University of Southern California Dissertations and Theses

Molecular mechanism of the recruitment of SWI/SNF chromatin remodeling complex and histone acetyltransferase to estrogen-responsive promoters

(USC Thesis Other)

Molecular mechanism of the recruitment of SWI/SNF chromatin remodeling complex and histone acetyltransferase to estrogen-responsive promoters

PDF

Download a page range

Download transcript

Copy asset link

Request this asset

Transcript (if available)

Content
MOLECULAR MECHANISM OF THE RECRUITMENT OF SWI/SNF CHROMATIN
REMODELING COMPLEX AND HISTONE ACETYLTRANSFERASE TO
ESTROGEN-RESPONSIVE PROMOTERS

by

KwangWon Jeong

--------------------------------------------------------------------------------------------

A Dissertation Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(BIOCHEMISTY AND MOLECULAR BIOLOGY)

May 2010

Copyright 2010 KwangWon Jeong

ii
ACKNOWLEDGEMENTS

Foremost, I would like to express my deepest gratitude to my mentor, Dr. Michael
Stallcup, for his excellent guidance, caring, patience, and providing me with an excellent
atmosphere for doing my research. I could not have imagined having a better advisor and
mentor for my Ph.D study.

Besides my advisor, I would like to thank the rest of my thesis committee: Prof. Woojin
An and Prof. Wei Li for their encouragement, insightful comments, and hard questions. I
would never have been able to finish my dissertation without the guidance of committee
members

I thank all my lab members, YoungHo, Rajas, Danielle, Dan, Irina, ChenYin, and Janet.
Special thanks to Dan Gerke and Kelly, who provided excellent technical supports. My
research would not have been possible without their helps. Also I thank KyungHwan, for
the stimulating discussions and for all the fun we have had in the last four years.

Finally, I would like to thank my wife, GunJe Lee and my daughter, Kate Jeong. They
were always there cheering me up and stood by me through the good times and bad. Most
gratefully, I would like to dedicate this work to my parents SungBong Jeong and
TaeBoon Choi, for their unconditional love and support.

iii
TABLE OF CONTENTS

Acknowledgements …………………………………………………………………….ii

List of Figures ……………………………………………………………………………iv

Abstract …………………………………………………………………………….v

Chapter 1: Introduction ……………………………………………………………..1

Chapter 2: Recruitment of the SWI/SNF chromatin remodeling complex
to steroid hormone-regulated promoters by nuclear
receptor coactivator Flightless-I …………………………………….17

Chapter 3: Recruitment of Tip60, a histone acetyltransferase,
to estrogen receptor regulated promoters …………………………….77

Chapter 4: Concluding Remarks …………………………………………………...116

Bibliography …………………………………………………………………………...124

iv
LIST OF FIGURES

Fig. 1-1. Nucleosome core particle …..…………..………………..………..……....4

Fig. 1-2. Schematic diagram of Tip60 protein domains………………….………..12

Fig. 1-3. Dynamics of cofactor recruitment directed by E2- liganded ……….…...15
hERα on the pS2 promoter

Fig. 2-1. Reduction of endogenous Fli-I attenuated the expression of ….….……..25
ER target genes

Fig. 2-2. Interaction of Fli-I with ER ................................................................…...30

Fig. 2-3. Fli-I interacts with BAF53 directly...…………………………….….…...33

Fig. 2-4. Fli-I binding to ER and to the SWI/SNF complex is important.………...36
for ER-mediated transcription

Fig. 2-5. Recruitment of ER, Fli-I, and BRG1 to the pS2 promoter ……………..42
in MCF-7 cells

Fig. 2-6. Role of Fli-I and BAF53 in cyclical, estrogen-dependent .……….……..49
recruitment of SWI/SNF complex to the pS2 promoter

Fig. 2-7. Role of Fli-I in cyclical, estrogen-dependent recruitment……….….…...56
of SWI/SNF complex to the GREB1 promoter

Fig. 2-8. Fli-I and BAF53 are required for estrogen-dependent ………..…..…….60
expression of ER target genes

Fig. 3-1. Reduction of endogenous Tip60 attenuated the …….………..……..….84
expression of ER target genes

Fig. 3-2. Recruitment of Tip60 to ER target gene promoters in MCF-7 cells ….....87

Fig. 3-3. Interaction with ER is important for coactivator function of Tip60 ….....92

Fig. 3-4. Interaction with ER is critical for Tip60 recruitment ……………......…..97

Fig. 3-5. Recruitment of Tip60 in BRG1 depleted MCF-7 cells …………....…..100

Fig. 3-6. Tip60 binds to methylated histone H3K4 …………………...…..…..…105

v

ABSTRACT

Estrogen receptor α (ER) is a member of the family of nuclear receptors and functions as
a transcriptional factor to induce gene expression by binding to specific DNA sequences
upon hormone treatment. It regulates cell growth, development and metabolic
homeostasis in multi-cellular organisms. Estrogen-mediated transcription has been
intensively studied genome-wide as well as on a small number of specific endogenous
target promoters. However, the exact mechanism by which ER coordinates the activities
of chromatin remodeling complexes and coactivators to facilitate initiation of
transcription remains elusive. Here, we show the molecular mechanisms of the
recruitment of the SWI/SNF chromatin remodeling complex by Fli-I, and recruitment of
Tip60, a histone acetyltransferase.
Fli-I can bind directly to both ER and BAF53, an actin-related component of the
SWI/SNF complex, suggesting that Fli-I may recruit SWI/SNF to ER target genes via
interaction with BAF53. Depletion of endogenous Fli-I or BAF53 specifically eliminated
part of the complex cyclical pattern of recruitment of SWI/SNF to estrogen-responsive
promoters in a way that indicates multiple roles and multiple mechanisms of recruitment
for SWI/SNF in estrogen-dependent target gene expression.
Tip60 interacts with ER in a hormone dependent manner, and point mutation of a
leucine-rich motif reduced Tip60 promoter occupancy at an estrogen-regulated promoter.
Tip60 also interacts with methylated histone H3K4 in vitro and the depletion of
endogenous BRG1 also impairs Tip60 recruitment significantly but not completely.

vi
These findings suggest a role for chromatin remodeling by SWI/SNF complex followed
by possible binding of Tip60 to methylated histones during the transcription initiation
process. These results suggest three steps that may contribute to Tip60 recruitment and
occupancy on estrogen-regulated promoters: 1) chromatin remodeling by SWI/SNF; 2)
interaction with ER; and 3) binding to methylated histones.
These results begin to establish the functional relationships and interdependencies
that coordinate the actions of the many coactivators participating in the transcriptional
activation process.

1
CHAPTER 1: Introduction

Estrogen Receptor Signaling Pathway

Estrogen regulates growth, differentiation, and other diverse functions in a broad range of
target tissues. The biological effects of estrogen are mediated through estrogen receptors
(ERs). ERs are members of the nuclear receptor superfamily which includes
glucocorticoid receptor (GR), androgen receptor (AR), progesterone receptor (PR),
thyroid hormone receptor (TR), retinoic acid receptor (RAR), retinoic X receptor (RXR),
and many orphan receptors (Gaub et al., 1990). There are two different forms of the
estrogen receptor, usually referred to as ERα and ERβ, and this thesis will focus on ERα.
The canonical mechanism of ER action involves binding of estrogen to estrogen receptor
followed by receptor dimerization and binding to specific response elements (ERE)
located in the promoter region of target genes (Nilsson et al., 2001). Estrogen binding
induces a conformational change in the C-terminal ligand binding domain, which allows
recruitment of coactivator proteins, such as p160 coactivators. p160 coactivators are
characterized by N-terminus bHLH-PAS domain, central nuclear receptor interacting
domain (NID), and C-terminus activation domains (AD1 and AD2) and function as a
scaffold protein to recruit other downstream coactivators. For example, the AD1 region
of p160 proteins recruits CBP/p300, whereas AD2 recruits CARM1, PRMT1, and β-
catenin. In various proteins, the bHLH-PAS domain is involved in DNA binding and
protein-protein interaction, and bHLH-PAS domain in p160 coactivator functions as AD3
which also recruits other coactivators, such as CoCoA, Fli-I, and GAC63 (Chen, Kim &

2
Stallcup, 2005; Kim, Li & Stallcup, 2003; Lee, Campbell & Stallcup, 2004).

The finding that around one third of the genes regulated by ER do not contain ERE-like
sequence (O'Lone et al., 2004) implies that ER can regulate gene expression, without
direct binding to specific DNA sequences, by modulating the function of other
transcriptional factors through protein-protein interaction in the nucleus (Gottlicher, Heck
& Herrlich, 1998). This was exemplified by the interaction of ER with the activator
protein 1 (AP-1) and the proposed ‘piggy-backing model’. Upon stimulation, ER
interacts with c-Fos and c-Jun at the AP-1 DNA binding site, which leads to activation of
target genes such as IGF-1, ovalbumin, collagenase, and cyclinD1 (Gaub et al., 1990;
Sabbah et al., 1999; Umayahara et al., 1994; Webb et al., 1995). Interestingly, it has been
found that some of estrogen effects are so rapid that they cannot depend on the activation
of gene expression. These actions of ER are known as non-genomic actions and are
believed to be mediated through membrane-associated ERs and ER binding to protein
kinases (Losel & Wehling, 2003). Non-genomic actions of estrogen include stimulation
of adenylate cyclase (Aronica, Kraus & Katzenellenbogen, 1994; Razandi et al., 1999),
activation of MAPK pathway (Migliaccio et al., 1996), and activation of PI3-kinase
signaling pathway (Chen et al., 1999b; Marino et al., 2002), which eventually activate
various downstream transcriptional factors such as Elk-1, CREB, AP-1, NF-kB, and
STAT. Recently, genome-wide analysis of estrogen receptor and RNA polymerase II
(PolII) binding sites have been conducted in combination with estrogen-regulated gene
expression (Carroll et al., 2006; Kininis et al., 2007; Rae et al., 2005). Thus, these data
will establish new resources for identifying important but unexpected actions of estrogen.

3

Chromatin and Transcription

Chromatin is the complex of DNA and histones that makes up chromosome in eukaryotic
nuclei. The basic structure of chromatin is the nucleosome which is approximately 146 bp
of DNA wrapped around an histone octamer containing two copies each of four histone
H2A, H2B, H3, and H4 (Luger et al., 1997) separated by linker DNA (Fig. 1-1). The
basic repeated element of chromatin is the nucleosome. In addition to core histone
proteins, linker histone H1 is also associated with DNA and allows forming a higher
order chromatin structure (Hansen, 2002). With addition of H1, the "beads-on-a-string"
structure in turn coils into a 30 nm diameter helical structure known as the 30 nm fiber or
filament. Chromatin structure contributes to transcription with multiple influences.
Basically, it restricts the access of promoter regions to the transcription factors or protein
complexes, however, this restriction is overcome by various modifications of histone tails.

Histone modifications include phosphorylation, acetylation, methylation, ubiquitination
and sumoylation of amino-terminal tails of the histone lysine, arginine, serine and
threonine residues and these reversible modifications are directed by kinases, histone
acetyltransferases, histone methyltransferase, ubiquitin ligases, and SUMO ligases (Gill,
2004; Narlikar, Fan & Kingston, 2002). It is known that histone tails have an important
role in the folding process of chromatin fibers (Carruthers & Hansen, 2000). Considering
this, acetylation of lysine residues in histones can antagonize the folding of chromatin

4

Fig. 1-1. Nucleosome core particle: 73-bp half. The view is down the superhelix axis
with the pseudodyad axis aligned vertically. The central base pair through which the dyad
passes is above the SHL0 label, 0 (SHL, superhelix axis location). Each SHL label
represents one further DNA double helix turn from SHL0. The complete histone proteins
primarily associated with the 73-bp superhelix half are shown (interparticle tail regions
are not shown). The two copies of each histone pair are distinguished as unprimed and
primed copies, where the histone fold of the unprimed copy is primarily associated with
the 73-bp DNA half and the primed copy with the 72-bp half. The 4-helix bundles are
labelled as H39 H3 and H2B H4; histone-fold extensions of H3 and H2B are labelled as
aN9, aN and aC, respectively; the interface between the H2A docking domain and the H4
C terminus as b; and Nand C-terminal tail regions as N or C (Karolin Luger et al., Nature
1997).

5

Fig. 1-1. Continued.

6
by change of electrostatics (Hansen, 2002). However, methylation of lysine residues does
not alter their charge. Thus, another hypothesis proposed that specific histone
modifications such as methylation, create or destroy binding sites for other protein that
mediate downstream effects (Jenuwein & Allis, 2001; Strahl & Allis, 2000; Turner,
2000).

Although some specific modifications of histones have been shown to be a major
mechanism of transcription regulation, (e.g. methylation of lysines H3K4 and H3K36 in
transcriptional activation and methylation of lysines H3K9 and H3K27 in transcriptional
repression), the complete understanding of the precise mechanism by which histone
modification influences overall transcription remains elusive. Thus, the term ‘histone
code’ suggests that transcriptional regulation is guided by combinatorial modification of
histone tails. This idea implies the importance of spatial combinations and the order of
transcription factor or coactivator action as well as the activity of individual proteins.

Chromatin Remodeling Complexes

Chromatin remodeling complexes are multiprotein complexes that utilize the energy
derived from ATP hydrolysis to alter chromatin architecture at the nucleosome level. The
four major classes of ATP-dependent chromatin remodeling complexes, SWI/SNF, ISWI,
Mi-2/NURD, and INO80, are characterized by the identity of their ATPase subunit
(Eberharter & Becker, 2004; Peterson & Workman, 2000; Trotter & Archer, 2007) and
SWI/SNF and ISWI complexes are the 2 most-studied and characterized classes.

7

The SWI/SNF families, identified by genetic screens in yeast, are important genes for
mating-type switching, and later, their functions in transcriptional activity have been
introduced (Hirschhorn et al., 1992; Laurent, Treitel & Carlson, 1991; Peterson, Dingwall
& Scott, 1994; Winston & Carlson, 1992). The nature of the remodeled chromatin state
generated by SWI/SNF complex has been extensively studied in vitro. (Eberharter &
Becker, 2004; Fan et al., 2003). Biochemical analysis revealed that the yeast SWI/SNF
complex alters nucleosomal structure and increases the accessibility of protein to DNA
templates assembled as chromatin in an ATP-dependent manner in vitro (Peterson &
Workman, 2000). Human SWI/SNF complex is a large multiprotein complex that
contains either BRG1 or hBRM as a core ATPase and 10-12 BRG1-associated factors
(BAFs) (Wang et al., 1996). Four subunit; BRG1, BAF155, BAF170, and BAF47 are
required for the minimal catalytic activity (Phelan et al., 1999). It appears that the BRG1-
containing complex and the hBRM-containing complex have similar activity in vitro
(Kadam & Emerson, 2003), but quite distinct biological function in vivo (Bultman et al.,
2000; Reyes et al., 1998). The BRG1 knockout mouse showed very early embryonic
death, whereas BRM null mice are viable.

The ISWI (Imitation of SWI) family was originally identified in Drosophila, and includes
NURF (nucleosome-remodeling factor), ACF (ATP-dependent chromatin assembly and
remodeling factor), and CHRAC (chromatin-accessibility complex) (Becker, Tsukiyama
& Wu, 1994; Tsukiyama, Becker & Wu, 1994). Like the SWI/SNF complex, the ISWI
complex contains the ISWI ATPase subunit, but fewer 2~4 other subunits. In addition

8
to the highly conserved ATPase domain, the ISWI protein contains a SANT (SWI3,
ADA2, N-CoR, and TFIIB) domain, a potential nucleosome interaction module (Boyer,
Latek & Peterson, 2004). Although they have a similar core ATPase subunit for their
functions, ISWI complexes demonstrate different functions on nucleosomes. CHRAC
and ACF tend to cluster and assemble nucleosomes and deactivate transcription
(Dirscherl & Krebs, 2004; Gavin, Horn & Peterson, 2001; Lusser & Kadonaga, 2003;
Vignali et al., 2000), whereas, the NURF complex acts to destabilize the chromatin
structure (Gavin et al., 2001; Langst & Becker, 2001; Lusser & Kadonaga, 2003). The
observation that the ISWI complex is preferentially associated with transcriptionally
silent chromatin regions in Drosophila suggests that the primary role of ISWI in vivo is
transcriptional repression and gene silencing (Deuring et al., 2000).

Flightless-I (Fli-I), a Nuclear Receptor Coactivator

Flightless-I was originally identified in Drosophila; where a hypomorphic mutation
resulted in a loss of flight ability, while severe mutations lead to impaired cellularization
and gastrulation of the embryo (Campbell et al., 1993). The fliI gene encodes a 1256
amino acid protein with molecular weight 143 kDa. By homology analysis, it has been
shown that the Fli-I is characterized by a leucine-rich repeat (LRR) region at the N-
terminus which is known to be involved in protein-protein or protein-lipid interactions
(Kajava, Vassart & Wodak, 1995; Kobe & Deisenhofer, 1995a; Kobe & Deisenhofer,
1995b). The C-terminus of Fli-I contains a gelsolin-like domain characteristic of actin-
binding proteins (Campbell et al., 1993). Like other gelsolin family proteins, Fli-I is

9
able to bind to actin or actin-related proteins (ARPs). In the cytoplasm, Fli-I specifically
co-localized with cytoskeletal structures (Davy et al., 2000; Davy et al., 2001). Several
binding proteins have been identified by yeast two-hybrid analysis and biochemical
approaches. FLAP (Fli-I LRR associated protein) has been discovered to bind to the LRR
region of Fli-I. There are two mammalian genes encoding FALP1/LRRFIP1 and
FLAP2/TRIP/LRRFIP2/ GCF2. Though FLAP is known as a dsRNA-binding protein, the
exact functions of FLAP remain unclear.

The coactivator function of Fli-I was discovered by Lee et al. in 2004 (Lee et al., 2004).
Fli-I has been shown to interact physically and functionally with p160 coactivators to
enhance nuclear receptor mediated transcription. In vitro and in vivo binding studies
showed that Fli-I efficiently interacts with ER and TR in the presence of hormone. The
interaction with CARM1 might be another mechanism by which Fli-I activates
transcription. Interestingly, it has been shown that the gelsolin-like domain of Fli-I
interacts with BAF53, which is an actin-related protein. Point mutation in the gelsolin-
like domain where BAF53 binds compromised its coactivation function, suggesting that
this interaction is necessary for the full coactivator function of Fli-I. BAF53 is a
component of the SWI/SNF complex, and thus these results suggest another possible
mechanism by which Fli-I can recruit the SWI/SNF complex to nuclear receptor-
regulated promoters.

10
Tip60, a Histone Acetyltransferase

Tip60, originally isolated as a HIV-1 Tat interactive protein (Kamine et al., 1996) is a
protein with multiple roles including regulation of transcription, cell cycle and DNA
repair regulation. Lysine acetyltransferases are categorized into seven groups and Tip60
is one of the best characterized MYST protein family members. Alternative splicing of
the HTATIP gene encoding Tip60 generates at least three variants, Tip60 isoform 1,
Tip60 isoform 2 (Tip60α), and Tip60 isoform 3 (Tip60β/PLIP). The predominant form is
isoform 2 with 60 kDa molecular weight . Isoform 1 is the largest form of Tip60 and
known to have distinct function (Legube & Trouche, 2003). Isoform 3 lacks exon 5 that
encodes a proline-rich region and appears to have similar function as Tip60 isoform 2
(Ran & Pereira-Smith, 2000; Sheridan et al., 2001). The cellular Tip60 protein level is
relatively low and tightly regulated by proteasome-dependent degradation (Legube et al.,
2002). Without stimulation, Tip60 is very unstable with a 30-190 min half-life, however,
DNA damage such as UV radiation, inhibits ubiquitination of Tip60 and the cellular level
is dramatically increased (Legube et al., 2002). Tip60 isoform 2 is the best characterized
variant. It consists of 513 amino acids, which contains an N-terminal chromodomain, and
C-terminal MYST domain (Sapountzi, Logan & Robson, 2006) (Fig. 1-2). The majority
of cellular Tip60 protein exists in a stable multiprotein complex that consists of at least
18 subunits (Ikura et al., 2000; Kusch et al., 2004). In addition to Tip60, the existence of
RuvBL1 and RuvBL2, the putative helicases, in the Tip60 complex supports the function
of Tip60 in DNA damage signaling and apotosis.

11
Tip60 is also known as a nuclear receptor coactivator. The interaction with AR has been
shown to activate AR-mediated transcription (Brady et al., 1999; Gaughan et al., 2001;
Gaughan et al., 2002). The mechanism for AR-mediated transcription involves Tip60
forming a trimeric complex of Tip60, AR, and HDAC1 upon the endogenous AR-
responsive PSA promoter, and acetylation of AR by Tip60 regulates AR activity by
competition with HDAC1 (Gaughan et al., 2002). The exact mechanism by which Tip60
coactivates other nuclear receptors (i.e ER) remains unclear.

