for 1, 2, 4, 6, 8, 10, 15, or 20
minutes. Following this, 100 p1 of nuclei was transferred to another tube containing 2.5 pl
of 0.5 M EDTA to quench the MN ase digestion. Then, the genomic DNA from each
sample was recovered with chloroform treatment and ethanol precipitation. The extent of

DNA digestion was analyzed on a 1.5% agarose gel.

c) Bulk digestion of nuclei by MNase and purification of histones.

Bulk nuclei were digested for the time determined to get most lO-nucleosome fi'agments
in the test digestion and then stopped by 10 pM EDTA. After digestion, the nuclei were
precipitated by centrifugation at 10,000 rpm in an SS34 rotor (12,000 X g) for 10 min at
4°C. The precipitated nuclei were subjected to lysis in 10 mL of 10 mM EDTA and 0.5
M NaCl. Then the lysed nuclei were clarified by centrifugation at 10,000 rpm in an SS34
rotor (12,000 X g) for 5 min at 4°C. Supernatant was collected, followed by measurement
of DNA absorbance (Abszm). Histones in the supernatant were separated in Beckrnan
SW28 rotor tubes with 36 mL of 5% to 30% linear sucrose gradient. Each sucrose
gradient was loaded with lysis supernatant of 500 AbS260 units or less. Then the loaded
sucrose gradients were centrifuged at 26,000 rpm in an SW28 rotor (89,500 X g) for 16
hr at 4°C. After centrifugation, the sucrose gradient was fractionated to 1.1 mL per
fraction at 4°C. Histones in each fraction from a representative gradient were checked by
electrophoresis on a 15% SDS-polyacrylamide gel and visualized by Coomassie blue

staining. Peak fiactions of core histones were pooled together in dialysis tubing and then

76

dialyzed overnight against 4 liters of Tris/EDTA solution (4 mM EDTA, 50 mM Tris, pH
7.9). The genomic DNA in histones was removed by passing through hydroxyapatite
column. The amount of DNA in the dialyzed histones was determined upon Ab8250. The
volume of hydroxyapatite column was prepared according to 1 mL of hydroxyapatite per
1.5 mg DNA. The preparation of hydroxyapatite column, as well as the sample loading,
washing, and fractionating were facilitated by an AKTAexplorer Chromatography
apparatus. Hydroxyapatite column was equilibrated in HC Buffer (1 mM DTT, 0.2 mM
PMSF, 40 mM NazHPO4, pH 6.8) before loading the sample. After loading the sample,
the hydroxyapatite column was washed with 3 to 4 column volumes of HC Buffer/0.35 M
NaCl. Core histones were eluted fi'om the hydroxyapatite column with HC Buffer/2.5 M
NaCl. The eluted core histones were collected as 1 mL fiactions. Histones in each
fiaction were checked by electrophoresis in a 15% SDS-polyacrylamide gel and
Coomassie blue staining visualization. The fractions with peak core histones were pooled
together, and the DNA in these fractions was analyzed on 1% agarose gel. The core
histones clarified from DNA were dialyzed overnight at 4°C against 4 liters of Core
Histone Storage buffer (10% glycerol, 1 mM EDTA, 10 mM KCl, 10 mM HEPES,
pH7.6). Finally, the concentration of core histones was measured by using Bradford
Protein Assay (Bio-Rad). The histone H1 preparation followed the same procedure as

used for core histones.

In vitro chromatin assembly
Chromatin assembly on U6 template, pU6/Hae/Ra.2, followed the protocols as described

in previous papers (3, 6). To start chromatin assembly on 1 pg of pU6/1-Iae/Ra.2 template,

77

 

50 pl of Drosophila S-l90 chromatin assembly extract was mixed with 0.96 pg of core
histones and 0.9 pg of histone H1 (optional) in Buffer R (1.5 mM MgC12, 1 mM DTT, 10
mM KCl, 10 mM B-glycerophosphate, 0.5 mM EGTA, 10% glycerol, 10 mM HEPES,
pH 7.5, 0.2 mM PMSF) to a final volume of 170 pl, followed with incubation at room
temperature for 30 minutes. After incubation, 28.5 pl of ATP mix (42.5 pl of 0.5 M
phosphocreatine, 4.25 pl of 0.5 M ATP, 22.7 pl of ddeO, 29.7 pl of 0.1 M MgClz, 0.84
pl of creatine phosphokinase solution) was added. Creatine phosphokinase solution was
composed of 50 mM NaCl, 10 mM potassium phosphate with pH 7.0, 50% (v/v) glycerol,
5 mg/mL creatine phosphokinase of type I (Sigma). Then 0.32 pg of Oct-1 and 1 pg of

pU6/Hae/Ra.2 were added. Finally, the chromatin assembly reactions were incubated at

 

27°C for 5 hours to complete assembly.