The goal of this thesis is to define the mechanistic contributions of diverse events induced
by chromatin-remodeling, histone modifications, and protein-protein interactions to
transcriptional activation, and to define the mechanisms that coordinate the activities of
multiple coactivator complexes on promoters of ER target genes. Including the SWI/SNF
complex and histone modification enzymes, hundreds of coactivators and protein
complexes have been discovered by yeast two-hybrid screens and biochemical
technologies. This provoked the realization that transcriptional regulation might be a
quite complicated and dynamic event rather than static process. The idea of the histone
code increased the complexity of proteins or protein complexes recruited during the gene
expression process. Since 2000, it has been demonstrated that an ordered, sequential, and
cyclical pattern of steady-state level occupancy by ER, various coregulators, and various
histone modifications occurs on ER-mediated promoters (Metivier et al., 2003; Shang et
al., 2000).

The kinetic of association of ERα and PolII on pS2 promoter show a periodicity of 40-

12

Fig. 1-2. Schematic diagram of Tip60 protein domains. Isoform 2 (Accession Number:
NP-006379) is the best characterized Tipo60 isoform. Isoform 1 (Accession Number:
NP-874369) encodes an additional 33 amino acids at the N-terminus, while isoform 3
(Accession Number: NP-874398) results from the exclusion of 52 amino acids between
the chromodomain and the MYST domain (Vasileia Sapountzi et al., 2006).

13

Fig. 1-2. Continued.

14
60 min (Metivier et al., 2003). Upon E2 treatment, three different types of cycle of ERα
to pS2 promoter occur. These are an initial transcriptionally unproductive cycle followed
by two different transcriptionally productive cycles. During the first transcriptionally
unproductive cycle, ERα initiates the association of the SWI/SNF chromatin remodeling
complex followed by PRMT1, p300 or Tip60. In the next two transcriptionally
productive cycles, p68 is initially recruited to pS2 promoter followed by HMTs, HATs,
and SRC1, leading the methylation and acetylation of the histone H3 and H4 (Fig 1-3).
Although these data describe the cyclical events that occur on the ER-regulated promoter
to initiate transcription, the exact mechanisms by which promoter assembles coregulator
complexes remain unclear.

In this thesis, I examine 1) how proteins or protein complexes are recruited by hormonal
activation of ER, 2) how the mechanism is precisely regulated in an ordered and cyclical
manner, and 3) how various events such as chromatin remodeling, histone modification,
and protein-protein interactions are inter-related for optimal recruitment of coactivator
complexes and setting up the promoter for the initiation of transcription. Here, I show the
molecular mechanism of SWI/SNF recruitment by a nuclear receptor coactivator, Fli-I as
well as the mechanism of the recruitment of a histone acetyltransferase, Tip60. These
results will begin to establish the functional relationships and interdependencies that
coordinate the actions of the many coactivators participating in the transcriptional
activation process. Moreover, these new insights into transcriptional attainment and
regulation will provide new opportunities to understand gene expression.

15

Fig. 1-3. Dynamics of cofactor recruitment directed by E2-liganded hERα on the pS2
promoter. Kinetic ChIP experiments were performed using specified antibodies as shown
within the images. After 2 hr treatment with 2.5 μM-amanitin, cells were washed and
placed in media supplemented with 2.5% dextrancharcoal treated FCS including 10 nM
Estradiol (E2). Then, chromatin was prepared on sampled cells at 5 minutes intervals.
The amount of immunoprecipitated pS2 promoter was quantified by real-time PCR.
Values, expressed as % of the inputs, are the mean of three separate experiments, and
have a SD < 2%. All ChIP were performed from a single chromatin preparation for each
time point (Metivier et al., 2003 Cell).

16
Fig. 1-3. Continued.

17
CHAPTER 2: Recruitment of the SWI/SNF chromatin remodeling
complex to steroid hormone-regulated promoters by nuclear receptor
coactivator Flightless-I

INTRODUCTION

Chromatin remodeling for transcriptional regulation involves protein complexes with two
distinct types of enzymatic activities, i.e. ATP-dependent chromatin-remodeling and
posttranslational histone modification. The four major classes of ATP-dependent
chromatin-remodeling complexes − SWI/SNF, ISWI, Mi-2/NuRD, and INO80 − are
characterized by the identity of their ATPase subunit (Eberharter & Becker, 2004;
Peterson & Workman, 2000; Trotter & Archer, 2007). Human SWI/SNF complexes
contain either human Brahma (hBRM) or Brahma-Related Gene 1 (BRG1) protein as the
catalytic ATPase subunit, along with approximately 10-12 BRG1-associated factors
(BAFs), the composition of which varies (Trotter & Archer, 2008). The nature of the
remodeled chromatin state generated by SWI/SNF complexes has been extensively
studied in vitro and is characterized by increased mobility of nucleosomes on DNA
templates and altered degree of association of DNA with histones within nucleosomes
(Eberharter & Becker, 2004; Fan et al., 2003).

The role of SWI/SNF complexes in nuclear receptor (NR)-mediated transcriptional
activation was first studied for the glucocorticoid receptor in a heterologous yeast model

18
system and later in mammalian cells (Muchardt & Yaniv, 1993; Trotter & Archer, 2007;
Yoshinaga et al., 1992). SWI/SNF complexes play critical roles in establishing
hypersensitive chromatin architecture associated with steroid hormone receptor binding
sites on DNA and are recruited to these sites in a hormone dependent manner (John et al.,
2008; Johnson et al., 2008; Trotter & Archer, 2008).

Including the SWI/SNF complex, more than 300 coactivators and corepressors for NRs
have been identified, but their mechanistic contributions to transcriptional activation and
the mechanisms that coordinate the activities of multiple coactivator complexes on
promoters of NR target genes are mostly unknown (Lonard & O'Malley, 2006; Roeder,
2005; Rosenfeld, Lunyak & Glass, 2006; Stallcup et al., 2003). Recent studies identified
Flightless-I (Fli-I) as a transcriptional coactivator for NRs, including estrogen receptor
(ER) α and thyroid hormone receptor, in transient reporter gene assays (Lee et al., 2004).
Fli-I has a leucine-rich repeat (LRR) region at its N-terminus and a C-terminal gelsolin-
like domain (Fig. 2-1A) which places it in the gelsolin protein family (Lee et al., 2004).
Like other members of the gelsolin family, Fli-I binds to actin and actin-related proteins
(Davy et al., 2000; Davy et al., 2001). Also like gelsolin, the C-terminal gelsolin-like
domain of Fli-I consists of two large tandem repeats, each of which contains three
smaller repeats; in gelsolin, each small repeat folds as an independent structural module
(Urosev et al., 2006). Although we previously showed that Fli-I is required for activation
of a transient reporter gene by steroid hormones (Lee et al., 2004), the mechanism by
which Fli-I contributes to transcriptional activation of NR target genes is unknown. Our
finding that Fli-I interacts directly with NRs and BAF53 (Lee et al., 2004), which is an

19
actin-related protein component of SWI/SNF complexes (Goodson & Hawse, 2002;
Trotter & Archer, 2008), suggests a possible functional relationship between Fli-I and
SWI/SNF. This led us to propose that Fli-I might be responsible for recruitment of the
SWI/SNF complex to steroid hormone-responsive promoters (Lee et al., 2004). In the
current study we therefore tested whether depletion of Fli-I by siRNA affected the
estrogen-dependent expression of endogenous ER target genes and recruitment of
SWI/SNF to the corresponding promoters of ER target genes. Our findings indicate an
important role for Fli-I in the transcription initiation process and the function of
SWI/SNF and support a model for multiple roles of SWI/SNF complexes in estrogen-
dependent transcription of ER target genes.

EXPERIMENTAL PROCEDURES

Plasmids. The following plasmids were described previously (Lee et al., 2004): pSG5-
ER(DBD-AF2); human Fli-I expression vectors pSG5-FLAG-LRR and pSG5-FLAG-
GelA; and GST fusion protein expression vector pGEX4T1-ER(LBD). pSG5-HA-BAF53
contains the BAF53 coding region inserted into XhoI and BglII sites of pSG5.HA (Chen
et al., 1999a). pSG5-FLAG (Lee et al., 2004) expression vectors encoding the following
proteins were constructed by inserting the appropriate PCR-amplified cDNA coding
regions into EcoRI and XhoI sites: pSG5-FLAG-G1 (Fli-I amino acids 495 to 597),
pSG5-FLAG-G2 (amino acids 598 to 709), pSG5-FLAG-G3 (amino acids 710 to 822),
pSG5-FLAG-GelB (amino acids 825-1269). GST-protein expression vectors encoding
the following proteins were constructed by inserting the appropriate cDNA coding

20
region into EcoRI and XhoI sites of the vector: pGEX4T1-LRR (amino acids 1 to 494),
pGEX4T1-GelA (amino acids 495 to 822), pGEX4T1-GelB (amino acids 825 to 1269),
pGEX4T1-G1 (amino acids 495 to 597), pGEX4T1-G3 (amino acids 710 to 822). His-
tagged full length Fli-I (pTriEX4-Fli-I) was created by PCR amplification of pCDNA-
Fli-I and subcloning into NcoI and XhoI sites of pTriEX4 vector (Novagen). pTriEX4-
Fli-I-ΔG1 lacking amino acids 495 to 597 was generated by two step PCR amplification
and insertion. The first PCR product comprised of the first 1410 bp of Fli-I cDNA was
inserted into NcoI and BamHI sites of pTriEX4, and the second PCR fragment comprised
of bp 1719-3801 was inserted into the XmaI site within the previous insert and the XhoI
site in pTriEX4 vector. Point mutations in Fli-I, GelA and G1 were introduced by site-
directed mutagenesis using the Quick-Change II Site-Directed Mutagenesis Kit
(Stratagene). BRG1 expression plasmid was kindly provided by Dr. Anthony N.
Imbalzano (University of Massachusetts Medical School).

Protein interaction assays and immunoblot. The procedure for GST-pulldown assays
was described previously (Lee et al., 2004). GST-fusion proteins were expressed in E.
coli BL21(DE3) strain and purified by incubation with glutathione Sepharose beads and
washing with NETN buffer (300 mM NaCl, 1 mM EDTA, 20 mM Tris-HCl (pH 8.0),
and 0.01% NP-40). His-tagged BAF53 and Fli-I were also expressed in E. coli
BL21(DE3) strain and purified with Ni-NTA agarose beads (Qiagen). FLAG-tagged Fli-I
fragments were synthesized by transcription and translation in vitro using the TNT-Quick
coupled reticulocyte lysate system (Promega) according to the manufacturer’s protocol.
For coimmunoprecipitation assay, 293T cells were plated at 1.5 X 10
6
cells per 10-cm

21
dish and transiently transfected using Lipofectamine 2000 (Invitrogen) and the indicated
amount of plasmids. At 48 h after transfection, cell extracts were prepared in 1.0 ml
RIPA buffer (50 mM Tris-Cl (pH 8.0), 150 mM NaCl, 1% NP-40, 1% sodium
deoxycholate, 0.1% sodium dodecyl sulfate, 2 mM EDTA). Immunoblotting was
performed as described previously (Lee et al., 2004), using the following antibodies: anti-
His, anti-ERα, anti-Fli-I, anti-BRG1, anti-BAF53, anti-β-actin, anti-GAL4DBD and
normal mouse or rabbit IgG (Santa Cruz Biotechnology); anti-HA (Roche Applied
Science); anti-FLAG (Sigma Aldrich).

Chromatin immunoprecipitation assay. Chromatin immunoprecipitation (ChIP) assays
were performed according to previously described protocols (Lee et al., 2004). Briefly,
MCF-7 cells were transfected with siRNA and then cultured for 3 days in phenol red-free
Dulbecco’s modified Eagle medium supplemented with 5% charcoal-dextran-stripped
fetal bovine serum. At approximately 90% confluency, cells were treated with 100 nM
estradiol (E2) or vehicle for the indicated time. After cross-linking with formaldehyde,
cell extracts were prepared from control and E2-treated MCF-7 cells.
Immunoprecipitation of sonicated chromatin solutions was conducted by overnight
incubation at 4°C with normal mouse or rabbit IgG, anti-ERα, anti-Fli-I, anti-BRG1,
anti-TRAP220 (Santa Cruz Biotechnology) or anti-RNA polymerase II (Millipore).
Cross-linking was reversed by heating and immunoprecipitated DNA was purified by
phenol-chloroform extraction and ethanol precipitation. The purified DNA was dissolved
in 100 μl of TE buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA) and analyzed by
quantitative PCR using the Stratagene Mx3000P system with SYBRGreen dye. The

22
primers used were: pS2 promoter region, 5’-GGCAGGCTCTGTTTGCTTAAAGAGCG-
3’ (forward) and 5’-GGCCATCTCTCACTATGAATCACTTCTGC-3’ (reverse); pS2
coding region, 5’-TGCCAGCTGTGGGGAGCTGAATAACTT-3’ (forward) and 5’-
CAGTTCGTTCTGTACACCGAGGCCACT-3’ (reverse); GREB1 promoter region, 5’-
GTGGCAACTGGGTCATTCTGA-3’ (forward) and 5’-
CGACCCACAGAAATGAAAAGG-3’ (reverse).

RNA interference and qRT-PCR. Small interfering RNA experiments were performed
according to previously published methods (Lee et al., 2004). The sequences of siRNA
used were: siFli-I, 5’-CAACCUGACCACGCUUCAUdTdT-3’ (sense), 5’-
AUGAAGCGUGGUCAGGUUGdTdT-3’ (anti-sense); siFli-I(2), 5’-
GCUGGAACACUUGUCUGUGdTdT-3’ (sense), 5’-
CACAGACAAGUGUUCCAGCdTdT-3’ (anti-sense) ;siBAF53, 5’-
GCUUUCCUUGAAAUGCACUdTdT-3’ (sense) and 5’-
AGUGCAUUUCAAGGAAAGCdTdT-3’ (anti-sense); siBAF53(2), 5’-
GGUACUUCAAGUGUCAGAUdTdT-3’ (sense) and 5’-
AUCUGACACUUGAAGUACCdTdT-3’ (anti-sense); siBRG1, 5’-
CAUGCACCAGAUGCACAAGdTdT-3’ (sense) and 5’-
CUUGUGCAUCUGGUGCAUGdTdT-3’ (anti-sense); siBRG1(2), 5’-
CCGUGGACUUCAAGAAGAUdTdT-3’ (sense) and 5’-
AUCUUCUUGAAGUCCACGGdTdT-3’ (anti-sense); siNS, 5’-
UUCUCCGAACGUGUCACGUdTdT-3’ (sense) and 5’-
ACGUGACACGUUCGGAGAAdTdT-3’ (anti-sense) (Takata et al., 2007).

23
Transfections in MCF-7 cells were performed using Lipofectamine 2000 (Invitrogen)
according to the manufacturer’s protocol. Total RNA was isolated from MCF-7 cells with
Trizol (Invitrogen) after hormone treatment as indicated and subjected to reverse
transcription (RT) by iScript cDNA synthesis kit (Bio-Rad). 5 μl of RT product was used
for qPCR analysis with the following primers: pS2, 5’-GAACAAGGTGATCTGCG-3’
(forward) and 5’-TGGTATTAGGATAGAAGCACCA-3’ (reverse); Cathepsin D, 5’-
GTACATGATCCCCTGTGAGAAGGT-3’ (forward) and 5’-
GTCACCGGAGTCCATCACGATG-3’ (reverse) (Oxelmark et al., 2006); GREB1, 5’-
CAAAGAATAACCTGTTGGCCCTGC-3’ (forward) and 5’-
GACATGCCTGCGCTCTCATACTTA-3’ (reverse) (Rae et al., 2005); BRG1, 5’-
CATCATCGTGCCTCTCTCAAC-3’ (forward) and 5’-
ACACGCACCTCGTTCTGCTG-3’ (reverse); BAF53, 5’-
GAGTTCCCAAGCTTCTACCTTCCT -3’ (forward) and 5’-
CATTCTACAAAAGATGGTCATTCTTTTC-3’ (reverse); β-actin, 5’-
ACCCCATCGAGCACGGCATCG-3’ (forward) and 5’-
GTCACCGGAGTCCATCACGATG-3’ (reverse) (Sun, Nawaz & Slingerland, 2007);
GAPDH, 5’-TCTGGTAAAGTGGATATTGTTG-3’ (forward) and 5’-
GATGGTGATGGGATTTCC-3’ (reverse) (Chen et al., 2005). Relative expression levels
were normalized to GAPDH mRNA levels.

24
RESULTS

2.1 Reduction of endogenous Fli-I attenuated the expression of ER target genes.

We previously showed that Fli-I enhances and is required for efficient NR mediated
transcription of transiently transfected reporter genes (Lee et al., 2004). Here we
examined endogenous ER target genes in MCF-7 cells. Compared with non-specific
siRNA (siNS), siRNA specific for Fli-I (siFli-I) effectively reduced Fli-I mRNA and
protein levels (Fig. 2-1B). Fli-I siRNA also significantly inhibited E2-dependent
expression of the endogenous pS2, GREB1 and Cathepsin D genes (Fig. 2-1C, showing
the mean and SD from results of seven independent experiments). For the seven
independent experiments, the reduction of Fli-I mRNA levels was 57 ± 13 %, and the
fold induction of ER target gene expression by E2 was reduced by 48 ± 14 % for pS2
mRNA (p < 0.01 by paired, two-tailed T-test), 47 ± 8 % for GREB1 mRNA (p < 0.01),
and 49 ± 21 % for Cathepsin D mRNA (p < 0.05) (Fig. 2-1C). Although knockdown
efficiency of Fli-I varied from experiment to experiment, there was a strong linear
correlation between reduction of Fli-I mRNA and reduction of E2-induced levels of pS2
mRNA with R
2
correlation coefficient of 0.83 (Fig. 2-1C). The reduction of Fli-I mRNA
level by another siRNA targeting a different part of the coding region of Fli-I caused a
similar inhibition of ER target gene expression (Fig. 2-1D). Thus, Fli-I is required for
hormonal induction of ER target genes in MCF-7 cells.

25

Fig. 2-1. Reduction of endogenous Fli-I attenuated the expression of ER target genes. (A)
Domains of Fli-I: LRR, N-terminal leucine rich repeats; GelA and GelB, the gelsolin-like
domain consisting of two large tandem repeats; the six numbered regions represent small
repeated modules homologous to the actin binding motifs of gelsolin. (B) Depletion of
Fli-I mRNA and protein by siRNA transfection. MCF-7 cells were transfected with
siRNA specific for Fli-I (siFli-I) or non-specific siRNA (siNS) and grown in hormone
free-media for 72 h. Total RNA was analyzed for Fli-I mRNA by qRT-PCR and
normalized to the level of GAPDH mRNA. Protein levels of Fli-I, ER, and β-actin were
assessed by immunoblot. (C) Effect of reduced Fli-I on expression of estrogen-responsive
genes. MCF-7 cells were transfected with siFli-I or siNS and grown in hormone free-
media. After 72 h cells were treated with 100 nM E2 or vehicle for 24 h. Total RNA was
analyzed by qRT-PCR. The levels of pS2, GREB1 and Cathepsin D mRNAs were
normalized to that of GAPDH mRNA. Results shown in the bar graphs are mean and SD
of data from seven independent experiments. P values were determined by paired, two-
tailed t-tests from the seven independent experiments, as indicated by the brackets in the
figures. The line graph shows the correlation between reduction of Fli-I and pS2 mRNA
levels. From the seven independent experiments, % reduction of pS2 mRNA was plotted
against % reduction of Fli-I mRNA, and the R
2
value was calculated.

26

Fig. 2-1. Continued.

27

Fig. 2-1. Continued. (D) Inhibition of the expression of ER target genes by siFli-I(2).
MCF-7 cells were transfected with siFli-I(2) or non-specific siRNA (siNS) and grown in
hormone-free media. After 72 h cells were treated with 100 nM E2 or vehicle for 24 h.
Total RNA was analyzed by qRT-PCR as in C. The levels of Fli-I, pS2, GREB1 and
Cathepsin D mRNAs were normalized to that of GAPDH mRNA. Fli-I mRNA was
measured without E2 treatment. Similar results were obtained in an independent
experiment using the same siRNA (data not shown).

28

Fig. 2-1. Continued.

D

29
2.2 Mapping interaction sites of Fli-I with ER.

We previously showed that Fli-I binds to full length ER (Lee et al., 2004). To further
explore possible functional relationships, we mapped the ER binding site on Fli-I. ER-
ligand binding domain (LBD) fused to glutathione S-transferase (GST) bound in vitro in
an E2-dependent manner to the GelA domain (amino acids 495-822) of Fli-I but not to
the LRR domain (amino acids 1-494) or the GelB domain (amino acids 825-1269) (Fig.
2-2A). In similar GST pulldown assays with each of the three repetitive subregions of
GelA (Fig. 2-1A), only the third repeat (G3, amino acids 710-822) bound ER-LBD, and
binding was E2-dependent, indicating that G3 is the major binding site for ER-LBD (Fig.
2-2B, upper panel). Conversely, GST-tagged G3 bound an in vitro translated ER
fragment consisting of the DNA binding domain (DBD) and LBD in a hormone-
dependent manner (Figure 2-2B, lower panel).
Interestingly, the G3 fragment contains an LXXLL motif (where L represents leucine and
X denotes any amino acid) which is observed in many coactivators that interact with
hormone activated NR. To determine whether the LXXLL motif in G3 is required for the
hormone-dependent interaction with ERα, a GST pulldown assay was performed using a
mutant GelA fragment with Leu769 and Leu770 changed to Ala (i.e. LXXLL to
LXXAA). This mutation eliminated binding of GelA to hormone activated ERα (Fig 2-
2C). Furthermore, the same mutation in His-tagged full length Fli-I almost completely
eliminated binding to ERα LBD, compared with wild type Fli-I (Fig. 2-2D), suggesting
that the ER-Fli-I interaction is direct, that the G3 gelsolin repeat is the primary site in Fli-
I for binding to ER LBD, and that the LXXLL motif is critical for this interaction.

30

Fig. 2-2. Interaction of Fli-I with ER. (A) GST pulldown assays were performed with in
vitro translated FLAG-tagged LRR, GelA or GelB incubated in the absence or presence
of E2 with GST-fused ER-LBD bound to glutathione-Sepharose beads. Bound proteins
were analyzed by immunoblot with anti-FLAG antibody. (B) GST pulldown assays were
performed with in vitro translated small repeating modules (G1, G2, and G3) of Fli-I
incubated with GST-fused ER-LBD in the absence or presence of E2 (upper panel). In
vitro translated ER fragment containing DBD and LBD (AF2) was incubated with GST-
fused G3 fragment in the absence or presence of E2 (lower panel). (C,D) Leucine rich
motif in G3 is involved in hormone dependent interaction with ER LBD. GST pulldown
assays were performed with in vitro translated GelA or GelA(LL/AA) mutant protein (C),
or with E. coli expressed his-tagged full length Fli-I or Fli-I(LL/AA) mutant protein (D),
incubated with GST-ER LBD in the absence or presence of E2.

31

Fig. 2-2. Continued.

32
2.3 Fli-I interacts directly with BAF53.

We previously demonstrated by coimmunoprecipitation that the GelA fragment of Fli-I
binds BAF53, an actin-like component of the SWI/SNF complex (Lee et al., 2004). In
further mapping studies, deletion of the G1 module (amino acids 495-597) from His-
tagged full length Fli-I substantially decreased co-immunoprecipitation of HA-tagged
BAF53 by anti-His-tag antibodies from total cell extracts of transiently transfected 293T
cells (Fig. 2-3A). Thus the G1 fragment is required for binding of BAF53 to full length
Fli-I. Furthermore, purified recombinant His-tagged BAF53 was bound by GST-tagged
GelA, but not by GST-LRR or GST-GelB, indicating that the GelA-BAF53 interaction is
direct (Fig. 2-3B). The G1 fragment of GelA was sufficient to bind BAF53 (Fig. 2-3C).

A Glu-to-Lys mutation in the first actin-binding motif of the protein gelsolin reduces
binding of gelsolin to actin (Way, Pope & Weeds, 1992). We previously showed that the
analogous E585K mutation in the G1 motif of Fli-I reduced binding of the GelA fragment
of Fli-I to BAF53 (Lee et al., 2004). The same mutation in full length Fli-I substantially
reduced binding to BAF53 (Fig. 2-3D). This result confirmed that G1 is the primary
BAF53 binding site in Fli-I and that Glu585 in full-length Fli-I is critically involved in
the interaction with BAF53. In contrast, purified recombinant BAF53 did not bind to
GST-ER-LBD (Fig. 2-3E); thus any association between BAF53 and ER would have to
be indirect and could potentially be mediated by Fli-I.

33

Fig. 2-3. Fli-I interacts with BAF53 directly. (A, D) Coimmunoprecipitation was
performed with 1 ml total cell extracts from 293T cells (100 mm dish) transfected with
pSG5-HA-BAF53 (2 μg) and pTriEX4-Fli-I, pTriEX4-Fli-IΔG1 (lacking amino acids
495 to 597) or pTriEX4-Fli-I(E/K) (2 μg). Fli-I fragments were immunoprecipitated with
1 μg of anti-His-tag antibody or normal mouse IgG, and BAF53 was detected in
immunoblots with anti-HA antibody. (B) GST pulldown assays were performed with
bacterially expressed His-tagged BAF53 incubated with GST-fused Fli-I fragments.
Bound protein was analyzed by immunoblot with anti-His-tag antibody (Upper panel).
GST proteins eluted from the beads were visualized by Coomassie Blue staining (Lower
panel). (C) GST pulldown assay performed with bacterially expressed His-tagged BAF53
incubated with GST-fused G1 fragment of Fli-I. (E) GST pulldown assays were
performed with in vitro translated HA-tagged BAF53 incubated with GST-fused ER-
LBD in the absence or presence of E2. No bound protein was detected by immunoblot
with anti-HA antibody.

34

Fig. 2-3. Continued.

35
2.4 Fli-I binding to ER and to the SWI/SNF complex is important for ER-mediated
transcription.

Components of the SWI/SNF complex form a very strong protein complex which is
stable even in 3M urea, and most SWI/SNF subunits (e.g. BRG1, hBRM, and BAFs)
exist in complexes in vivo (Zhao et al., 1998). We failed to observe direct interaction of
BRG1 with Fli-I (data not shown), but we found that the GelA fragment of Fli-I binds to
BAF53 directly (Fig. 2-3B). Therefore, we speculated that Fli-I recruits the SWI/SNF
complex via interaction with BAF53. Among the three actin binding motifs in the GelA
region, the G1 fragment showed strong autonomous transcriptional activation when fused
to Gal4 DBD (Fig. 2-4A), and we also found that the G1 fragment is sufficient to interact
with BAF53 (Fig. 2-3C), suggesting that the recruitment of the SWI/SNF complex by G1
may account for the transcriptional activation. The observation that the G1(E585K)
mutant protein which does not bind to BAF53 showed much weaker transcriptional
activation (Fig. 2-4B) supports our hypothesis that Fli-I recruits the SWI/SNF complex
via the interaction with BAF53 to activate ER-mediated transcription.
To assess whether Fli-I requires interaction with ER or the SWI/SNF chromatin
remodeling complex for its coactivator function, we performed ER-mediated reporter
gene assay in SW13 cells. The SW13 cell lines contain no detectable BRG1 or hBRM
proteins (the ATPase core subunits of SWI/SNF), but nonetheless contains all the BAF
subunits of the SWI/SNF complex (Dunaief et al., 1994; Muchardt & Yaniv, 1993). ER-
mediated activation of estrogen responsive genes was diminished in these cells; but
reintroduction of the BRG1 protein induces formation of a functional SWI/SNF

36

Fig. 2-4. Fli-I binding to ER and to the SWI/SNF complex is important for ER-
mediated transcription. (A&B) G1 motif of Fli-I has automous activation function.
CV-1 cells were transfected with pGK1-luc reporter plasmid (200 ng) controlled by
Gal4 responsive elements, and expression plasmids encoding Gal4 DBD or Gal4 DBD
fused to each gelsolin-like motif (G1 through G6) or to the E585K mutant of the G1
fragment (200 ng). Transfected cells were grown for 2 days and harvested for
luciferase assays (bar graphs) and immunoblots using antibodies against Gal4 (upper
panel of B).

37

Fig. 2-4. Continued.

38

Fig. 2-4. Continued. (C) SW13 cells were tranfected with MMTV-ERE luciferase
reporter plasmid (200 ng) and expression plasmids encoding ERα (0.1 ng), BRG1 (10 ng
or 50 ng), and GelA, GelA(E585K), or GelA(LL/AA) (100 ng), as indicated. Transfected
cells were grown with E2 for 48 hours and luciferase activities of the transfected-cell
extracts were determined by luminometer (bar graph). Expression of wild type and
mutant GelA was monitored by immunoblot using antibodies against the FLAG epitope
(inset).

39

Fig. 2-4. Continued.

40
complex and restores estrogen stimulated gene expression, indicating that SWI/SNF
activity is essential for estrogen receptor mediated transcription. In our study wild type
GelA, GelA(E/K) or GelA(LL/AA) were transiently expressed with ERα in the absence
or presence of co-expressed BRG1. In the absence of BRG1, ERα failed to activate
transcription efficiently in the presence of E2 (Fig. 2-4C lanes 1-2). However, expression
of BRG1 in SW13 cells by transient transfection restored hormone dependent activation
of the reporter gene (lanes 3-4). Interestingly, the level of transcription was
synergistically enhanced by co-expression of BRG1 with GelA (lanes 6-7), which can
bind to ER through the G3 motif and to BAF53 in the SWI/SNF complex through the G1
motif. However, GelA alone did not activate reporter gene expression in the absence of
BRG1 (lane 5). In contrast, the GelA(E585K) mutant which does not bind to BAF53, and
the GelA(LL/AA) mutant which does not bind to ERα failed to cooperate functionally
with BRG1 and ERα (Fig. 2-4C lanes 8-13). These results further support the ability of
Fli-I to recruit the SWI/SNF complex to ER target promoters by the interaction of GelA
with ERα through the leucine-rich motif (LXXLL) in G3 and by the interaction of GelA
with BAF53 through the G1 motif.

41
2.5 Fli-I and the SWI/SNF chromatin-remodeling complex are recruited to the
promoter of an ER target gene, pS2.

Results from our protein interaction and transient reporter gene assays (Figs. 2-2 ~2-4)
indicated that the interaction of Fli-I with ER and with the SWI/SNF subunit BAF53 are
critical for the coactivator function of Fli-I. Therefore, we tested whether Fli-I is involved
in recruitment of SWI/SNF to the promoter region of an endogenous ER target gene. E2
treatment results in cyclical recruitment of ER and various coregulators to the pS2
promoter in MCF-7 cells (Burakov et al., 2002; Metivier et al., 2003; Shang et al., 2000).
ERα and Fli-I were associated with the pS2 promoter at 30 and 60 min after E2 treatment
of MCF-7 cells (Fig. 2-5A), but Fli-I was not recruited to the pS2 coding region (Fig. 2-
5B), demonstrating regional specificity of Fli-I recruitment. A more detailed time course
of Fli-I occupancy on the pS2 promoter during E2 treatment found that there were two
peaks of Fli-I occupancy around 20 and 80 min of E2 treatment (Fig. 2-5C). E2 also
induced occupancy of BRG1 on the pS2 promoter at 30 min; BRG1 occupancy was
reduced to the uninduced level at 60 min (Fig. 2-5A), consistent with the different timing
previously observed for recruitment of BRG1 versus ER (Metivier et al., 2003).

2.6 Role of Fli-I and BAF53 in cyclical, estrogen-dependent recruitment of
SWI/SNF complex to the pS2 promoter

Next, transfection with siRNA was used to test whether Fli-I is required for recruitment
of BRG1 to the pS2 promoter in response to E2. The siRNA against Fli-I reduced the

42

Fig. 2-5. Recruitment of ER, Fli-I and BRG1 to the pS2 promoter in MCF-7 cells.
Chromatin immunoprecipitation assays were performed with MCF-7 cells in 150 mm
dishes treated with 100 nM E2 or vehicle for 30 or 60 min. After immunoprecipitation of
cross-linked chromatin fragments with the indicated antibody, the amount of pS2
promoter region (A) or coding region (B) present was determined by qPCR. The data are
plotted as percentage of total input before immunoprecipitation and are from a single
experiment which is representative of at least two independent experiments. Error bars
represent range of variation of duplicate PCR reactions.

43

Fig. 2-5. Continued.

44

Fig. 2-5. Continued. (C) Recruitment of Fli-I to the pS2 promoter in MCF-7 cells.
Chromatin immunoprecipitation assays were performed with MCF-7 cells in 150 mm
dishes treated with 100 nM E2 for the indicated times. After immunoprecipitation of
cross-linked chromatin fragments with antibody against Fli-I, the amount of pS2
promoter region (left panel) or coding region (right panel) present was determined by
qPCR. The data are plotted as percentage of total input before immunoprecipitation.

45

Fig. 2-5. Continued.

C

46
endogenous cellular level of Fli-I protein and mRNA, but had no effect on the level of
ERα, BRG1, BAF53 or actin protein (Fig. 2-6D, two right panels). Using MCF-7 cells
transfected with non-specific or Fli-I-specific siRNA, we analyzed the occupancy of ER,
selected coregulators, and RNA polymerase II on the pS2 promoter at time points
between 0 and 120 min after adding E2. Although the peaks are small, we consistently
observed three distinct cycles of ERα recruitment to the pS2 promoter during 120 min of
E2 treatment, consistent with previous reports (Metivier et al., 2003); moreover, this
pattern of ERα occupancy of the pS2 promoter was the same in cells transfected with
non-specific siRNA or siRNA against Fli-I (Fig. 2-6A, left panel). We observed a large,
early peak of BRG1 recruitment to the pS2 promoter at or before 30 min of E2 treatment,
and a second smaller peak at approximately 90 min; this was also consistent with a
previous finding that the first peak of BRG1 recruitment overlaps with but reaches its
maximum before the initial peak of ER recruitment, and that subsequent peaks of BRG1
recruitment were out of phase with peaks of ER recruitment (Metivier et al., 2003). When
endogenous Fli-I protein level was reduced, the early peak of BRG1 recruitment was
eliminated, but the later peak was not (Fig. 2-6A, middle panel). The recruitment of
RNA polymerase II reached a peak at 60 min after the beginning of hormone treatment in
siNS transfected MCF7 cells and reached another peak at 120 min or later (right panel).
The two peaks of RNA polymerase II recruitment correspond to the second and third
peaks of ERα recruitment observed here and previously (Metivier et al., 2003). Depletion
of endogenous Fli-I substantially compromised the hormone-dependent recruitment of
RNA polymerase II at both 60 min and 120 min (Fig. 2-6A right panel).

47
Although these results were reproducible in multiple independent experiments, the small
number of time points in the first peak of BRG1 recruitment led us to examine in more
detail the early time period after E2 addition. We observed a broad peak of ER occupancy
on the pS2 promoter with a maximum value at approximately 40 min, while Fli-I and
BRG1 occupancy peaked at approximately 10-15 min (Fig. 2-6B). Transfection with
siRNA against Fli-I had no effect on recruitment of ER, but the early peaks of Fli-I and
BRG1 recruitment were eliminated. The reduction of Fli-I level by another siRNA
targeting a different part of the coding region of Fli-I caused a similar inhibition of BRG1
occupancy on the pS2 promoter (Fig. 2-6C). Thus endogenous Fli-I is required for the
initial peak of recruitment of BRG1, and presumably the SWI/SNF complex, to the pS2
promoter in response to E2.

In contrast to the effect on BRG1 recruitment, reduction of endogenous Fli-I had no
effect on the E2-dependent recruitment of Med1/TRAP220, a component of the Mediator
complex, to the pS2 promoter (Fig. 2-6D), demonstrating a specific role for Fli-I in
recruitment of BRG1. The Mediator complex is involved in recruitment of RNA
polymerase II to many promoters. Although the ability of Fli-I to bind BAF53 suggests a
possible mechanism for recruitment of SWI/SNF, Fli-I could potentially also interact
with other components of the SWI/SNF complex, such as actin, to which it is known to
bind (Davy et al., 2000; Davy et al., 2001); or Fli-I could influence SWI/SNF recruitment
by an indirect mechanism. To explore the role of BAF53 in Fli-I mediated recruitment of
SWI/SNF, additional ChIP assays were performed in MCF-7 cells with reduced levels of
BAF53. As when Fli-I levels were reduced, depletion of BAF53 by siRNA eliminated

48
the initial E2-induced peak of BRG1 recruitment to the pS2 promoter but did not affect
the second smaller peak of BRG1 recruitment (Fig. 2-6E). Thus, BAF53 as well as Fli-I
is required for the initial peak of BRG1 recruitment, consistent with a mechanism of
SWI/SNF recruitment by Fli-I which involves interaction of Fli-I with BAF53.

49

Fig. 2-6. Role of Fli-I and BAF53 in cyclical, estrogen-dependent recruitment of
SWI/SNF complex to the pS2 promoter. (A,B) Chromatin immunoprecipitation assays
were performed with MCF-7 cells as in Fig. 2-5 after transfection with siRNA against
Fli-I (siFli-I) or non-specific siRNA (siNS) and treatment with E2 for the indicated times.
Results shown are from a single experiment and are representative of at least two
independent experiments.

50

Fig. 2-6. Continued.

51

Fig. 2-6. Continued. (C) Recruitment of ERα and BRG1 to ER target gene promoters in
MCF-7 cells. Chromatin immunoprecipitation assays were performed with MCF-7 cells
as in Fig. 2-6A after transfection with siFli-I(2) or non-specific siRNA (siNS) and
treatment with E2 for the indicated times. Recruitment of ERα and BRG1 to the pS2
promoter (upper panel) and GREB1 (lower panel) promoter were determined by qPCR.

52

Fig. 2-6. Continued.

C

53

Fig. 2-6. Continued. (D&E) Role of Fli-I and BAF53 in the recruitment of SWI/SNF
complex and Mediator complex to the pS2 promoter. (D) ChIP assays were performed in
MCF-7 cells transfected 72 h previously with siFli-I or siNS. E2 or vehicle was added 45
or 90 min before crosslinking of chromatin, ChIP was performed with the indicated
antibodies, and the presence of the pS2 promoter region in the immunoprecipitates was
determined by qPCR (First two panels). Fli-I, ERα, BRG1, BAF53 and β-actin protein
levels from total cell lysates of MCF-7 cells were determined by immunoblot (Third
panel). Fli-I mRNA levels were determined by qRT-PCR (Fourth panel). (E) ChIP
assays were performed as in (A), but siRNA against BAF53 was substituted for siRNA
against Fli-I (Third panel). BAF53 mRNA levels were determined by qRT-PCR (First
panel). BAF53, ERα, Fli-I and BRG1 protein levels from total cell lysate of MCF-7 cells
were determined by immunoblot (Middle panel).

54

Fig. 2-6. Continued.

D
E

55
2.7 Role of Fli-I in E2-dependent recruitment of BRG1 to the GREB1 promoter.

We examined another ER target gene, GREB1, which has three putative estrogen
response elements. The most robust recruitment of ERα occurs at a site 1.6 kb upstream
from the transcription initiation site, and recruitment of ER occurs there in a cyclical
fashion, as with the pS2 promoter (Sun et al., 2007). We observed a cyclical recruitment
of BRG1 to the GREB1 promoter, with peaks at approximately 15 min and 90 min,
similar to what we observed on the pS2 promoter; but in contrast to the pS2 promoter, the
second peak of BRG1 recruitment was higher than the first peak on the GREB1 promoter
(compare Fig. 2-7A with Fig. 2-6A and E). As on the pS2 promoter, depletion of Fli-I by
siRNA transfection eliminated the first peak of BRG1 recruitment but had no effect on
the second peak (Fig. 2-7A). A more detailed analysis of the first 60 minutes of E2
treatment confirmed that depletion of Fli-I had no effect on ER recruitment but
eliminated the first peak of BRG1 recruitment (Fig. 2-7B). Similar results were obtained
with another siRNA targeting a different part of the coding region of Fli-I (Fig. 2-6C).
Thus Fli-I is required for recruitment of BRG1 to the GREB1 promoter as well as the pS2
promoter, suggesting a broader role for Fli-I in estrogen regulation of various ER target
genes.

56

Fig. 2-7. Role of Fli-I in cyclical, estrogen-dependent recruitment of SWI/SNF complex
to the GREB1 promoter. Recruitment of ER, BRG1, and Fli-I to the GREB1 promoter
was determined by ChIP assay as in Fig. 2-6 using specific primers for GREB1 promoter
region.

57

Fig. 2-7. Continued.

58
2.8 BAF53 and BRG1, as well as Fli-I, are required for expression of ER target
genes.

The requirement of both Fli-I and BAF53 for estrogen-dependent recruitment of BRG1 to
E2-responsive promoters leads us to propose that Fli-I recruits SWI/SNF through the
interaction of Fli-I with BAF53. According to this hypothesis, BAF53 and BRG1 as well
as Fli-I should be required for E2-dependent expression of ER target genes. Furthermore,
since Fli-I and BAF53 are required for the first but not subsequent peaks of BRG1
recruitment to ER target genes, reduction of endogenous Fli-I or BAF53 might affect the
kinetics of ER target gene expression in response to E2. We therefore performed a 48-
hour time course of ER target gene expression after adding E2 to siRNA-transfected
MCF-7 cells. Fli-I and BAF53 mRNA levels were effectively reduced in MCF7 cells by
the appropriate siRNA (Fig. 2-8A). Depletion of either Fli-I or BAF53 compromised the
E2-induced increase in mRNA levels of three ER target genes (pS2, GREB1, and
Cathepsin D) but had no effect on the level of β-actin mRNA, relative to GAPDH mRNA
(Fig. 2-8A). The reduction of target gene expression was consistent throughout the 48-
hour time period examined. The reduction of Fli-I or BAF53 level by another siRNA
targeting a different part of the coding region of Fli-I or BAF53 caused a similar
inhibition of ER target gene expression (Fig. 2-1D and 2-8D). Furthermore, depletion of
BRG1 by siRNA caused a similar reduction in E2-induced expression of ER target genes
(Fig. 2-8B), as expected, suggesting that loss of the initial peak of BRG1 recruitment
caused a sustained deficiency in E2-dependent transcription on these promoters. A
similar result was observed with a second siRNA targeting a different part of the coding

59
region of BRG1 (Fig. 2-8E). Thus both Fli-I and BAF53 are required for optimal
recruitment of BRG1 to E2-responsive promoters, and all three of these proteins are
required for E2-enhanced expression of ER target genes.