MNase digestion to detect chromatin assembly on pU6/Hae/Ra.2

50 p1 of assembled chromatin was transferred to each of four 1.5 mL microcentrifuge
tubes containing 1.5 pl of 0.1 M CaClz. 5 pl of the MNase solutions with different
concentrations, 1.3 units/mL, 4 units/mL, 12 units/mL and 36 units/mL was added
respectively to the tubes to start digestion at room temperature for 10 minutes. Then the
digestion was stopped by adding 5 pl of MNase stop buffer (0.24 mM EDTA, 29 pg/mL
RNase). The digestion samples were treated with 100 pl of0. 125 mg/mL Proteinase K
solution prepared in transcription stop solution (1% SDS, 0.2 M NaCl, 0.25 mg/mL
glycogen Type IX, 20 mM EDTA, pH 8.0). DNA in the digestion samples was recovered
by phenol extraction and ethanol precipitation. The precipitated DNA fragments were

separated by electrophoresis on a 1.25 % agarose gel for EtBr visualization.

78

Results

Histone preparation

Nuclei for histone preparation were prepared from 150 g of Drosophila melanogaster
embryos with age from 0 to 12 hours. The optimal time of MNase digestion was
measured in a test digestion. As shown in Fig. A-l, the digestion for 4 minutes produced
most DNA fiagments of 10 nucleosomes in length. This length of chromatin provides
adequate separation from contaminating proteins during the subsequent gradient
treatment. After the determination of the optimal digestion time, nuclei were subjected to
bulk digestion. Nuclei were then lysed to release the histones. Histones were separated by
linear sucrose gradients. After separation, the gradients were fiactionated in 1.1 mL
fractions. The histone distribution in fi'actions from a representative sucrose gradient
(No.1) was tested on a 15% SDS-PAGE. As shown in Fig. A-2, pure core histones
distributed only from fraction 1 to fraction 22. Histone H1 appeared from fraction 23 to
fraction 35. Two fractions, 15 and 25th, fiom another gradient (No.5) demonstrated
similar histone distribution. The peak core histones were pooled together (pool 3 in Fig.
A-2) for further histone concentration and DNA removal by passing through
hydroxyapatite column. After chromatography process, histone distribution and genomic
DNA in the fractions fi'om flowthrough, wash, and elution fractions were analyzed on a
SDS-PAGE and agarose gel, respectively. As shown in Fig. A-3, Core histones were
concentrated in 3 fractions: 15, 16 and 17th (Fig. A-3). As shown in Fig. A-4, there is no
visible DNA in any samples collected after passing through hydroxyapatite column. In
contrast, a DNA ladder in the sample from pool 3 before passing through hydroxyapatite

column was observed, suggesting that the concentrated histones are free of DNA. The

79

 

Fig. A-l. Optimization of Micrococcal Nuclease (MNase) digestion. DNA ladders
were made after nuclei were partially digested by MNase for varying time points as
indicated (see materials and methods). MN ase digested nuclei were separated by 1.5%
agarose gel electrophoresis and visualized by EtBr staining. Nuclei digested for 4 minutes

were used for following histone preparation.

Fig. A-2. Histone distribution among fractions after fractionated from sucrose
gradients. 40 pl of each selected fiaction was boiled with 10 ul 5X laemli buffer.
Histones in each sample were separated by 15% SDS-PAGE and then visualized by
Coomassie blue staining. Chicken total histones were used as an indicator. Pool 3 with
peak core histones was used for the core histone preparation. Pool 5 with peak histone H1

was used for the histone H1 preparation.

80

Fig. A-1

DNA ladder 13"
(hp)

4000
3000

1650 “a .-

1000 ’*
850A ;:

400* ~
300‘

200$

 

100*

Digestion t1me(min): 1 2 4 6 8 1015 20

Fig. A-2

Bottom —> ----

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fraction 1 2 3 4 S 6 7 8 9 10 11 121314151617181920212223 24252627 28293031 32 33 355—155—25
\ J \

 

Pool 1 Pool 2 Pool 3 Pool 4 Pool 5

* Chicken histones

81

Fig. A-3. Core histones were concentrated by hydroxyapatite column

chromatography. 20 pl of each representative sample from flow through, wash and
fractionation was boiled with 5 pl of 5 X Iaemmli buffer. Core histones in each sample
were separated by 15% SDS-PAGE and then visualized by Coomassie blue staining.
a...»