60

Fig. 2-8. Fli-I and BAF53 are required for estrogen-dependent expression of ER target
genes. (A) Endogenous Fli-I or BAF53 mRNA levels in MCF7 cells were determined
from total RNA preparations by qRT-PCR after transient transfection with the indicated
siRNAs. MCF7 cells transfected with non-specific siRNA or siRNA against Fli-I or
BAF53 were treated with 100 nM E2 for the indicated times, and mRNA levels for the
indicated ER target genes or β-actin gene were analyzed by qRT-PCR and normalized to
the level of GAPDH mRNA. Mean and range of variation for duplicate PCR reactions are
shown.

61

Fig. 2-8. Continued.

62

Fig. 2-8. Continued. (B) BRG1 is required for estrogen-dependent expression of ER
target genes. MCF7 cells were transfected with non-specific siRNA or siRNA against
BRG1, and mRNA levels for the indicated ER target genes were determined at various
times after the beginning of E2 treatment as in (A).

63

Fig. 2-8. Continued.

64

Fig. 2-8. Continued. (C) Proposed model for recruitment of SWI/SNF complex by Fli-I.
Fli-I is recruited to ER target genes in response to estradiol via binding to ER-LBD (via
the G3 module in the gelsolin-like domain of Fli-I) or binding to the N-terminal region of
GRIP1 or another p160 coactivator (via the leucine rich repeat region in the N-terminal
domain of Fli-I). Fli-I recruits the SWI/SNF complex by binding to BAF53 (via the G1
module in the gelsolin-like domain of Fli-I). Fli-I is required only for the initial peak of
recruitment of SWI/SNF after adding estradiol. Subsequent recruitment of SWI/SNF is
mediated by interactions of SWI/SNF with other undefined components of the
transcription complex. Thus, upon hormone treatment, Fli-I mediates the initial
recruitment of SWI/SNF to facilitate remodeling of chromatin structure.

65

Fig. 2-8. Continued.

66

Fig. 2-8. Continued. (D) Inhibition of the expression of ER target genes by siBAF53(2).
MCF-7 cells were transfected with siBAF53(2) or non-specific siRNA (siNS) and grown
in hormone free-medium. After 72 h cells were treated with 100 nM E2 or vehicle for 24
h. Total RNA was analyzed by qRT-PCR as in Fig. 2-8A. The levels of BAF53, pS2,
GREB1 and Cathepsin D mRNAs were normalized to that of GAPDH mRNA. BAF53
mRNA was measured without E2 treatment.

67

Fig. 2-8. Continued.

D

68

Fig. 2-8. Continued. (E) Inhibition of the expression of ER target genes by siBRG1(2).
MCF-7 cells were transfected with siBRG1(2) or non-specific siRNA (siNS) and grown
in hormone free-media. After 72 h cells were treated with 100 nM E2 or vehicle for 24 h.
Total RNA was analyzed by qRT-PCR as in Fig. 2-8B. The levels of BRG1, pS2 and
GREB1 mRNAs were normalized to that of GAPDH mRNA. BRG1 mRNA was
measured without E2 treatment.

69

Fig. 2-8. Continued.

E

70
DISCUSSION

Role of Fli-I in recruitment of SWI/SNF chromatin-remodeling complexes to
estrogen-responsive gene promoters.

While Fli-I was previously shown to enhance transient reporter gene activation by
nuclear receptors and was required for hormone dependent expression of the transient
reporter gene (Lee et al., 2004), we show here that Fli-I is required for efficient hormonal
induction of endogenous ER target genes (Figs. 2-1 & 2-8). In multiple independent
experiments, there was a strong linear correlation between the degree of reduction in Fli-I
mRNA and the degree of reduction in target gene expression. Furthermore, the slope for
the plot of % reduction of pS2 mRNA versus % reduction of Fli-I mRNA was close to 1
(Table 1-1), suggesting that Fli-I has a very important role in hormonal activation of ER
target genes.

SWI/SNF chromatin-remodeling complexes are required for ER-mediated transcriptional
activity (Chiba et al., 1994; DiRenzo et al., 2000). Guided by the findings that Fli-I binds
actin and actin related SWI/SNF component BAF53 (Davy et al., 2000; Davy et al.,
2001) and that Fli-I requires the SWI/SNF complex to activate ER-mediated transcription
in transient transfection assays (Fig. 2-4), we demonstrated that Fli-I plays an important
role in recruitment of BRG1-containing SWI/SNF complexes to two different ER target
genes (Figs. 2-6 & 2-7). Our results therefore suggest a model whereby SWI/SNF is
recruited to estrogen-responsive promoters by the interaction of its BAF53 subunit with

71
Fli-I (Fig. 2-8C). This result was unexpected, given the previous demonstrations that
various subunits of the SWI/SNF complex can interact with ERα and with the
glucocorticoid receptor (Belandia et al., 2002; Garcia-Pedrero et al., 2006; Hsiao et al.,
2003; Muchardt & Yaniv, 1993; Nie et al., 2000; Yoshinaga et al., 1992), and the implicit
assumption that these interactions were responsible for recruitment of SWI/SNF to
steroid hormone responsive promoters. While our results demonstrate that Fli-I is
required (presumably by bridging ER or ER-associated coactivators to BAF53) for
recruitment of SWI/SNF to estrogen responsive promoters, it is possible that interactions
of ER with additional subunits of SWI/SNF also contribute to SWI/SNF recruitment, as
discussed further below.

Fli-I, through the G3 module of its gelsolin-like domain, binds directly to the LBD of ER
in a hormone-dependent manner (Fig. 2-2), and the Fli-I G1 module binds directly to
BAF53 (Fig. 2-3). These findings support a model for Fli-I to recruit the SWI/SNF
complex to DNA-bound ER (Fig. 2-8C) and also provide an explanation for the
previously described ability of the GelA fragment of Fli-I to enhance transcriptional
activation by ERα in transient reporter gene assays (Lee et al., 2004) (Fig. 2-4C). Indeed,
the G1 module within the GelA fragment of Fli-I has a strong autonomous activation
domain when fused to Gal4 DBD (Fig. 2-4A), and this activity may be due to its ability
to recruit BAF53 and the associated SWI/SNF complex, since the E585K mutation in Fli-
I eliminated binding to BAF53 (Fig. 2-3D) and most of the transactivation activity of G1
(Fig. 2-4B). In contrast to CV-1 cells (Lee et al., 2004), GelA did not show coactivator
activity in SW13 cells, which lack BRG1 and hBRM. However, upon reintroduction of

72
BRG1 by transient transfection, GelA regained its coactivator function in a BRG1-
dependent manner, suggesting that Fli-I requires the SWI/SNF complex for its
coactivator function. Moreover, we found that the interactions with BAF53 and ER are
critical for GelA to activate ER-mediated transcription (Fig. 2-4C). Neither the
GelA(E585K) nor the GelA(LL/AA) mutant protein was able to activate transcription as
efficiently as wild type GelA in SW13 cells transfected with BRG1. However, in addition
to ER and BAF53, Fli-I can also bind to the p160 coactivator GRIP1 and to the protein
methyltransferase CARM1 (Lee et al., 2004); which of these interactions is responsible
for recruitment of Fli-I to estrogen-responsive promoters still remains to be determined.
Multiple contacts with ER and other coactivators may contribute to stable recruitment of
Fli-I to the promoter.

Roles of BAFs in chromatin-remodeling complexes.

It has been proposed that β-actin and actin-related proteins are required for the maximum
ATPase activity of SWI/SNF (Rando et al., 2002; Shen et al., 2003) and for the stable
association of chromatin-remodeling complexes with chromatin (Olave, Reck-Peterson &
Crabtree, 2002). Recently, it has been demonstrated that BAF53b, a neuron-specific
BAF53-related component of SWI/SNF, is specifically required for targeting SWI/SNF
to the promoter of genes essential for dendritic growth (Wu et al., 2007). Our findings
indicate an important role for BAF53 in E2-stimulated expression of ER target genes (Fig.
2-8). The essentially identical reductions in E2-dependent expression of ER target genes
caused by reduction of BAF53 or Fli-I (Fig. 2-8), coupled with the direct binding of

73
BAF53 to Fli-I in vitro (Fig. 2-3), further support the model that the interaction of Fli-I
with BAF53 is responsible for recruitment of SWI/SNF to promoters of ER target genes.
It was recently demonstrated that BAF53 is required for expression and inter-
chromosomal interactions between two ER target genes in response to E2 (Hu et al.,
2008). Our findings provide an apparent molecular mechanism for the involvement of
BAF53 in the reorganization of chromatin domains by showing that Fli-I is responsible
for linking BAF53 to the transcription complex formed by hormone-activated ER.

Other subunits of SWI/SNF complexes have previously been shown to bind steroid
hormone receptors. BAF57 binds to ERα and to p160 coactivators and is required for E2-
dependent expression of transiently transfected reporter genes and endogenous ER target
genes (Belandia et al., 2002; Garcia-Pedrero et al., 2006). Interaction of the
glucocorticoid receptor with SWI/SNF was reported to be mediated by BAF60a in a
ligand independent manner (Hsiao et al., 2003) and by BAF250 in a ligand dependent
manner (Nie et al., 2000). Similarly, the interaction of SWI/SNF complexes with
androgen receptor is mediated by direct interaction of androgen receptor with BAF57 and
BAF155 (Hong et al., 2005; Link et al., 2005). However, whether any of these
interactions is required for recruitment of SWI/SNF to steroid hormone-regulated target
genes has not been determined. Since SWI/SNF complexes are very large (~ 2 Mdal),
multiple protein-protein interactions might be involved in its association with the
transcription initiation complex assembled by ER.

74
Role of Fli-I in the complex temporal pattern of SWI/SNF recruitment, and
implications for the mechanism of transcriptional activation.

Chromatin immunoprecipitation studies defined an ordered, sequential, and cyclical
pattern of steady-state level occupancy by ER, various coregulators, and various histone
modifications on the pS2 promoter upon exposure of MCF-7 cells to E2 (Burakov et al.,
2002; Metivier et al., 2003; Shang et al., 2000). Multiple peaks of steady state ER
recruitment are observed, separated by intervals of approximately 40 min. After the
beginning of E2 treatment, BRG1 occupancy increases in parallel with ER recruitment
but reaches its first peak of occupancy at 10 min, before the first ER peak of occupancy;
subsequent peaks of BRG1 occupancy occur in between the peaks of ER occupancy and
coincide with recruitment of HDAC1 and HDAC7 (Metivier et al., 2003). It was
therefore proposed that the first peak of BRG1 occupancy helps to establish a chromatin
conformation permissive for transcription initiation, while the subsequent peaks of BRG1
recruitment facilitate clearance of ER and the transcription complexes from the promoter.

We observed a similar E2-induced cyclical pattern of pS2 and GREB1 promoter
occupancy by ER and BRG1 in MCF-7 cells, with the first peak of BRG1 recruitment
overlapping with but reaching a maximum before the first peak of ER occupancy and the
second peak of BRG1 recruitment occurring between peaks of ER occupancy (Figs. 2-6
& 2-7). Remarkably, we observed that only the initial peak of recruitment of BRG1 was
eliminated by reducing cellular levels of Fli-I or BAF53, while the second peak of BRG1
occupancy at 90 min still occurred normally (Figs. 2-6 & 2-7). These results suggest

75
that BRG1 and SWI/SNF complexes are recruited by Fli-I interaction with BAF53 during
the initial peak of BRG1 occupancy, but SWI/SNF uses a different mechanism of
recruitment that does not require Fli-I or BAF53 in subsequent peaks of recruitment. This
conclusion apparently fits well with the previous conclusion that the BRG1-based
SWI/SNF complex is required for promoting transcription during its first peak of
occupancy, but is involved with clearing ER and the transcription complex from the
promoter during subsequent peaks of recruitment (Metivier et al., 2003). In any case,
inhibition of only the initial, but not subsequent, peaks of BRG1 and SWI/SNF
recruitment by depletion of Fli-I or BAF53 has a profound effect on the E2-induced
expression of ER target genes (Fig. 2-8A). While the actual peak of Fli-I occupancy
occurs slightly after the first peak of BRG1 occupancy at 10 min, there may be sufficient
Fli-I on the promoter at 10 min to recruit BRG1. Alternatively, Fli-I may facilitate BRG1
recruitment by an indirect mechanism that is not currently apparent. In any case, our data
demonstrate that Fli-I is required for recruitment of SWI/SNF complexes to the promoter
to help establish the initial transcription initiation complex (Fig. 2-8C), while subsequent
rounds of promoter occupancy by SWI/SNF involve another mechanism of recruitment
which is yet undefined.

Implications for coordination of SWI/SNF and Mediator functions.

Whether chromatin remodeling by SWI/SNF-type complexes and histone modifying
enzymes is required for recruitment of the Mediator complex (which is implicated in
recruitment of RNA polymerase II), or vice-versa, has been a topic of considerable

76
debate and may depend on promoter context (Govind et al., 2005; He, Battistella &
Morse, 2008; Lemieux & Gaudreau, 2004; Wang et al., 2005; Yoon et al., 2003).
Sequential recruitment of the p160 coactivator complex (which contains several histone
modifying enzymes), Mediator complexes, and SWI/SNF-type complexes has been
proposed to account for their combinatorial and complementary actions in mediating
transcriptional activation (Burakov et al., 2002; Ito & Roeder, 2001; Metivier et al., 2003).
Our observations indicate that the recruitment of Mediator complex to the pS2 promoter
is not dependent on the initial recruitment of SWI/SNF, since depletion of Fli-I with the
accompanying elimination of the first peak of SWI/SNF recruitment had no effect on
recruitment of Mediator component Med1/TRAP220 (Fig. 2-6C). However, depletion of
Fli-I compromised recruitment of RNA polymerase II in response to hormone (Fig. 2-6A),
suggesting that both Fli-I and Mediator contribute to recruitment of RNA polymerase II
to the pS2 promoter after hormone treatment. We also recently showed that another
coactivator, CCAR1, is required for recruiting the Mediator complex to the pS2 promoter
(Kim et al., 2008). Since both Fli-I (this study) and CCAR1 (Kim et al., 2008) can
interact with ER and with components of the p160 complex, Fli-I and CCAR1 would
appear to provide a possible mechanism for coordination of the complementary functions
of three important coactivator complexes, i.e. p160, Mediator, and SWI/SNF. Thus, we
have begun to establish the functional relationships and interdependencies among the
many coactivators required for transcriptional activation.

77
CHAPTER 3: Recruitment of Tip60, a histone acetyltransferase, to
estrogen receptor regulated promoters.

INTRODUCTION

MYST (MOZ, YBF2, SAS2, and Tip60) family proteins have diverse cellular functions,
such as transcriptional regulation, DNA damage repair and apoptosis. (Ikura et al., 2000;
Kusch et al., 2004; Utley & Cote, 2003). All members in this family share the catalytic
MYST domain and a zinc finger domain and acetylate free histones in vitro. Tip60 was
originally isolated as a HIV-1 Tat Interacting Protein (Kamine et al., 1996) and later
identified as a histone acetyltransferase (Yamamoto & Horikoshi, 1997). The Tip60
complex was isolated and characterized in 2000 (Cai et al., 2003; Ikura et al., 2000). It
consists of at least 14 distinct subunits and displays HAT activity, ATPase activity, and
DNA helicase activity, suggesting a role of Tip60 in DNA repair. Most recently, it has
been shown that the direct interaction between the chromodomain of Tip60 and histone
H3K9me3 at DNA double-strand break (DSB) activates the acetyltransferase activity of
Tip60 (Sun et al., 2009). Recombinant Tip60 acetylates core histones H2A, H3 and H4 in
vitro (Kimura & Horikoshi, 1998; Yamamoto & Horikoshi, 1997) and, in the stable
multi-protein complex, it can acetylate nucleosomes even when linker histones are
present (Ikura et al., 2000). Apart from histones, several examples of non-histone protein
acetylation by Tip60 have been reported. Tip60 acetylates and activates ATM in HeLa
cells (Sun et al., 2005). Acetylation of p53 DNA-binding domain by Tip60 regulates
apoptosis induction in 293 and H1299 cells (Sykes et al., 2006; Tang et al., 2006).

78
MYC is another protein which is the substrate of the Tip60 (Patel et al., 2004).

In most cases, Tip60 acts as a coactivator for transcription. Tip60 is able to interact with
E2F1 and MYC at the target promoter and to enhance transactivation (Frank et al., 2003;
Taubert et al., 2004). Tip60 is also involved in the NF-kB signaling pathway. Without
stimulation, the KAI1 promoter is repressed by a corepressor complex consisting of N-
CoR/TAB2/HDAC3. IL-1β signaling activates MEKK1-dependent translocation of this
corepressor complex allowing the Tip60 complex to access to the promoter and activate
transcription (Baek et al., 2002).

Tip60 was identified as a binding protein for the ligand binding domain of AR in a yeast
two-hybrid screen, and it has been demonstrated that Tip60 coactivates AR, ER, and PR-
mediated transcription in transient transfection assay with LnCAP cell and Cos-1 cells
(Brady et al., 1999). Later, it was reported that the LXXLL motif of Tip60 is required and
sufficient for AR interaction (Gaughan et al., 2001). The mechanism for AR-mediated
transcription involves Tip60 forming a trimeric complex upon the endogenous AR-
responsive PSA promoter, and acetylation of AR by Tip60 regulates AR activity by
competition with HDAC1 (Gaughan et al., 2002). However, little is known about the
mechanism by which Tip60 is recruited upon hormonal activation in NR-mediated
transcription.

In the current study, we sought to determine the molecular mechanism of the recruitment
of Tip60 to ER-mediated promoters during transcription initiation. Thus, we tested

79
whether Tip60 interacts with ER or histone tails through C-terminal NR box or N-
terminal chromodomain. Finally, we investigated whether inhibition of chromatin
remodeling or disruption of Tip60 interaction with ER affects the optimal recruitment of
Tip60 to the pS2 promoter and subsequent transcription. Our findings indicate the
function of Tip60 in endogenous ER target gene expression and provide a model for three
key steps that may contribute to Tip60 recruitment and occupancy on estrogen-regulated
promoters.

EXPERIMENTAL PROCEDURES

Plasmids. pSG5-ERα and GST-protein expression vectors pGEX4T-ER(LBD) were
described previously (Jeong, Lee & Stallcup, 2009). Tip60 expression plasmid was
kindly provided by Dr. Anastasia Kralli (Scripps). pSG5-FLAG expression vectors
encoding the following proteins were constructed by inserting the appropriate cDNA
coding region into EcoRI and XhoI sites: pSG5-FLAG-Tip60 (1-100), pSG5-FLAG-
Tip60 (101-430), Point mutations in Tip60 were introduced by site-directed mutagenesis
kit (Stratagene).

Protein interaction assays and immunoblot. The procedure for GST-pulldown assays
was described previously (Jeong et al., 2009; Lee et al., 2004). GST-fusion proteins were
expressed in E. coli BL21(DE3) strain and purified by incubation with glutathione
Sepharose beads and washing with NETN buffer (300 mM NaCl, 1 mM EDTA, 20 mM
Tris-HCl (pH 8.0), and 0.01% NP-40). FLAG-tagged Tip60 fragments were

80
synthesized by transcription and translation in vitro using the TNT-Quick coupled
reticulocyte lysate system (Promega) according to the manufacturer’s protocol. For
coimmunoprecipitation assay, Cos-7 cells were plated at 1.5 X 10
6
cells per 10-cm dish
and transiently transfected using Lipofectamine 2000 (Invitrogen) and the indicated
amount of plasmids. At 48 h after transfection, cell extracts were prepared in 1.0 ml
RIPA buffer (50 mM Tris-Cl (pH 8.0), 150 mM NaCl, 1% NP-40, 1% sodium
deoxycholate, 0.1% sodium dodecyl sulfate, 2 mM EDTA). Immunoblotting was
performed as described previously (Jeong et al., 2009), using the following antibodies:
anti-ERα, anti-BRG1, anti-Tip60, and anti-β-actin and normal mouse or rabbit IgG
(Santa Cruz Biotechnology); anti-FLAG (Sigma Aldrich).

Chromatin immunoprecipitation assay. Chromatin immunoprecipitation (ChIP) assays
were performed according to previously described protocols (Jeong et al., 2009). Briefly,
MCF-7 cells were transiently transfected with plasmid encoding FLAG-tagged Tip60
wild type or mutant protein transiently and then cultured for 2 days in phenol red-free
Dulbecco’s modified Eagle medium (DMEM) supplemented with 5% charcoal-dextran-
stripped fetal bovine serum. Cells were treated with E2 for the indicated time, and ChIP
was performed with antibodies against FLAG epitope, and precipitated DNA was
analyzed by qRT-PCR. For the experiment involving siRNA treatment followed by ChiP,
MCF-7 cells were transfected with siRNA and then cultured for 3 days in phenol red-free
DMEM supplemented with 5% charcoal-dextran-stripped fetal bovine serum. At
approximately 90% confluency, cells were treated with 100 nM estradiol (E2) or vehicle
for the indicated time. After cross-linking with formaldehyde, cell extracts were

81
prepared from control and E2-treated MCF-7 cells. Immunoprecipitation of sonicated
chromatin solutions was conducted by overnight incubation at 4°C with normal mouse or
rabbit IgG, anti-ERα, anti-Fli-I, anti-BRG1, anti-TRAP220 (Santa Cruz Biotechnology)
or anti-RNA polymerase II (Millipore). Cross-linking was reversed by heating and
immunoprecipitated DNA was purified by phenol-chloroform extraction and ethanol
precipitation. The purified DNA was dissolved in 100 μl of TE buffer (10 mM Tris-HCl
(pH 8.0), 1 mM EDTA) and analyzed by quantitative PCR using the Stratagene Mx3000P
system with SYBRGreen dye. The primers used were: pS2 (ERE I), 5’-
GGCAGGCTCTGTTTGCTTAAAGAGCG-3’ (forward) and 5’-
GGCCATCTCTCACTATGAATCACTTCTGC-3’ (reverse); pS2 (ERE II), 5’-
CCTCCCCAGCTCACGTTGT-3’ (forward) and 5’-GGGTTGCATTTAAGGGACCTT-
3’ (reverse); pS2 (ERE III), 5’-GTCGTTGCCAGCGTTTCC-3’ (forward) and 5’-
CTTCTCCACGCCCTGTAAATTT-3’ (reverse); GREB1 promoter region, 5’-
GTGGCAACTGGGTCATTCTGA-3’ (forward) and 5’-
CGACCCACAGAAATGAAAAGG-3’ (reverse); Cathepsin D (ERE I), 5’-
GGAGCGGAGGGTCCATTC-3’ (forward) and 5’-TCCAGACATCCTCTCTGGAA-3’
(reverse); Cathepsin D (ERE II), 5’-CCTCCTCAACTGCTCTTGCA-3’ (forward) and
5’-GCGGCTGAGATGCTGAGTCA-3’ (reverse).

RNA interference and qRT-PCR. Small interfering RNA experiments were performed
according to previously published methods (Jeong et al., 2009; Lee et al., 2004). The
sequences of siRNA used were: siTip60, 5’-CGUCCAUUACAUUGACUUCdTdT-3’
(sense), 5’-GAAGUCAAUGUAAUGGAUGdTdT-3’ (anti-sense); siBRG1, 5’-

82
GCUUUCCUUGAAAUGCACUdTdT-3’ (sense) and 5’-
AGUGCAUUUCAAGGAAAGCdTdT-3’ (anti-sense); siNS, 5’-
UUCUCCGAACGUGUCACGUdTdT-3’ (sense) and 5’-
ACGUGACACGUUCGGAGAAdTdT-3’ (anti-sense) (Takata et al., 2007).
Transfections in MCF-7 cells were performed using Oligofectamine (Invitrogen)
according to the manufacturer’s protocol. Total RNA was isolated from MCF-7 cells with
Trizol (Invitrogen) after hormone treatment as indicated and subjected to reverse
transcription (RT) by iScript cDNA synthesis kit (Bio-Rad). 5 μl of RT product was used
for qPCR analysis with the following primers: pS2, 5’-GAACAAGGTGATCTGCG-3’
(forward) and 5’-TGGTATTAGGATAGAAGCACCA-3’ (reverse); Cyclin D1, 5’-
AAGCTCAAGTGGAACCT-3’ (forward) and 5’-AGGAAGTTGTTGGGGC-3’
(reverse) (Oxelmark et al., 2006); GREB1, 5’-CAAAGAATAACCTGTTGGCCCTGC-
3’ (forward) and 5’-GACATGCCTGCGCTCTCATACTTA-3’ (reverse) (Rae et al.,
2005); BRG1, 5’-CATCATCGTGCCTCTCTCAAC-3’ (forward) and 5’-
ACACGCACCTCGTTCTGCTG-3’ (reverse); β-actin, 5’-
ACCCCATCGAGCACGGCATCG-3’ (forward) and 5’-
GTCACCGGAGTCCATCACGATG-3’ (reverse) (Sun et al., 2007); GAPDH, 5’-
TCTGGTAAAGTGGATATTGTTG-3’ (forward) and 5’-GATGGTGATGGGATTTCC-
3’ (reverse) (Chen et al., 2005). Relative expression levels were normalized to GAPDH
mRNA levels.

Peptide interaction assays. Peptide pulldown assays were performed according to
previously described protocols (Collins et al., 2008). Briefly, 1.0 μg of biotinylated

83
histone H3 (1-21, Millipore) or H4 (1-19, Millipore) were bound to streptavidine-
sepharose beads (Amersham) for 1hr. Beads were then washed three times with binding
and washing buffer (25mM Tris, pH 8.0, 140mM NaCl, 3mM KCl and 0.1% (v/v)
Nonidet P-40) and incubated with in vitro translated Tip60 proteins overnight at 4
o
C in a
total volume of 500 μl of binding and washing buffer. Beads were then washed three
times each in 1 ml binding and washing buffer at 4
o
C. Bound proteins were eluted
directly by boiling in SDS-PAGE loading buffer and analyzed by western
immunoblotting.

RESULTS

3.1 Reduction of Endogenous Tip60 Attenuated the Expression of ER Target
Genes.

It has been reported that Tip60 enhances AR-mediated transactivation of transient
reporter gene in LnCAP cells and Cos-1 cells, and also enhances ER-mediated
transactivation a of vitellogenin reporter gene in transient transfection in LnCAP cells
(Brady et al., 1999; Gaughan et al., 2002). Here we tested the function of Tip60 in CV-1
cells with an estrogen responsive reporter gene with modified MMTV promoter, MMTV-
ERE reporter plasmid (Fig. 3-1A). Co-transfection of CV-1 cells with MMTV-ERE-luc,
ER, and Tip60 in the presence of estradiol led to a 3-fold increase in ER-mediated
transcription without any effect on basal levels of transcription. Next, we investigated the
function of Tip60 on endogenous ER target genes in MCF7 cells. Compared with non-

84

Fig. 3-1. Reduction of endogenous Tip60 attenuated the expression of ER target genes.
(A) CV-1 cells were transfected in 12-well plates with MMTV-ERE-luc reporter plasmid
(200 ng), pSG5-ERα (0.2 ng), and pCDNA-Tip60 (10 ng) as indicated and grown in
media containing 100nM E2. Cell extracts were assayed for luciferase activity. (B)
Depletion of Tip60 mRNA and protein by siRNA transfection. MCF-7 cells were
transfected with siRNA specific for Tip60 (siTip60) or non-specific siRNA (siNS) and
grown in hormone free-media for 72 h. Total RNA was analyzed for Tip60 mRNA by
qRT-PCR and normalized to the level of GAPDH mRNA. Protein levels of Tip60, ER,
BRG1, PRMT1, and β-actin were assessed by immunoblot. (C) Effect of reduced Tip60
on the expression of estrogen-responsive genes. MCF-7 cells were transfected with
siTip60 or siNS and grown in hormone free-media. After 72 h cells were treated with 100
nM E2 or vehicle for 24 h. Total RNA was analyzed by qRT-PCR. The levels of pS2,
GREB1, CyclinD1, and β-actin mRNAs were normalized to that of GAPDH mRNA.
These results are from a single experiment which is representative of three independent
experiments.

85

Fig. 3-1. Continued.

86
specific siRNA (siNS), siRNA specific for Tip60 (siTip60) effectively reduced Tip60
mRNA and protein level (Fig. 3-1B). The increases of pS2 and GREB1 gene expression
induced by E2 treatment were inhibited by knockdown of Tip60. The expression of
CyclinD1 was also significantly inhibited by depletion of Tip60 (Fig 3-1C). In contrast to
pS2 gene, the induction of CyclinD1 transcription was dramatically inhibited by
knockdown of Tip60, which was even below the basal transcriptional level in the absence
of E2, suggesting that Tip60 might regulate cyclinD1 gene expression in different manner.

3.2 Tip60 is recruited to ER-target gene promoters upon hormone treatment.

Previously, chromatin immunoprecipitation studies defined an ordered and cyclical
pattern of steady-state level occupancy by ER and various coactivators (Metivier 2003).
The recruitment of Tip60 follows that of ERα, BRG1, and PRMT1 during the first cycle
of ER recruitment and cycles with 45~60 min interval. This finding suggests that
chromatin remodeling followed by histone modification occurs at the very beginning
upon hormone treatment to open up chromatin structure and to provide binding sites for
other cofactors as well as protein complexes, including histone-modifying enzymes.

To further explore whether Tip60 is also recruited to other ER target gene promoters, we
investigated its recruitment to GREB1, Cathepsin D as well as the enhancer region of pS2
promoters induced by estradiol stimulation. In addition to the ERE at the proximal region,
the pS2 promoter has two ER binding sites located distal (~ -10 kb) from the transcription
start site (TSS), and it has been shown that ERs occupying at distal sites can recruit

87

Fig. 3-2. Recruitment of Tip60 to ER target gene promoters in MCF-7 cells. (A)
Chromatin immunoprecipitation assays were performed with MCF-7 cells in 150 mm
dishes treated with 100 nM E2 or vehicle for the indicated time. After
immunoprecipitation of cross-linked chromatin fragments with the indicated antibody,
the amount of pS2 promoter region present was determined by qPCR. The data are
plotted as percentage of total input before immunoprecipitation and are from a single
experiment which is representative of at least two independent experiments. Error bars
represent range of variation of duplicate PCR reactions.

88

Fig. 3-2. Continued.

89

Fig. 3-2. Continued. (B&C) Recruitment of Tip60 to GREB1 and Cathepsin D
promoters in MCF-7 cells. Chromatin immunoprecipitation assays were performed as in
Fig 3-2A.

90
coactivators and the RNA polymerase transcription machinery and mediate specific
structural changes to chromatin (Pan et al., 2008). Treatment of MCF-7 cells results in
cyclical recruitment of Tip60 to all three ERE sites of the pS2 promoter (Fig. 3-2A),
although we observed a slight difference in occupancy level. We observed an early peak
of Tip60 recruitment to the pS2 promoter at 15 min of E2 treatment and a second peak at
45 min.

There are three consensus EREs located at -21.2, -9.5, and -1.6 kb upstream of the TSS of
the GREB1 gene, which appear to mediate long-range gene activation by estrogen
receptor (Sun et al., 2007). Bretschneider et al. have reported that E2-mediated activation
of Cathepsin D expression involves looping of a distal enhancer element at 9 kb upstream
of the TSS (Bretschneider et al., 2008). E2 treatment to MCF-7 cells led to significant
recruitment of Tip60 to the Cathepsin D promoter as well as the GREB1 promoter (Fig.
3-2B). In contrast to the pS2 promoter, the recruitment of Tip60 to the Cathepsin D
promoter showed regional specificity between different EREs within one promoter.
Robust recruitment of Tip60 was observed at the distal enhancer region of the Cathepsin
D gene in a cyclical pattern, whereas, we failed to observe the recruitment on the
proximal region (Fig. 3-2C). This might suggest that different EREs might recruit
different coactivators within the same promoter by an unknown mechanism.

91
3.3 Estrogen receptor interacts with Tip60 in vitro and in vivo through an NR box
in a hormone dependent manner.

It has been reported that the LXXLL motif of Tip60 is required for AR activation
(Gaughan et al., 2001). Therefore, we speculated that Tip60 might be recruited to ER-
target gene promoters through direct interaction with ER. The ER-ligand binding domain
(LBD) fused to GST bound in vitro to full length Tip60 but not to the N-terminus and
middle region of Tip60, and the interaction was increased by E2 treatment (Fig. 3-3A).
These results suggest that C-terminal region containing the NR box is the major
interacting site for ER. In further in vitro binding studies, FLAG-tagged Tip60
coimmunoprecipitated with estrogen receptor in the presence and absence of E2, but E2
enhanced binding (Fig 3-3B). To assess whether Tip60 requires the LXXLL motif for its
interaction with ER, we repeated the binding assay using a mutant Tip60 with Leu-492
and Leu-493 changed to Ala (i.e. LXXLL to LXXAA). This mutation eliminated E2-
dependent binding to ER LBD in vitro (Fig. 3-2B) and in vivo (Fig. 3-3C and D)
compared with wild type Tip60, suggesting that the leucine-rich motif at the C-terminus
of Tip60 is involved in the hormone dependent interaction with ERα. To assess whether
the interaction through the LXXLL motif is functionally required for activation of ER
mediated-transcription, we performed ER-mediated reporter genes assays with wild type
and mutant Tip60. The level of transcription of the reporter gene was significantly
decreased by coexpression of mutant Tip60 (Fig. 3-3E). This result further supports that
the interaction with ER through the LXXLL motif is important for the coactivator
function of Tip60.

92

Fig. 3-3. Interaction with ER is important for coactivator function of Tip60. (A and B)
GST pulldown assays were performed with in vitro translated FLAG-tagged wild type
Tip60 or mutant Tip60 incubated in the absence or presence of E2 with GST-fused ER-
LBD bound to glutathione-Sepharose beads. Bound proteins were analyzed by
immunoblot with anti-FLAG antibody. (C and D) Coimmunoprecipitation assay were
performed with 1 ml total cell extracts from Cos-7 cells (100 mm dish) transfected with
pSG5-ERα (2 μg) and pSG5-FLAG-Tip60 or pSG5-FLAG-Tip60 (LL/AA) (2 μg).
Immunoprecipitation was performed with 1 μg of anti-FLAG antibody or normal mouse
IgG, and ERα was detected in immunoblots with anti-HA antibody.

93

Fig. 3-3. Continued.

94

Fig. 3-3. Continued. (E) CV-1 cells were tranfected with MMTV-ERE luciferase
reporter plasmid (200 ng) and expression plasmids encoding ERα (0.2 ng), and wild type
pCDNA-FLAG-Tip60 (wild type) or mutant Tip60 (10 ng), as indicated. Transfected
cells were grown with E2 for 48 hours, and luciferase activities of the transfected-cell
extracts were determined by luminometer (bar graph). Expression of wild type and
mutant Tip60 in CV-1 cells was monitored by immunoblot using antibodies against
FLAG epitope.

95

Fig. 3-3. Continued.

96
3.4 NR box plays a critical role for the recruitment of Tip60.

Our finding that the LXXLL motif of Tip60 is necessary for ER interaction and ER-
mediated transcription led us to hypothesize that the interaction through the LXXLL
motif is a possible mechanism for recruitment of Tip60 to ER target genes. To test our
hypothesis, chromatin immunoprecipitation was performed. We transiently
overexpressed FLAG–tagged wild type or mutant Tip60 in MCF7 cells. Cells were
treated with E2 for 20 and 40 min followed by cross-linking, and the recruitment of
exogenously expressed FLAG-tagged Tip60 was determined by pulling down chromatin
with anti FLAG antibody followed by qRT-PCR of the precipitated DNA. Upon E2
treatment, we observed a 4-fold increase of wild type Tip60 to the proximal region of the
pS2 promoter at 20 min and a 2-fold increase at 40 min (Fig. 3-4). In contrast to wild type
Tip60, the recruitment of mutant Tip60 was substantially decreased to almost the level
observed with non-transfected cells, which represents baseline. Western immunoblotting
results demonstrated that the expression levels of wild type and mutant Tip60 by transient
transfection were similar. Thus, the difference of recruitment was not due to a difference
in cellular protein levels. These results indicate that interaction with ER is required for
the promoter occupancy of Tip60 induced by E2, and the leucine rich motif at the C-
terminus of Tip60 plays a critical role.

97

Fig. 3-4. Interaction with ER is critical for Tip60 recruitment. FLAG-tagged Tip60 wild
type or LXXAA mutant Tip60 was transiently expressed in MCF-7 cells and grown in
hormone-free media. After 48 hours, chromatin immunoprecipitation assays were
performed with 100 nM E2 or vehicle for the indicated time. After immunoprecipitation
of cross-linked chromatin fragments with anti-FLAG antibody, the amount of pS2
promoter region present was determined by qPCR. Expression of wild type and mutant
Tip60 in MCF-7 cells was monitored by immunoblot using antibodies againt FLAG
epitope.

98

Fig. 3-4. Continued.

99
3.5 Chromatin remodeling is also required for the recruitment of Tip60.

Although it appears that ER plays an important role for the recruitment of Tip60 to ER
target gene promoters, we also observed that knockdown of BRG1, a core subunit in the
SWI/SNF complex, resulted in a decrease of Tip60 recruitment, although ER was still
associated with the pS2 promoter at 20 and 40 min (Fig. 3-5B). These findings led us to
investigate in more detail the early period after E2 treatment (Fig. 3-5C). Transient
transfection of siRNA against BRG1 effectively reduced endogenous BRG1 level, but
had little effect on Tip60 level (Fig. 3-5A). After the beginning of E2 treatment, Tip60
was associated with the pS2 promoter at 10 to 20 min and we observed another peak of
recruitment at 40 to 50 min in MCF-7 cells transfected with non-specific siRNA (siNS).
Again, when the endogenous BRG1 level was reduced, the two peaks of Tip60
recruitment were dramatically reduced (Fig. 3-5C). Thus, endogenous BRG1 protein,
presumably as part of the SWI/SNF chromatin-remodeling complex, is also required for
promoter occupancy by Tip60. Interestingly, it appears that Tip60 was recruited
effectively at the very beginning of each cycle (i.e. at 10 min and 40 min) even after
depletion of BRG1, but the extent and duration of occupancy was dramatically reduced.
This implies that an alternative mechanism must be involved in the stable occupancy of
Tip60 on the pS2 promoter (e.g. interaction with ER).

100

Fig. 3-5. Recruitment of Tip60 in BRG1 depleted MCF-7 cells. (A) Endogenous BRG1
was depleted by transfection of siRNA against BRG1 (siBRG1) in MCF-7 cells. BRG1
and Tip60 level were determined by qRT-PCR and western immunoblotting. (B)
Chromatin immunoprecipitation assays were performed with MCF-7 cells as in Fig. 3-2
after treatment with E2 for the indicated times.

101

Fig. 3-5. Continued.

102

Fig. 3-5. Continued. (C) Kinetic ChIP experiment of recruitment of Tip60 in BRG1
depleted MCF-7 cells. Chromatin immunoprecipitation assays were performed with
MCF-7 cells as in Fig. 3-5(B) after transfection with siRNA against BRG1 (siBRG1) or
non-specific siRNA (siNS) and treatment with E2 for the indicated times.

103

Fig. 3-5. Continued.

104
3.6 Tip60 interacts with methylated H3 in vitro.

It has been reported that the initial transcription process is promoted through the
engagement of the SWI/SNF complex followed by the recruitment of histone
methyltransferases (HMTs) and histone acetyltransferases (HATs) (Metivier et al., 2003),
In addition, the N-terminus of Tip60 contains a chromodomain, and chromodomain of
some other proteins are known to bind methylated histones (Jacobs & Khorasanizadeh,
2002; Nielsen et al., 2002; Sapountzi et al., 2006). Based on these observations, we
hypothesized that Tip60 binding to methylated histone tails, which is triggered by
chromatin remodeling and subsequent histone methylation by HMTs, is the alternative
mechanism for Tip60 recruitment mentioned in the previous section. Thus, we
determined whether Tip60 binds to any methylated histone tails. Recently, it has been
reported that Tip60 binds to histone H3K9me3 during the DNA double-strand break
repair process, and this interaction is required to activate the acetyltransferase activity of
Tip60 (Sun et al., 2009). Here, we determined whether Tip60 binds to active methylation
marks of histone tails, such as histone H3K4, H3R17, and H4R3. To identify the binding
site on H3 or H4, biotinylated peptides corresponding to the N-terminal tails of histone
H3 or H4 and containing various methylation modifications were used for peptide
pulldown assays (Fig. 3-6A). In vitro translated Tip60 was incubated with methylated H3
or H4 peptide bound on affinity resin. Tip60 specifically bound to mono- and di-
methylated histone H3K4, but failed to bind to methylated H3R17 and H4R3. In
chromatin immunoprecipitation assays, it appears that the recruitment of BRG1 precedes
Tip60 recruitment (Fig. 3-6B). Therefore, these results imply that the recruitment of

105

Fig. 3-6. Tip60 binds to methylated histone H3K4. (A) In vitro translated Tip60 protein
was incubated with beads (B), or beads containing histone H3 that is unmodified (U),
mono- (me1), or di- (me2) methylated on Lys 4 or Arg 17 or beads containing histone
H4 dimethylated on Arg 3. Bound Tip60 was detected by SDS-PAGE with anti-FLAG
antibody.