Lane “P3” is core histones before passing through hydroxyapatite column. Lane is

the chicken histones as an indicator of molecular weight.

Fig. A-4. Genomic DNA was depleted from core histones by hydroxyapatite column
chromatography. 20 pl of each representative sample from flow through, wash and
fractionation was resolved by 1% agarose gel and then visualized by EtBr staining. Lane
“P3” indicates the genomic DNA in the core histones before passing through the

hydroxyapatite column.

82

 

 

 

 

 

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83

fractions with concentrated core histones were pooled together for dialysis against
storage buffer. Finally, the concentration of total core histones was measured by Bradford
assay. The core histone concentration was 1 pg/ pl. The histone H1 preparation followed
the same procedure used for core histone preparation, except using the pool 5 (Fig. A-2).

The concentration of purified histone H1 was 2 pg/pl.

Chromatin assembly on pU6/Hae/Ra.2 template
A nucleosome is positioned between the PSE and the DSE on U6 snRNA promoter,

which facilitates the U6 snRNA transcription. The positioned nucleosome mediates a

 

coordinate binding of Oct-1 POU domain and SNAPc on the U6 promoter (6). RB
interacts with many histone modifiers and chromatin remodeling factors (1). To test
whether RB represses U6 snRN A transcription by affecting the nucleosome structure on
the U6 snRNA promoter, it is necessary to obtain an in vitro chromatinized U6 snRNA
template. Chromatin assembly on a U6 snRN A template was performed as described in
Materials and Methods. The quality of in vitro chromatinized template was checked by
partial MNase digestion and agarose gel electrophoresis. As shown in Fig. A-5 B,
regularly spaced DNA ladders were seen in all sets of chromatin assembly upon MNase
digestion. Chromatin assembled with core histone only showed a DNA ladder with about
160 bp spacing between two consecutive bands (Fig. A-S B), reflecting the length of
DNA fiagments surrounding the core nucleosome. Whereas, a DNA ladder with about
200 bp spacing between two consecutive bands was displayed in assembled samples

containing both core histones and histone H1 (Fig. A-S B), reflecting the length of DNA

84

Fig. A-5. Chromatin was assembled on pU6/Hae/Ra.2 and the Oct-l POU domain
did not interrupt the overall chromatin assembly. (A) Schematic representation of the
procedure of chromatin assembly (see materials and methods). (B) The assembled
chromatin was partially digested by Micrococcal Nuclease (MN ase) with different
concentrations: 1.3, 4, 12 and 36 U/mL, for 5 minutes. After digestion, the DNA
fragments were separated on a 1.25% agarose gel and visualized by EtBr staining. In the
left panel, chromatin was assembled by core histones alone, with (lanes 6-9) or without
(lanes 2-5) Oct-l POU domain. In the right panel, chromatin was assembled by core
histones and histone H1, with (lanes 3,5,7 and 9) or without (lanes 2, 4, 6 and 8) Oct-1

POU domain.

85

Fig. A-5

A

+_s-190,3°_"““.+ATP . __.+DNA,_ _
Histones R.T. regeneration mlx Transcrlptlon factors

5 hr
27 °C

 

Chromatin assembly

 

MNase Digestion

(hp)
2000 ..

1650 a

1000 ‘
850 "‘
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400 ‘0
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12345 6789 123456789
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Histone H1

   

 

86

fiagments surrounding the core nucleosome plus the linker DNA. Comparing the DNA
ladder between samples with the Oct-1 POU domain (Fig. A-S B left panel lanes 6 to 9,
right panel lanes 3,5,7and 9) and samples without the Oct-l POU domain (Fig. A-S B left
panel lanes 2 to 5, right panel lanes 2,4,6 and 8), the length of DNA fragment contained
in a nucleosome was not changed by the Oct-1 POU domain. This suggests that the
transcription factor Oct-1 POU domain does not interrupt the overall chromatin assembly
on pU6/Hae/Ra.2, which is consistent with the previous report (6). The chromatinized

pU6/I-lae/Ra.2 will be used in firture transcriptional assays.

87

 

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