106

Fig. 3-6. Continued. (B) BRG1 and Tip60 recruitment to the pS2 promoter. Chromatin
immunoprecipitation assays were performed with MCF-7 cells as in Fig. 3-2 after
treatment with E2 for the indicated times.

107

Fig. 3-6. Continued. (C) Proposed model for recruitment of Tip60 to ER-mediated target
gene promoter. SWI/SNF complex is recruited in response to estradiol via binding to Fli-I
which binds to ER ligand binding domain. SWI/SNF complex remodels chromatin
structure to allow histone methyltransferases to be recruited and methylate histone H3 tail.
Initially, Tip60 is recruited by the interaction with ERα via LXXLL motif at C-terminus,
then it binds to methylated histone H3K4.

108

Fig. 3-6. Continued.

109
Tip60 to the pS2 promoter might be regulated through at least three key steps (Fig. 3-6C).
First, hormone-bound ER recruits Tip60 to pS2 promoter region through the interaction
with LXXLL motif at the C-terminus of Tip60. Second, SWI/SNF complexes remodel
chromatin structure and recruit HMTs, and then 3) histone methylation by HMTs is
required to make an additional contact with Histone H3 tails, and this leads to formation
of a stable Tip60 complex at the promoter region.

110
DISCUSSION

Role of Tip60 in Nuclear receptor mediated transcription

Tip60 binds to and acetylates androgen receptor in a hormone-dependent manner to
generate hyperacetylated, active AR (Gaughan et al., 2002). It also has been shown that
Tip60 coactivates ER-mediated transcription in transient transfection assays (Brady et al.,
1999; Gaughan et al., 2001). However, in contrast to its function in the DNA double
strand break repair process, little is known about the exact role of Tip60 in the nuclear
receptor signaling pathway. Here, we show that Tip60 is required for efficient hormonal
induction of endogenous ER target genes (Fig. 3-1), confirming the important function of
Tip60 on ER-mediated transcription of endogenous genes. The induction of a subset of
ER target gene by E2 treatment was compromised by depletion of Tip60 in MCF-7 cells.
Interestingly, Tip60 appears to regulate both basal expression and hormone-responsive
expression of Cyclin D1, implying a distinct role for Tip60 on different ER target genes.

Differential Recruitment and Actions of Tip60 on Different ER Target Genes.

It is known that Tip60 is recruited after hormone treatment to the proximal region of the
pS2 promoter (Metivier et al., 2003). However, the recruitment Tip60 to other ER target
gene promoters and the mechanism by which Tip60 is recruited to ER-regulated
promoters are not known. We showed the cyclical occupancy of Tip60 on enhancer
region of the pS2 promoter, which includes the recently reported distal ERE sites (Pan

111
et al., 2008), as well as the proximal ERE. Although we observed a slight difference in
occupancy level, Tip60 showed cyclical occupancy at all of three ERE sites with an
initial peak at 15 min and second peak at 45 min after the beginning of E2 treatment (Fig.
3-2A). Tip60 recruitment on the Cathepsin D promoter showed a dramatic contrast in
ERE specificity. The ERE II site, -8.7 kb upstream from the TSS, recruited Tip60 in
response to E2 treatment; however, we failed to observe significant recruitment on the
ERE I site at -0.1 kb from the TSS, although there is no severe difference of ER
occupancy on these two ERE sites (Bretschneider et al., 2008). These results suggest that
ER on different EREs might recruit different subset of coactivators, leading to different
regulation on different target genes. Recently, it has been reported that DNA sequences at
GR binding sites, differing by as little as a single base pair, differently affect GR
conformation and regulatory activity (Meijsing et al., 2009). In light of these findings,
Tip60 might be recruited by different mechanisms to different EREs or in different
promoter contexts; this could lead to different contributions by Tip60 to expression of
different target genes. Interestingly, knockdown of Tip60 had little or no effect on
Cathepsin D gene expression induced by E2 (data not shown), in contrast to a strong
requirement for Tip60 for pS2 or GREB1 gene expression, supporting the proposed for
different function of Tip60 on different ER target genes.

Role of the SWI/SNF complex in recruitment of Tip60 to Estrogen-responsive gene
promoters

BRG1 is one of the first enzymatic proteins to be recruited to the pS2 promoter after

112
initiation of E2 treatment, followed by histone-modifying enzymes, and general
transcriptional factors (Metivier et al., 2003). The exact mechanism by which promoters
assemble various proteins or protein complexes in a precise manner has not been
determined yet; however, it is believed that the SWI/SNF chromatin-remodeling complex
probably generates permissive chromatin structure to make an accessible environment for
the recruitment of other cofactors. I have been intrigued by the fact that 1) the
recruitment of BRG1 to the pS2 promoter precedes the recruitment of Tip60; 2) extensive
histone modification events occur after BRG1 recruitment including histone methylation
and acetylation; 3) Tip60 contains a chromodomain which (in other chromodomain
proteins) is known to bind to methylated lysine residues of histone tails; and 4) most
recently, it has been reported that the chromodomain of Tip60 binds to histone H3K9me3
during the DNA double-strand break (DSB) repair process (Sun et al., 2009). Therefore,
we investigated whether Tip60 requires chromatin remodeling and modified histone tails
for its optimal recruitment to the pS2 promoter. Interestingly, knockdown of BRG1, a
core subunit in the SWI/SNF complex, resulted in decrease of Tip60 recruitment
although ER was still associated with the pS2 promoter at 20 and 40 min (Fig. 3-4).
These results suggest that, in addition to its interaction with ER, there might be another
mechanism for Tip60 recruitment which is dependent on the chromatin-remodeling by
the SWI/SNF complex. To confirm our results, we examined in more detail the early
period after initiation of E2 treatment (Fig. 3-5). We observed a broad peak of Tip60
occupancy on the pS2 promoter at 10 ~ 20 min and second peak at 45 min. Depletion of
BRG1 substantially compromised the E2-dependent Tip60 recruitment at 20 and 40 min.
Although the promoter occupancy of Tip60 was affected by the inhibition of SWI/SNF

113
function at 15 and 20 min in early peak and 45 ~ 60 min in second peak, we observed a
minor recruitment of Tip60 at the very beginning of each peak at 10 and 40 min. These
results suggest that an alternative mechanism might be involved at the beginning of each
Tip60 recruitment cycle. For example, association with ER activated by E2 might be
responsible for initial recruitment of Tip60 during each cycle of occupancy, while the
SWI/SNF chromatin remodeling complex is also required for the continued occupancy by
Tip60 as well as the continued interaction of Tip60 with ERα.

Interaction of Tip60 with methylated histone H3.

By a large-scale RNAi screen in embryonic stem cells (ESCs), it has been revealed that
Tip60-p400 complex, a histone acetyltransferase and nucleosome remodeling complex, is
necessary for maintenance of ESC identity, and p400 localization strongly correlated with
H3K4me3 at active genes in ESCs (Fazzio, Huff & Panning, 2008). But many H3K4me3
marks are not associated with the Tip60-p400 complex. It appears that H3K4me3 is
necessary, but not sufficient, for targeting of Tip60-p400. Nanog and H3K4me3 appear to
act independently to promoter binding of Tip60-p400 to its targets. Here, we demonstrate
that Tip60 directly interacts with methylated histone H3K4 in vitro, but not with other
active histone marks. This result suggests that methylated histone H3K4 also might
contribute to Tip60 targeting during hormonal activation of ER target genes. The finding
that the initial part of each cyclical recruitment of Tip60 was still detectable even after
depletion of the SWI/SNF complex (Fig. 3-5C) may support the idea that interaction of
ER LBD with the LXXLL motif of Tip60 is critically required for initial recruitment of

114
Tip60 at the beginning of each cycle of occupancy (Fig. 3-4). It appears that Tip60 is
initially recruited via the interaction with ERα through the LXXLL motif of Tip60 in
response to E2, and this interaction is critical for overall recruitment. Then SWI/SNF
complex is recruited to promoter via binding to Fli-I, a nuclear receptor coactivator
which binds to the ER ligand binding domain in response to estradiol (Jeong et al., 2009).
Thus, I propose that the SWI/SNF complexes remodel chromatin structure to allow
histone methylatransfeases to be recruited and methylate histone H3 tail. Tip60, which is
initially recruited by ERα, now binds to methylated histone H3K4 to make a strong
contact (Fig. 3-6C).

This idea is supported by the recently proposed model, ‘recruitment and stabilization’
(Ruthenburg, Allis & Wysocka, 2007). In this model, the recognition of a histone
modification is likely not sufficient for stable protein or protein complex recruitment;
rather a sequence specific recruitment is mediated by a site-specific DNA binding
transcription factor and stabilized subsequently by interaction with a modified histone.
This was exemplified with NURF, which is initially recruited by GAGA transcription
factor. Once recruited, NURF occupancy is stabilized through the interaction with H3K4
methyl-lysine mark.

Six MLL family proteins including MLL1, MLL2, MLL3, MLL4, SET1A, and SET1B,
have been documented as histone H3K4 methyltransferases. SET7/SET9 is another
member of the SET domain-containing family in humans, and it also specifically
methylates Lys4 on histone H3 (Nishioka et al., 2002). Global analysis of histone

115
H3K4 methylation status and gene expression revealed that dimethyl- and trimethyl
histone H3K4 marks are enriched at active genes (Bernstein et al., 2002; Bernstein et al.,
2005; Ng et al., 2003; Santos-Rosa et al., 2002). Further studies will provide us
knowledge on whether promoter occupancy by Tip60 involves interaction with
methylated histone H3 and which HMT is involved in Tip60 recruitment.

116
CHAPTER 4: Concluding Remarks

Differential expression of the eukaryotic genome is achieved by precise modulation of its
spatial organization as well as change of chromatin structure (Cabal et al., 2006; Casolari
et al., 2005; Spector, 2003). To overcome the structural restriction, modification of
chromatin structures including chromatin remodeling and specific posttranslational
modifications of histone are directed at the specific transcription site by various enzymes.
The mechanisms of transcriptional regulation by nuclear receptors have been extensively
studied over the past 30 years. Recently, advances in microarray technologies and
analysis methods for large-scale genomic expression studies have generated an extensive
amount of information about nuclear receptor mediated transcription (Carroll et al., 2006;
Frasor et al., 2003; Kininis et al., 2007; Lin et al., 2007; Rae et al., 2005). Moreover,
location analysis of nuclear receptor binding by chromatin immunoprecipitation
combined with microarray (ChIP-on-chip) provided massive data on global nuclear
receptor binding site associated with NR target genes.

However, the mechanistic contributions of diverse events induced by chromatin-
remodeling, histone modifications, and protein-protein interactions to transcriptional
activation and the mechanisms that coordinate the activities of multiple coactivator
complexes on promoters of ER target genes remain elusive. In this thesis, we sought to
elucidate the molecular mechanisms by which various chromatin modification events are
tightly regulated and by which nuclear receptors coordinate the activity of different
coregulator complexes, thus eventually leading to assembly of transcription initiation

117
complexes. Here, studies on Fli-I and Tip60 have contributed substantially to our
understanding of these processes.

A yeast two-hybrid screen for proteins that bind to CARM1 identified the protein
Flightless I (Fli-I), an actin-binding protein, which has essential roles in Drosophila and
mouse development. Since cofilin was identified as the first actin-binding protein in 1987,
(Nishida et al., 1987), other members have been discovered including proflilin, thymosin
β4, CapG, gelsolin, Flightless I, myopodin, α−actinins, plastins, supervillin and filaminA.
Although most of them are predominantly localized in cytoplasm, they can be
translocated into the nucleus under certain conditions such as stress, differentiation or cell
stimulation by hormone. Recently, some members of the gelsolin family of actin-binding
proteins are known to be involved in nuclear receptor-mediated signaling. Supervillin and
gelsolin are capable of interacting with AR in an androgen dependent manner and
enhance AR-mediated expression of transient reporter gene (Nishimura et al., 2003; Ting
et al., 2002). Although the mechanism of transcriptional activation is not clear yet, these
results support the function of Fli-I in nuclear receptor mediated transcription.

Fli-I makes multiple contacts and synergistically enhances transcription with CARM1,
nuclear receptors, and GRIP1, thus qualifying it as a nuclear receptor coactivator (Lee et
al., 2004). Interestingly, the C-terminus gelsolin-like domain of Fli-I enhanced ER-
mediated transcription and the interaction with BAF53 was required for ER activity (Lee
et al., 2004). Based on these results, we hypothesized that Fli-I facilitates interaction of
nuclear receptors or the p160 coactivator complex with BAF53 containing protein

118
complexes in the nucleus.

Here, we showed a series of results supporting our hypothesis. First, depletion of cellular
Fli-I by siRNA compromised expression of a subset of ER-regulated target genes induced
by E2 treatment. The effect of Fli-I depletion was remarkable and reproducible so that
the difference between non-specific siRNA transfected cells and cells transfected with
siRNA targeting Fli-I was statistically significant in a paired t-test. Our recent microarray
experiments demonstrated that around one third of E2-regulated genes are affected by
depletion of Fli-I in MCF-7 cells (data not shown), suggesting a critical role for Fli-I in
ER-mediated transcription. Secondly, we demonstrated that Fli-I interacts with BAF53 as
well as ER via different part of the gelsolin-like C-terminal region of BAF53, and both
interactions are critically required for Fli-I coactivator function. Especially, transient
transfection assays with SW13 cells clearly show that Fli-I requires the SWI/SNF
complex to activate ER-driven transcription. Our finding was highlighted in chromatin
immunoprecipitation experiments in Fli-I depleted MCF-7 cells. Depletion of
endogenous Fli-I or BAF53 specifically eliminated part of the complex cyclical pattern of
recruitment of SWI/SNF to estrogen-responsive promoters, suggesting a critical role of
Fli-I and BAF53 interaction for SWI/SNF recruitment.

Here, a question arises as to what the role of other BAF proteins is in the SWI/SNF
complex? Previously, other subunits of SWI/SNF complexes have been shown to bind
steroid hormone receptors. BAF57, a HMG family protein is known to bind to ER and
AR (Belandia et al., 2002; Garcia-Pedrero et al., 2006). BAF60a and BAF250 were

119
reported to mediate SWI/SNF interaction to GR in a ligand independent or dependent
manner (Hsiao et al., 2003), (Nie et al., 2000). However, it is still not clear whether any
of these interactions is required for recruitment of SWI/SNF to steroid hormone-regulated
target genes. Since SWI/SNF complexes are very large (~ 2 Mdal), multiple protein-
protein interactions might be involved in its association with the transcription initiation
complex assembled by ER.

Another interesting question relates to the function of BRM-containing SWI/SNF
complexes in ER-mediated expression and how they are recruited to the promoter.
Human SWI/SNF chromatin remodeling complexes contain either BRG1 or hBRM as a
core ATPase subunit. Although BRG1 and hBRM are evolutionally conserved with a
considerable degree of amino acid sequence homology, their functions appear to be
somewhat different. BRG1
-/-
knockout mice are embryonically lethal and heterozygous
BRG1
-/-
mice are viable but cancer-prone (Bultman et al., 2000). However, BRM
-/-
mice
develop normally with increased body weight and are not cancer-prone (Reyes et al.,
1998). Moreover, BRG1 and hBRM are differentially expressed in various normal tissues.
BRG1 is known to be predominantly expressed in proliferating or self-renewaling cells,
whereas the BRM protein level increases during cellular differentiation (Reisman et al.,
2005). Differential requirement of SWI/SNF for androgen receptor activity on different
target genes has been reported (Marshall et al., 2003). Complete ligand-dependent
activation of two distinct AR target promoters (PSA and probasin) require SWI/SNF
function. Interestingly, AR stimulation on the probasin promoter could be partially
induced with BRG1, but hBRM strongly stimulated AR activity, whereas, the PSA

120
promoter was only induced by the restoration of hBRM, but not with BRG1. However,
the reason why different promoters have distinct preferences for different SWI/SNF
complexes still remains unclear.

The recruitment of the SWI/SNF complex to the pS2 promoter is quite interesting for
several reasons. First, it is the first known enzymatic complex to be recruited to the
promoter after E2 treatment. It has been proposed that the first peak of BRG1 occupancy
helps to establish a chromatin conformation permissive for transcription initiation.
Secondly, only BRG1-containing, but not hBRM-containing, SWI/SNF complexes are
recruited at the initial cycle, although they contain most of the BAF subunits in common.
Thirdly, subsequent peaks of BRG1 occupancy on the pS2 promoter occur in between the
peaks of ER occupancy and coincide with recruitment of HDAC1 and HDAC7 (Metivier
et al., 2003). This implies that SWI/SNF complexes might have at least two different
functions on the promoter and use different mechanisms of recruitment for each peak.
Our data showing that depletion of Fli-I affects only initial recruitment of SWI/SNF
complex but not subsequent peaks, clearly support this idea.

Given that SRC family coactivators and the Mediator complex appear to be capable of
interacting with a common binding site in NR, sequential recruitment to target gene
promoters by thyroid hormone receptor has been proposed by Ito and Roeder (Ito &
Roeder, 2001). A combinatorial model also has been proposed by Huang et al.: all the
chromatin remodeling, histone modification and Mediator complexes can be jointly
recruited by the chromatin-bound AR or TR via an adaptor molecule, and histone

121
modification by one coactivator (p300) has a role in the recruitment of some complexes
(SWI/SNF and Mediator). (Huang et al., 2003). This idea contributed to understanding of
the kinetics of transcriptional activation through the definition of an ordered sequence of
recruitment required to complete and activate the RNA polymerase II (PolII) complex
(Berk, 1999; McKenna & O'Malley, 2002; Metivier et al., 2008). The largest advance on
understanding of the kinetics of transcription complex assembly was achieved using ERα
transcriptional regulation on the pS2 gene promoter (Metivier et al., 2003). In the
presence of hormone, there are at least three distinct cycles of ER occupancy: an initial
transcriptionally unproductive cycle followed by at least two different transcriptionally
productive cycles (Metivier et al., 2003). It was believed that the first unproductive cycle
is required to generate a permissive chromatin structure for binding of other cofactors;
and during the transcriptionally productive cycle, the recruitment of liganded ERα
initiates the process of a sequential assembly of components to initiate transcription of
the pS2 gene.

The finding that only the initial peak of recruitment of BRG1 was eliminated by reducing
the cellular levels of Fli-I or BAF53 was surprising to us. These results support not only
that the SWI/SNF complexes use different mechanisms for recruitment at each cycle, but
also the notion of a combinatorial model which means different protein assemblies occur
on the same promoter at different time to achieve effective expression of the pS2 gene.
Besides recruitment and activation of PolII, coregulator recruitment during the
transcriptional productive cycle is correlated with the subsequent complete removal of
the PolII machinery (Metivier et al., 2003). Indeed, it is HDACs, in association with the

122
SWI/SNF complex that restricts the engagement of ERα and cofactors with the promoter
at the end of the first productive cycle. Although only the initial recruitment of SWI/SNF
was affected by knockdown of Fli-I, we observed significant decrease of pS2 gene
expression induced by E2, implying the critical role of the unproductive cycle for overall
gene expression.

During the initial cycle, PRMT1 is known to be recruited after BRG1, followed by p300
or Tip60, but not CARM1 or other HATs. PRMT1 is known to methylate histone H4R3,
whereas CARM1 methylates Histone H3R17, and both of them are marks for actively
transcribed gene. The finding that the recruitment of HMTs precedes that of HATs
suggests a possible mechanism for the requirement of histone methylation marks for
Tip60 recruitment. Although its function is not clear yet, Tip60 contains a chromodomain,
which is known to bind to methylated lysine residues in some other chromodomain
proteins (i.e HP-1). This led us to investigate the binding of Tip60 to methylation marks
on Histone H3 and H4 that are generally associated with active chromatin. Interestingly
Tip60 interacts with Histone H3 tails containing mono- and dimethyl-lysine at Lys 4
residue in peptide pulldown assays, but Tip60 didn’t bind to other active marks such as
Histone H3R17 or Histone H4R3. These results suggest a new function for the Tip60
chromodomain involved in coregulator recruitment and activation of transcription
initiation.

Tip60 is also able to interact with ER through its C-terminus LXXLL motif in a hormone
dependent manner. Introduction of point mutations in the LXXLL motif compromised

123
promoter occupancy by Tip60. However, it appears that recruitment of Tip60 also
requires chromatin-remodeling activity generated by SWI/SNF complexes, since
depletion of BRG1 also partially inhibits Tip60 promoter occupancy even though the
association of ER was not changed by knockdown of BRG1.

Thus, the accumulated data suggest three steps that may contribute to Tip60 recruitment
and occupancy on estrogen-regulated promoters: 1) chromatin remodeling by SWI/SNF;
2) interaction with ER; and 3) binding to methylated histones. Further studies are
required to identify the HMT which is responsible for Tip60 recruitment. (e. g. MLLs,
SET1 or SET9/7).

In conclusion, our study on Fli-I and Tip60 has opened new insight into the dynamic,
cyclical nature of transcription complex assembly and has presented new perspectives
and challenges. Our further study on the biological function of Fli-I and Tip60 will allow
us to better understand the molecular mechanism of transcription initiation by nuclear
receptors and coactivators.

124
BIBLIOGRAPHY

ARONICA, S. M., KRAUS, W. L. & KATZENELLENBOGEN, B. S. (1994). Estrogen action via
the cAMP signaling pathway: stimulation of adenylate cyclase and cAMP-regulated gene
transcription. Proc Natl Acad Sci U S A 91, 8517-21.

BAEK, S. H., OHGI, K. A., ROSE, D. W., KOO, E. H., GLASS, C. K. & ROSENFELD, M. G.
(2002). Exchange of N-CoR corepressor and Tip60 coactivator complexes links gene
expression by NF-kappaB and beta-amyloid precursor protein. Cell 110, 55-67.

BECKER, P. B., TSUKIYAMA, T. & WU, C. (1994). Chromatin assembly extracts from
Drosophila embryos. Methods Cell Biol 44, 207-23.

BELANDIA, B., ORFORD, R. L., HURST, H. C. & PARKER, M. G. (2002). Targeting of
SWI/SNF chromatin remodelling complexes to estrogen-responsive genes. EMBO J 21,
4094-103.

BERK, A. J. (1999). Activation of RNA polymerase II transcription. Curr Opin Cell Biol
11, 330-5.

BERNSTEIN, B. E., HUMPHREY, E. L., ERLICH, R. L., SCHNEIDER, R., BOUMAN, P., LIU, J.
S., KOUZARIDES, T. & SCHREIBER, S. L. (2002). Methylation of histone H3 Lys 4 in
coding regions of active genes. Proc Natl Acad Sci U S A 99, 8695-700.

BERNSTEIN, B. E., KAMAL, M., LINDBLAD-TOH, K., BEKIRANOV, S., BAILEY, D. K.,
HUEBERT, D. J., MCMAHON, S., KARLSSON, E. K., KULBOKAS, E. J., 3RD, GINGERAS, T. R.,
SCHREIBER, S. L. & LANDER, E. S. (2005). Genomic maps and comparative analysis of
histone modifications in human and mouse. Cell 120, 169-81.

BOYER, L. A., LATEK, R. R. & PETERSON, C. L. (2004). The SANT domain: a unique
histone-tail-binding module? Nat Rev Mol Cell Biol 5, 158-63.

BRADY, M. E., OZANNE, D. M., GAUGHAN, L., WAITE, I., COOK, S., NEAL, D. E. &
ROBSON, C. N. (1999). Tip60 is a nuclear hormone receptor coactivator. J Biol Chem 274,
17599-604.

BRETSCHNEIDER, N., SARA, K., SEIFERT, M., REID, G., GANNON, F. & DENGER, S. (2008).
E2-mediated cathepsin D (CTSD) activation involves looping of distal enhancer elements.
Mol Oncol 2, 182-90.

BULTMAN, S., GEBUHR, T., YEE, D., LA MANTIA, C., NICHOLSON, J., GILLIAM, A.,
RANDAZZO, F., METZGER, D., CHAMBON, P., CRABTREE, G. & MAGNUSON, T. (2000). A
Brg1 null mutation in the mouse reveals functional differences among mammalian
SWI/SNF complexes. Mol Cell 6, 1287-95.

125
BURAKOV, D., CROFTS, L. A., CHANG, C. P. & FREEDMAN, L. P. (2002). Reciprocal
recruitment of DRIP/mediator and p160 coactivator complexes in vivo by estrogen
receptor. J Biol Chem 277, 14359-62.

CABAL, G. G., GENOVESIO, A., RODRIGUEZ-NAVARRO, S., ZIMMER, C., GADAL, O., LESNE,
A., BUC, H., FEUERBACH-FOURNIER, F., OLIVO-MARIN, J. C., HURT, E. C. & NEHRBASS,
U. (2006). SAGA interacting factors confine sub-diffusion of transcribed genes to the
nuclear envelope. Nature 441, 770-3.

CAI, Y., JIN, J., TOMOMORI-SATO, C., SATO, S., SOROKINA, I., PARMELY, T. J., CONAWAY,
R. C. & CONAWAY, J. W. (2003). Identification of new subunits of the multiprotein
mammalian TRRAP/TIP60-containing histone acetyltransferase complex. J Biol Chem
278, 42733-6.

CAMPBELL, H. D., SCHIMANSKY, T., CLAUDIANOS, C., OZSARAC, N., KASPRZAK, A. B.,
COTSELL, J. N., YOUNG, I. G., DE COUET, H. G. & MIKLOS, G. L. (1993). The Drosophila
melanogaster flightless-I gene involved in gastrulation and muscle degeneration encodes
gelsolin-like and leucine-rich repeat domains and is conserved in Caenorhabditis elegans
and humans. Proc Natl Acad Sci U S A 90, 11386-90.

CARROLL, J. S., MEYER, C. A., SONG, J., LI, W., GEISTLINGER, T. R., EECKHOUTE, J.,
BRODSKY, A. S., KEETON, E. K., FERTUCK, K. C., HALL, G. F., WANG, Q., BEKIRANOV, S.,
SEMENTCHENKO, V., FOX, E. A., SILVER, P. A., GINGERAS, T. R., LIU, X. S. & BROWN, M.
(2006). Genome-wide analysis of estrogen receptor binding sites. Nat Genet 38, 1289-97.

CARRUTHERS, L. M. & HANSEN, J. C. (2000). The core histone N termini function
independently of linker histones during chromatin condensation. J Biol Chem 275,
37285-90.

CASOLARI, J. M., BROWN, C. R., DRUBIN, D. A., RANDO, O. J. & SILVER, P. A. (2005).
Developmentally induced changes in transcriptional program alter spatial organization
across chromosomes. Genes Dev 19, 1188-98.

CHEN, D., MA, H., HONG, H., KOH, S. S., HUANG, S. M., SCHURTER, B. T., ASWAD, D. W.
& STALLCUP, M. R. (1999a). Regulation of transcription by a protein methyltransferase.
Science 284, 2174-7.

CHEN, Y. H., KIM, J. H. & STALLCUP, M. R. (2005). GAC63, a GRIP1-dependent nuclear
receptor coactivator. Mol Cell Biol 25, 5965-72.

CHEN, Z., YUHANNA, I. S., GALCHEVA-GARGOVA, Z., KARAS, R. H., MENDELSOHN, M. E.
& SHAUL, P. W. (1999b). Estrogen receptor alpha mediates the nongenomic activation of
endothelial nitric oxide synthase by estrogen. J Clin Invest 103, 401-6.

126
CHIBA, H., MURAMATSU, M., NOMOTO, A. & KATO, H. (1994). Two human homologues
of Saccharomyces cerevisiae SWI2/SNF2 and Drosophila brahma are transcriptional
coactivators cooperating with the estrogen receptor and the retinoic acid receptor. Nucleic
Acids Res 22, 1815-20.

COLLINS, R. E., NORTHROP, J. P., HORTON, J. R., LEE, D. Y., ZHANG, X., STALLCUP, M. R.
& CHENG, X. (2008). The ankyrin repeats of G9a and GLP histone methyltransferases are
mono- and dimethyllysine binding modules. Nat Struct Mol Biol 15, 245-50.

DAVY, D. A., BALL, E. E., MATTHAEI, K. I., CAMPBELL, H. D. & CROUCH, M. F. (2000).
The flightless I protein localizes to actin-based structures during embryonic development.
Immunol Cell Biol 78, 423-9.

DAVY, D. A., CAMPBELL, H. D., FOUNTAIN, S., DE JONG, D. & CROUCH, M. F. (2001). The
flightless I protein colocalizes with actin- and microtubule-based structures in motile
Swiss 3T3 fibroblasts: evidence for the involvement of PI 3-kinase and Ras-related small
GTPases. J Cell Sci 114, 549-62.

DEURING, R., FANTI, L., ARMSTRONG, J. A., SARTE, M., PAPOULAS, O., PRESTEL, M.,
DAUBRESSE, G., VERARDO, M., MOSELEY, S. L., BERLOCO, M., TSUKIYAMA, T., WU, C.,
PIMPINELLI, S. & TAMKUN, J. W. (2000). The ISWI chromatin-remodeling protein is
required for gene expression and the maintenance of higher order chromatin structure in
vivo. Mol Cell 5, 355-65.

DIRENZO, J., SHANG, Y., PHELAN, M., SIF, S., MYERS, M., KINGSTON, R. & BROWN, M.
(2000). BRG-1 is recruited to estrogen-responsive promoters and cooperates with factors
involved in histone acetylation. Mol Cell Biol 20, 7541-9.

DIRSCHERL, S. S. & KREBS, J. E. (2004). Functional diversity of ISWI complexes.
Biochem Cell Biol 82, 482-9.

DUNAIEF, J. L., STROBER, B. E., GUHA, S., KHAVARI, P. A., ALIN, K., LUBAN, J.,
BEGEMANN, M., CRABTREE, G. R. & GOFF, S. P. (1994). The retinoblastoma protein and
BRG1 form a complex and cooperate to induce cell cycle arrest. Cell 79, 119-30.

EBERHARTER, A. & BECKER, P. B. (2004). ATP-dependent nucleosome remodelling:
factors and functions. J Cell Sci 117, 3707-11.

FAN, H. Y., HE, X., KINGSTON, R. E. & NARLIKAR, G. J. (2003). Distinct strategies to
make nucleosomal DNA accessible. Mol Cell 11, 1311-22.

FAZZIO, T. G., HUFF, J. T. & PANNING, B. (2008). An RNAi screen of chromatin proteins
identifies Tip60-p400 as a regulator of embryonic stem cell identity. Cell 134, 162-74.

127
FRANK, S. R., PARISI, T., TAUBERT, S., FERNANDEZ, P., FUCHS, M., CHAN, H. M.,
LIVINGSTON, D. M. & AMATI, B. (2003). MYC recruits the TIP60 histone
acetyltransferase complex to chromatin. EMBO Rep 4, 575-80.

FRASOR, J., DANES, J. M., KOMM, B., CHANG, K. C., LYTTLE, C. R. &
KATZENELLENBOGEN, B. S. (2003). Profiling of estrogen up- and down-regulated gene
expression in human breast cancer cells: insights into gene networks and pathways
underlying estrogenic control of proliferation and cell phenotype. Endocrinology 144,
4562-74.

GARCIA-PEDRERO, J. M., KISKINIS, E., PARKER, M. G. & BELANDIA, B. (2006). The
SWI/SNF chromatin remodeling subunit BAF57 is a critical regulator of estrogen
receptor function in breast cancer cells. J Biol Chem 281, 22656-64.

GAUB, M. P., BELLARD, M., SCHEUER, I., CHAMBON, P. & SASSONE-CORSI, P. (1990).
Activation of the ovalbumin gene by the estrogen receptor involves the fos-jun complex.
Cell 63, 1267-76.

GAUGHAN, L., BRADY, M. E., COOK, S., NEAL, D. E. & ROBSON, C. N. (2001). Tip60 is a
co-activator specific for class I nuclear hormone receptors. J Biol Chem 276, 46841-8.

GAUGHAN, L., LOGAN, I. R., COOK, S., NEAL, D. E. & ROBSON, C. N. (2002). Tip60 and
histone deacetylase 1 regulate androgen receptor activity through changes to the
acetylation status of the receptor. J Biol Chem 277, 25904-13.

GAVIN, I., HORN, P. J. & PETERSON, C. L. (2001). SWI/SNF chromatin remodeling
requires changes in DNA topology. Mol Cell 7, 97-104.

GILL, G. (2004). SUMO and ubiquitin in the nucleus: different functions, similar
mechanisms? Genes Dev 18, 2046-59.

GOODSON, H. V. & HAWSE, W. F. (2002). Molecular evolution of the actin family. J Cell
Sci 115, 2619-22.

GOTTLICHER, M., HECK, S. & HERRLICH, P. (1998). Transcriptional cross-talk, the second
mode of steroid hormone receptor action. J Mol Med 76, 480-9.

GOVIND, C. K., YOON, S., QIU, H., GOVIND, S. & HINNEBUSCH, A. G. (2005).
Simultaneous recruitment of coactivators by Gcn4p stimulates multiple steps of
transcription in vivo. Mol Cell Biol 25, 5626-38.

HANSEN, J. C. (2002). Conformational dynamics of the chromatin fiber in solution:
determinants, mechanisms, and functions. Annu Rev Biophys Biomol Struct 31, 361-92.

128
HE, Q., BATTISTELLA, L. & MORSE, R. H. (2008). Mediator requirement downstream of
chromatin remodeling during transcriptional activation of CHA1 in yeast. J Biol Chem
283, 5276-86.

HIRSCHHORN, J. N., BROWN, S. A., CLARK, C. D. & WINSTON, F. (1992). Evidence that
SNF2/SWI2 and SNF5 activate transcription in yeast by altering chromatin structure.
Genes Dev 6, 2288-98.

HONG, C. Y., SUH, J. H., KIM, K., GONG, E. Y., JEON, S. H., KO, M., SEONG, R. H., KWON,
H. B. & LEE, K. (2005). Modulation of androgen receptor transactivation by the SWI3-
related gene product (SRG3) in multiple ways. Mol Cell Biol 25, 4841-52.

HSIAO, P. W., FRYER, C. J., TROTTER, K. W., WANG, W. & ARCHER, T. K. (2003).
BAF60a mediates critical interactions between nuclear receptors and the BRG1
chromatin-remodeling complex for transactivation. Mol Cell Biol 23, 6210-20.

HU, Q., KWON, Y. S., NUNEZ, E., CARDAMONE, M. D., HUTT, K. R., OHGI, K. A., GARCIA-
BASSETS, I., ROSE, D. W., GLASS, C. K., ROSENFELD, M. G. & FU, X. D. (2008).
Enhancing nuclear receptor-induced transcription requires nuclear motor and LSD1-
dependent gene networking in interchromatin granules. Proc Natl Acad Sci U S A 105,
19199-204.

HUANG, Z. Q., LI, J., SACHS, L. M., COLE, P. A. & WONG, J. (2003). A role for cofactor-
cofactor and cofactor-histone interactions in targeting p300, SWI/SNF and Mediator for
transcription. EMBO J 22, 2146-55.

IKURA, T., OGRYZKO, V. V., GRIGORIEV, M., GROISMAN, R., WANG, J., HORIKOSHI, M.,
SCULLY, R., QIN, J. & NAKATANI, Y. (2000). Involvement of the TIP60 histone acetylase
complex in DNA repair and apoptosis. Cell 102, 463-73.

ITO, M. & ROEDER, R. G. (2001). The TRAP/SMCC/Mediator complex and thyroid
hormone receptor function. Trends Endocrinol Metab 12, 127-34.

JACOBS, S. A. & KHORASANIZADEH, S. (2002). Structure of HP1 chromodomain bound to
a lysine 9-methylated histone H3 tail. Science 295, 2080-3.

JENUWEIN, T. & ALLIS, C. D. (2001). Translating the histone code. Science 293, 1074-80.

JEONG, K. W., LEE, Y. H. & STALLCUP, M. R. (2009). Recruitment of the SWI/SNF
chromatin remodeling complex to steroid hormone-regulated promoters by nuclear
receptor coactivator flightless-I. J Biol Chem 284, 29298-309.

JOHN, S., SABO, P. J., JOHNSON, T. A., SUNG, M. H., BIDDIE, S. C., LIGHTMAN, S. L., VOSS,
T. C., DAVIS, S. R., MELTZER, P. S., STAMATOYANNOPOULOS, J. A. & HAGER, G. L.
(2008). Interaction of the glucocorticoid receptor with the chromatin landscape. Mol Cell
29, 611-24.

129
JOHNSON, T. A., ELBI, C., PAREKH, B. S., HAGER, G. L. & JOHN, S. (2008). Chromatin
remodeling complexes interact dynamically with a glucocorticoid receptor-regulated
promoter. Mol Biol Cell 19, 3308-22.

KADAM, S. & EMERSON, B. M. (2003). Transcriptional specificity of human SWI/SNF
BRG1 and BRM chromatin remodeling complexes. Mol Cell 11, 377-89.

KAJAVA, A. V., VASSART, G. & WODAK, S. J. (1995). Modeling of the three-dimensional
structure of proteins with the typical leucine-rich repeats. Structure 3, 867-77.

KAMINE, J., ELANGOVAN, B., SUBRAMANIAN, T., COLEMAN, D. & CHINNADURAI, G.
(1996). Identification of a cellular protein that specifically interacts with the essential
cysteine region of the HIV-1 Tat transactivator. Virology 216, 357-66.

KIM, J. H., LI, H. & STALLCUP, M. R. (2003). CoCoA, a nuclear receptor coactivator
which acts through an N-terminal activation domain of p160 coactivators. Mol Cell 12,
1537-49.

KIM, J. H., YANG, C. K., HEO, K., ROEDER, R. G., AN, W. & STALLCUP, M. R. (2008).
CCAR1, a key regulator of mediator complex recruitment to nuclear receptor
transcription complexes. Mol Cell 31, 510-9.

KIMURA, A. & HORIKOSHI, M. (1998). Tip60 acetylates six lysines of a specific class in
core histones in vitro. Genes Cells 3, 789-800.

KININIS, M., CHEN, B. S., DIEHL, A. G., ISAACS, G. D., ZHANG, T., SIEPEL, A. C., CLARK,
A. G. & KRAUS, W. L. (2007). Genomic analyses of transcription factor binding, histone
acetylation, and gene expression reveal mechanistically distinct classes of estrogen-
regulated promoters. Mol Cell Biol 27, 5090-104.

KOBE, B. & DEISENHOFER, J. (1995a). Proteins with leucine-rich repeats. Curr Opin
Struct Biol 5, 409-16.

KOBE, B. & DEISENHOFER, J. (1995b). A structural basis of the interactions between
leucine-rich repeats and protein ligands. Nature 374, 183-6.

KUSCH, T., FLORENS, L., MACDONALD, W. H., SWANSON, S. K., GLASER, R. L., YATES, J.
R., 3RD, ABMAYR, S. M., WASHBURN, M. P. & WORKMAN, J. L. (2004). Acetylation by
Tip60 is required for selective histone variant exchange at DNA lesions. Science 306,
2084-7.

LANGST, G. & BECKER, P. B. (2001). ISWI induces nucleosome sliding on nicked DNA.
Mol Cell 8, 1085-92.

130
LAURENT, B. C., TREITEL, M. A. & CARLSON, M. (1991). Functional interdependence of
the yeast SNF2, SNF5, and SNF6 proteins in transcriptional activation. Proc Natl Acad
Sci U S A 88, 2687-91.

LEE, Y. H., CAMPBELL, H. D. & STALLCUP, M. R. (2004). Developmentally essential
protein flightless I is a nuclear receptor coactivator with actin binding activity. Mol Cell
Biol 24, 2103-17.

LEGUBE, G., LINARES, L. K., LEMERCIER, C., SCHEFFNER, M., KHOCHBIN, S. & TROUCHE,
D. (2002). Tip60 is targeted to proteasome-mediated degradation by Mdm2 and
accumulates after UV irradiation. EMBO J 21, 1704-12.

LEGUBE, G. & TROUCHE, D. (2003). Identification of a larger form of the histone acetyl
transferase Tip60. Gene 310, 161-8.

LEMIEUX, K. & GAUDREAU, L. (2004). Targeting of Swi/Snf to the yeast GAL1 UAS G
requires the Mediator, TAF IIs, and RNA polymerase II. EMBO J 23, 4040-50.

LIN, C. Y., STROM, A., LI KONG, S., KIETZ, S., THOMSEN, J. S., TEE, J. B., VEGA, V. B.,
MILLER, L. D., SMEDS, J., BERGH, J., GUSTAFSSON, J. A. & LIU, E. T. (2007). Inhibitory
effects of estrogen receptor beta on specific hormone-responsive gene expression and
association with disease outcome in primary breast cancer. Breast Cancer Res 9, R25.

LINK, K. A., BURD, C. J., WILLIAMS, E., MARSHALL, T., ROSSON, G., HENRY, E.,
WEISSMAN, B. & KNUDSEN, K. E. (2005). BAF57 governs androgen receptor action and
androgen-dependent proliferation through SWI/SNF. Mol Cell Biol 25, 2200-15.

LONARD, D. M. & O'MALLEY, B. W. (2006). The expanding cosmos of nuclear receptor
coactivators. Cell 125, 411-4.

LOSEL, R. & WEHLING, M. (2003). Nongenomic actions of steroid hormones. Nat Rev
Mol Cell Biol 4, 46-56.

LUGER, K., MADER, A. W., RICHMOND, R. K., SARGENT, D. F. & RICHMOND, T. J. (1997).
Crystal structure of the nucleosome core particle at 2.8 A resolution. Nature 389, 251-60.

LUSSER, A. & KADONAGA, J. T. (2003). Chromatin remodeling by ATP-dependent
molecular machines. Bioessays 25, 1192-200.

MARINO, M., ACCONCIA, F., BRESCIANI, F., WEISZ, A. & TRENTALANCE, A. (2002).
Distinct nongenomic signal transduction pathways controlled by 17beta-estradiol regulate
DNA synthesis and cyclin D(1) gene transcription in HepG2 cells. Mol Biol Cell 13,
3720-9.

131
MARSHALL, T. W., LINK, K. A., PETRE-DRAVIAM, C. E. & KNUDSEN, K. E. (2003).
Differential requirement of SWI/SNF for androgen receptor activity. J Biol Chem 278,
30605-13.

MCKENNA, N. J. & O'MALLEY, B. W. (2002). Combinatorial control of gene expression
by nuclear receptors and coregulators. Cell 108, 465-74.

MEIJSING, S. H., PUFALL, M. A., SO, A. Y., BATES, D. L., CHEN, L. & YAMAMOTO, K. R.
(2009). DNA binding site sequence directs glucocorticoid receptor structure and activity.
Science 324, 407-10.

METIVIER, R., HUET, G., GALLAIS, R., FINOT, L., PETIT, F., TIFFOCHE, C., MEROT, Y.,
LEPERON, C., REID, G., PENOT, G., DEMAY, F., GANNON, F., FLOURIOT, G. & SALBERT, G.
(2008). Dynamics of estrogen receptor-mediated transcriptional activation of responsive
genes in vivo: apprehending transcription in four dimensions. Adv Exp Med Biol 617,
129-38.

METIVIER, R., PENOT, G., HUBNER, M. R., REID, G., BRAND, H., KOS, M. & GANNON, F.
(2003). Estrogen receptor-alpha directs ordered, cyclical, and combinatorial recruitment
of cofactors on a natural target promoter. Cell 115, 751-63.

MIGLIACCIO, A., DI DOMENICO, M., CASTORIA, G., DE FALCO, A., BONTEMPO, P., NOLA,
E. & AURICCHIO, F. (1996). Tyrosine kinase/p21ras/MAP-kinase pathway activation by
estradiol-receptor complex in MCF-7 cells. EMBO J 15, 1292-300.

MUCHARDT, C. & YANIV, M. (1993). A human homologue of Saccharomyces cerevisiae
SNF2/SWI2 and Drosophila brm genes potentiates transcriptional activation by the
glucocorticoid receptor. EMBO J 12, 4279-90.

NARLIKAR, G. J., FAN, H. Y. & KINGSTON, R. E. (2002). Cooperation between complexes
that regulate chromatin structure and transcription. Cell 108, 475-87.

NG, H. H., ROBERT, F., YOUNG, R. A. & STRUHL, K. (2003). Targeted recruitment of Set1
histone methylase by elongating Pol II provides a localized mark and memory of recent
transcriptional activity. Mol Cell 11, 709-19.

NIE, Z., XUE, Y., YANG, D., ZHOU, S., DEROO, B. J., ARCHER, T. K. & WANG, W. (2000).
A specificity and targeting subunit of a human SWI/SNF family-related chromatin-
remodeling complex. Mol Cell Biol 20, 8879-88.

NIELSEN, P. R., NIETLISPACH, D., MOTT, H. R., CALLAGHAN, J., BANNISTER, A.,
KOUZARIDES, T., MURZIN, A. G., MURZINA, N. V. & LAUE, E. D. (2002). Structure of the
HP1 chromodomain bound to histone H3 methylated at lysine 9. Nature 416, 103-7.

132
NILSSON, S., MAKELA, S., TREUTER, E., TUJAGUE, M., THOMSEN, J., ANDERSSON, G.,
ENMARK, E., PETTERSSON, K., WARNER, M. & GUSTAFSSON, J. A. (2001). Mechanisms of
estrogen action. Physiol Rev 81, 1535-65.

NISHIDA, E., IIDA, K., YONEZAWA, N., KOYASU, S., YAHARA, I. & SAKAI, H. (1987).
Cofilin is a component of intranuclear and cytoplasmic actin rods induced in cultured
cells. Proc Natl Acad Sci U S A 84, 5262-6.

NISHIMURA, K., TING, H. J., HARADA, Y., TOKIZANE, T., NONOMURA, N., KANG, H. Y.,
CHANG, H. C., YEH, S., MIYAMOTO, H., SHIN, M., AOZASA, K., OKUYAMA, A. & CHANG,
C. (2003). Modulation of androgen receptor transactivation by gelsolin: a newly
identified androgen receptor coregulator. Cancer Res 63, 4888-94.

NISHIOKA, K., CHUIKOV, S., SARMA, K., ERDJUMENT-BROMAGE, H., ALLIS, C. D.,
TEMPST, P. & REINBERG, D. (2002). Set9, a novel histone H3 methyltransferase that
facilitates transcription by precluding histone tail modifications required for
heterochromatin formation. Genes Dev 16, 479-89.

O'LONE, R., FRITH, M. C., KARLSSON, E. K. & HANSEN, U. (2004). Genomic targets of
nuclear estrogen receptors. Mol Endocrinol 18, 1859-75.

OLAVE, I. A., RECK-PETERSON, S. L. & CRABTREE, G. R. (2002). Nuclear actin and actin-
related proteins in chromatin remodeling. Annu Rev Biochem 71, 755-81.

OXELMARK, E., ROTH, J. M., BROOKS, P. C., BRAUNSTEIN, S. E., SCHNEIDER, R. J. &
GARABEDIAN, M. J. (2006). The cochaperone p23 differentially regulates estrogen
receptor target genes and promotes tumor cell adhesion and invasion. Mol Cell Biol 26,
5205-13.

PAN, Y. F., WANSA, K. D., LIU, M. H., ZHAO, B., HONG, S. Z., TAN, P. Y., LIM, K. S.,
BOURQUE, G., LIU, E. T. & CHEUNG, E. (2008). Regulation of estrogen receptor-mediated
long range transcription via evolutionarily conserved distal response elements. J Biol
Chem 283, 32977-88.

PATEL, J. H., DU, Y., ARD, P. G., PHILLIPS, C., CARELLA, B., CHEN, C. J., RAKOWSKI, C.,
CHATTERJEE, C., LIEBERMAN, P. M., LANE, W. S., BLOBEL, G. A. & MCMAHON, S. B.
(2004). The c-MYC oncoprotein is a substrate of the acetyltransferases hGCN5/PCAF
and TIP60. Mol Cell Biol 24, 10826-34.

PETERSON, C. L., DINGWALL, A. & SCOTT, M. P. (1994). Five SWI/SNF gene products
are components of a large multisubunit complex required for transcriptional enhancement.
Proc Natl Acad Sci U S A 91, 2905-8.

PETERSON, C. L. & WORKMAN, J. L. (2000). Promoter targeting and chromatin
remodeling by the SWI/SNF complex. Curr Opin Genet Dev 10, 187-92.

133
PHELAN, M. L., SIF, S., NARLIKAR, G. J. & KINGSTON, R. E. (1999). Reconstitution of a
core chromatin remodeling complex from SWI/SNF subunits. Mol Cell 3, 247-53.

RAE, J. M., JOHNSON, M. D., SCHEYS, J. O., CORDERO, K. E., LARIOS, J. M. & LIPPMAN,
M. E. (2005). GREB 1 is a critical regulator of hormone dependent breast cancer growth.
Breast Cancer Res Treat 92, 141-9.

RAN, Q. & PEREIRA-SMITH, O. M. (2000). Identification of an alternatively spliced form
of the Tat interactive protein (Tip60), Tip60(beta). Gene 258, 141-6.

RANDO, O. J., ZHAO, K., JANMEY, P. & CRABTREE, G. R. (2002). Phosphatidylinositol-
dependent actin filament binding by the SWI/SNF-like BAF chromatin remodeling
complex. Proc Natl Acad Sci U S A 99, 2824-9.

RAZANDI, M., PEDRAM, A., GREENE, G. L. & LEVIN, E. R. (1999). Cell membrane and
nuclear estrogen receptors (ERs) originate from a single transcript: studies of ERalpha
and ERbeta expressed in Chinese hamster ovary cells. Mol Endocrinol 13, 307-19.

REISMAN, D. N., SCIARROTTA, J., BOULDIN, T. W., WEISSMAN, B. E. & FUNKHOUSER, W.
K. (2005). The expression of the SWI/SNF ATPase subunits BRG1 and BRM in normal
human tissues. Appl Immunohistochem Mol Morphol 13, 66-74.

REYES, J. C., BARRA, J., MUCHARDT, C., CAMUS, A., BABINET, C. & YANIV, M. (1998).
Altered control of cellular proliferation in the absence of mammalian brahma
(SNF2alpha). EMBO J 17, 6979-91.

ROEDER, R. G. (2005). Transcriptional regulation and the role of diverse coactivators in
animal cells. FEBS Lett 579, 909-15.

ROSENFELD, M. G., LUNYAK, V. V. & GLASS, C. K. (2006). Sensors and signals: a
coactivator/corepressor/epigenetic code for integrating signal-dependent programs of
transcriptional response. Genes Dev 20, 1405-28.

RUTHENBURG, A. J., ALLIS, C. D. & WYSOCKA, J. (2007). Methylation of lysine 4 on
histone H3: intricacy of writing and reading a single epigenetic mark. Mol Cell 25, 15-30.

SABBAH, M., COURILLEAU, D., MESTER, J. & REDEUILH, G. (1999). Estrogen induction of
the cyclin D1 promoter: involvement of a cAMP response-like element. Proc Natl Acad
Sci U S A 96, 11217-22.

SANTOS-ROSA, H., SCHNEIDER, R., BANNISTER, A. J., SHERRIFF, J., BERNSTEIN, B. E.,
EMRE, N. C., SCHREIBER, S. L., MELLOR, J. & KOUZARIDES, T. (2002). Active genes are
tri-methylated at K4 of histone H3. Nature 419, 407-11.

SAPOUNTZI, V., LOGAN, I. R. & ROBSON, C. N. (2006). Cellular functions of TIP60. Int J
Biochem Cell Biol 38, 1496-509.

134
SHANG, Y., HU, X., DIRENZO, J., LAZAR, M. A. & BROWN, M. (2000). Cofactor dynamics
and sufficiency in estrogen receptor-regulated transcription. Cell 103, 843-52.

SHEN, X., RANALLO, R., CHOI, E. & WU, C. (2003). Involvement of actin-related proteins
in ATP-dependent chromatin remodeling. Mol Cell 12, 147-55.

SHERIDAN, A. M., FORCE, T., YOON, H. J., O'LEARY, E., CHOUKROUN, G., TAHERI, M. R.
& BONVENTRE, J. V. (2001). PLIP, a novel splice variant of Tip60, interacts with group
IV cytosolic phospholipase A(2), induces apoptosis, and potentiates prostaglandin
production. Mol Cell Biol 21, 4470-81.

SPECTOR, D. L. (2003). The dynamics of chromosome organization and gene regulation.
Annu Rev Biochem 72, 573-608.

STALLCUP, M. R., KIM, J. H., TEYSSIER, C., LEE, Y. H., MA, H. & CHEN, D. (2003). The
roles of protein-protein interactions and protein methylation in transcriptional activation
by nuclear receptors and their coactivators. J Steroid Biochem Mol Biol 85, 139-45.

STRAHL, B. D. & ALLIS, C. D. (2000). The language of covalent histone modifications.
Nature 403, 41-5.

SUN, J., NAWAZ, Z. & SLINGERLAND, J. M. (2007). Long-range activation of GREB1 by
estrogen receptor via three distal consensus estrogen-responsive elements in breast cancer
cells. Mol Endocrinol 21, 2651-62.

SUN, Y., JIANG, X., CHEN, S., FERNANDES, N. & PRICE, B. D. (2005). A role for the Tip60
histone acetyltransferase in the acetylation and activation of ATM. Proc Natl Acad Sci U
S A 102, 13182-7.

SUN, Y., JIANG, X., XU, Y., AYRAPETOV, M. K., MOREAU, L. A., WHETSTINE, J. R. &
PRICE, B. D. (2009). Histone H3 methylation links DNA damage detection to activation
of the tumour suppressor Tip60. Nat Cell Biol 11, 1376-82.

SYKES, S. M., MELLERT, H. S., HOLBERT, M. A., LI, K., MARMORSTEIN, R., LANE, W. S.
& MCMAHON, S. B. (2006). Acetylation of the p53 DNA-binding domain regulates
apoptosis induction. Mol Cell 24, 841-51.

TAKATA, H., MATSUNAGA, S., MORIMOTO, A., ONO-MANIWA, R., UCHIYAMA, S. &
FUKUI, K. (2007). H1.X with different properties from other linker histones is required
for mitotic progression. FEBS Lett 581, 3783-8.

TANG, Y., LUO, J., ZHANG, W. & GU, W. (2006). Tip60-dependent acetylation of p53
modulates the decision between cell-cycle arrest and apoptosis. Mol Cell 24, 827-39.

135
TAUBERT, S., GORRINI, C., FRANK, S. R., PARISI, T., FUCHS, M., CHAN, H. M.,
LIVINGSTON, D. M. & AMATI, B. (2004). E2F-dependent histone acetylation and
recruitment of the Tip60 acetyltransferase complex to chromatin in late G1. Mol Cell Biol
24, 4546-56.

TING, H. J., YEH, S., NISHIMURA, K. & CHANG, C. (2002). Supervillin associates with
androgen receptor and modulates its transcriptional activity. Proc Natl Acad Sci U S A 99,
661-6.

TROTTER, K. W. & ARCHER, T. K. (2007). Nuclear receptors and chromatin remodeling
machinery. Mol Cell Endocrinol 265-266, 162-7.

TROTTER, K. W. & ARCHER, T. K. (2008). The BRG1 transcriptional coregulator. Nucl
Recept Signal 6, e004.

TSUKIYAMA, T., BECKER, P. B. & WU, C. (1994). ATP-dependent nucleosome disruption
at a heat-shock promoter mediated by binding of GAGA transcription factor. Nature 367,
525-32.

TURNER, B. M. (2000). Histone acetylation and an epigenetic code. Bioessays 22, 836-45.

UMAYAHARA, Y., KAWAMORI, R., WATADA, H., IMANO, E., IWAMA, N., MORISHIMA, T.,
YAMASAKI, Y., KAJIMOTO, Y. & KAMADA, T. (1994). Estrogen regulation of the insulin-
like growth factor I gene transcription involves an AP-1 enhancer. J Biol Chem 269,
16433-42.

UROSEV, D., MA, Q., TAN, A. L., ROBINSON, R. C. & BURTNICK, L. D. (2006). The
structure of gelsolin bound to ATP. J Mol Biol 357, 765-72.

UTLEY, R. T. & COTE, J. (2003). The MYST family of histone acetyltransferases. Curr
Top Microbiol Immunol 274, 203-36.

VIGNALI, M., HASSAN, A. H., NEELY, K. E. & WORKMAN, J. L. (2000). ATP-dependent
chromatin-remodeling complexes. Mol Cell Biol 20, 1899-910.

WANG, G., BALAMOTIS, M. A., STEVENS, J. L., YAMAGUCHI, Y., HANDA, H. & BERK, A. J.
(2005). Mediator requirement for both recruitment and postrecruitment steps in
transcription initiation. Mol Cell 17, 683-94.

WANG, W., XUE, Y., ZHOU, S., KUO, A., CAIRNS, B. R. & CRABTREE, G. R. (1996).
Diversity and specialization of mammalian SWI/SNF complexes. Genes Dev 10, 2117-30.

WAY, M., POPE, B. & WEEDS, A. G. (1992). Are the conserved sequences in segment 1 of
gelsolin important for binding actin? J Cell Biol 116, 1135-43.

136
WEBB, P., LOPEZ, G. N., UHT, R. M. & KUSHNER, P. J. (1995). Tamoxifen activation of
the estrogen receptor/AP-1 pathway: potential origin for the cell-specific estrogen-like
effects of antiestrogens. Mol Endocrinol 9, 443-56.

WINSTON, F. & CARLSON, M. (1992). Yeast SNF/SWI transcriptional activators and the
SPT/SIN chromatin connection. Trends Genet 8, 387-91.

WU, J. I., LESSARD, J., OLAVE, I. A., QIU, Z., GHOSH, A., GRAEF, I. A. & CRABTREE, G. R.
(2007). Regulation of dendritic development by neuron-specific chromatin remodeling
complexes. Neuron 56, 94-108.

YAMAMOTO, T. & HORIKOSHI, M. (1997). Novel substrate specificity of the histone
acetyltransferase activity of HIV-1-Tat interactive protein Tip60. J Biol Chem 272,
30595-8.

YOON, S., QIU, H., SWANSON, M. J. & HINNEBUSCH, A. G. (2003). Recruitment of
SWI/SNF by Gcn4p does not require Snf2p or Gcn5p but depends strongly on SWI/SNF
integrity, SRB mediator, and SAGA. Mol Cell Biol 23, 8829-45.

YOSHINAGA, S. K., PETERSON, C. L., HERSKOWITZ, I. & YAMAMOTO, K. R. (1992). Roles
of SWI1, SWI2, and SWI3 proteins for transcriptional enhancement by steroid receptors.
Science 258, 1598-604.

ZHAO, K., WANG, W., RANDO, O. J., XUE, Y., SWIDEREK, K., KUO, A. & CRABTREE, G. R.
(1998). Rapid and phosphoinositol-dependent binding of the SWI/SNF-like BAF
complex to chromatin after T lymphocyte receptor signaling. Cell 95, 625-36.

Abstract (if available)

Abstract Estrogen receptor α (ER) is a member of the family of nuclear receptors and functions as a transcriptional factor to induce gene expression by binding to specific DNA sequences upon hormone treatment. It regulates cell growth, development and metabolic homeostasis in multi-cellular organisms. Estrogen-mediated transcription has been intensively studied genome-wide as well as on a small number of specific endogenous target promoters. However, the exact mechanism by which ER coordinates the activities of chromatin remodeling complexes and coactivators to facilitate initiation of transcription remains elusive. Here, we show the molecular mechanisms of the recruitment of the SWI/SNF chromatin remodeling complex by Fli-I, and recruitment of Tip60, a histone acetyltransferase.

Linked assets

University of Southern California Dissertations and Theses

Conceptually similar

PDF

The mechanism of recruitment of Tip60 to ER target genes

PDF

Functional role of chromatin remodeler proteins in cancer biology

PDF

The role of Hic-5 in glucocorticoid receptor binding to chromatin

PDF

Regulation of Aurora kinase B and its effect on phosphorylation of G9a/GLP

PDF

The role of histone H4 lysine 20 monomethylation in gene expression and differentiation

PDF

Impacts of post-translational modifications on interactions between G9a and its N-terminus binding partners

PDF

Determinination of the causal potential of histone modifications on transcription and chromatin structure

PDF

The transcriptional regulation of the pro-survival protein Grp78 by activating transcription factors and chromatin-modifying enzymes: its upregulation in response to HDAC-inhibitor treatment and ...

PDF

Multiple functions of the PR-Set7 histone methyltransferase: from transcription to the cell cycle

PDF

Elucidating the functional role of CHD7 associated nuclear PDH complex and other associated proteins on neural crest development

PDF

Determining the mechanism and necessity of matrix metalloproteinase 2 (MMP-2) nuclear localization for proficient skeletal muscle differentiation

PDF

Mysm1, a histone de-ubiquitinase, is essential for dendritic cell development and function

Asset Metadata

Creator Jeong, KwangWon (author)

Core Title Molecular mechanism of the recruitment of SWI/SNF chromatin remodeling complex and histone acetyltransferase to estrogen-responsive promoters

School Keck School of Medicine

Degree Doctor of Philosophy

Degree Program Biochemistry and Molecular Biology

Degree Conferral Date 2010-05

Publication Date 04/27/2010

Defense Date 12/18/2009

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag chromatin remodeling,coactivator,estrogen receptor,flightless,Fli-I,histone,nuclear receptor,OAI-PMH Harvest,SWI/SNF,Tip60

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Stallcup, Michael R. (committee chair), An, Woojin (committee member), Li, Wei (committee member)

Creator Email kjeong@usc.edu,mirabo5244@hotmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m2960

Unique identifier UC1180478

Identifier etd-Jeong-3577 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-314818 (legacy record id),usctheses-m2960 (legacy record id)

Legacy Identifier etd-Jeong-3577.pdf

Dmrecord 314818

Document Type Dissertation

Rights Jeong, KwangWon

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Repository Name Libraries, University of Southern California

Repository Location Los Angeles, California

Repository Email cisadmin@lib.usc.edu