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The mechanism of recruitment of Tip60 to ER target genes
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The mechanism of recruitment of Tip60 to ER target genes
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Content
THE MECHANISM OF RECRUITMENT OF TIP60 TO ER TARGET GENES
by
Janet M. Lee
A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(BIOCHEMISTRY AND MOLECULAR BIOLOGY)
May 2011
Copyright 2011 Janet M. Lee
ii
ACKNOWLEDGEMENTS
First and foremost, I would like to thank Dr. Michael Stallcup for giving me the
opportunity to learn and grow as a scientist in his laboratory. Throughout my master’s
course, he has been so patient, supportive, and truly inspiring. I believe that I have
matured into the scientist that I am today thanks to him and I thank him for being such an
incredible advisor, mentor, and friend to me. I would like to thank Dr. Zoltan Tokes for
always being there to help and encourage me along, for believing in me, and overall for
being a wonderful advisor. I would also like to thank Dr. Judd Rice for his time and
suggestions for this thesis.
I would especially like to thank Dr. Kwangwon Jeong who has been the best
teacher and friend. I would like to thank my colleagues Danielle Bittencourt, Kelly
Chang, Rajas Chodankar, Daniel Gerke, Irina Ianculescu, Chenyin Ou, Daniel Purcell,
and Daiying Wu for all their helpful discussions and friendships.
Lastly, I would like to thank my family, especially my mom and dad. Thank you
for always encouraging my curiosities and for giving my dreams the wings to fly.
iii
TABLE OF CONTENTS
Acknowledgements………………………………………………………………... ii
List of Tables..…………………………………………………………………….. iv
List of Figures……………………………………………………………………... v
List of Abbreviations……………………………………………………………… vi
Abstract……………………………………………………………………………. vii
Chapter 1: Introduction………………………………………………………... 1
Chapter 2: Materials and Methods……………………………………………. 7
Chapter 3: Results Section…………………………………………………….. 14
Chapter 4: Discussion Section………………………………………................ 38
Bibliography……………………………………………………………………….. 42
iv
LIST OF TABLES
Table 1 Sequences of siRNA……………………………………………… 10
Table 2 Sequences of primers used for qRT-PCR………………………… 10
Table 3 Sequences of primers used for ChIP……………………………… 12
v
LIST OF FIGURES
Fig. 1 Schematic Diagrams of steroid receptors and Tip60....................... 3
Fig. 2 Tip60 is recruited to the estrogen responsive elements and
enhancer regions of various ER target genes……………............... 15
Fig. 3 Tip60 affects the expression of a subset of the hormonally
induced ER target genes…………………………………………… 21
Fig. 4 Tip60 interacts with estrogen receptor in a hormone dependent
Manner…………………………………………………………….. 23
Fig. 5 Tip60 interacts with estrogen receptor through its NR box in a
hormone-dependent manner both in vivo and in vitro…………….. 26
Fig. 6 LXXAA mutation of Tip60 compromises its recruitment to
pS2 EREs………………………………………………………….. 31
Fig. 7 Protein Purification of His-tagged Tip60 chromodomain
fragment…………………………………………………………… 33
Fig. 8 Tip60 chromodomain preferentially interacts with mono-
methylated and di-methylated H3K4……………………………… 34
Fig. 9 The SWI/SNF complex is required for the coactivator function
of Tip60 with ERα………………………………………………… 35
Fig. 10 Dominant Negative Effect of Tip60 chromodomain fragment……. 37
Fig. 11 Model of the Mechanism of Recruitment of Tip60 to ER
Target Genes………………………………………………………. 41
vi
LIST OF ABBREVIATIONS
AF-1/AF-2 – activation function
DBD – DNA binding domain
ERE – estrogen response element
GST – glutathione S-transferase
HAT – histone acetyltransferase
LBD – ligand binding domain
MMTV-ERE – mammary mouse tumor virus-estrogen response element
NR – nuclear receptor
NR box – nuclear receptor box
Tip60 – Tat interactive protein 60
vii
ABSTRACT
The steroid receptors in the nuclear receptor superfamily are responsible for regulating
many different functions including transcription. However, in order to completely and
correctly activate transcription, the steroid receptors must recruit not only the general
initiation factors but also other coregulatory factors, such chromatin remodelers and
coactivators. Tip60, a 60 kD protein with an N-terminal chromodomain, a central
intrinsic histone acetyltransferase (HAT) activity, and a C-terminal nuclear receptor
binding domain (NR box) was previously reported to be a coactivator for androgen
receptor, in addition to being able to bind to the estrogen receptor in vivo. Subsequently,
Tip60 was identified to be recruited very early on to the pS2 estrogen response element1
(ERE1) site, hormone binding enhancer elements involved in transactivation, upon
hormone treatment. Here, it is shown that the interaction between Tip60 and the estrogen
receptor, both in vivo and in vitro, requires ligand binding to estrogen receptor and also
interacts via the C-terminal NR box, specifically the LXXLL motif. Furthermore, the
abolishment of the LXXLL motif compromises Tip60’s recruitment to certain estrogen
response elements (EREs) of ER target genes. In addition, the N-terminal chromodomain
is shown to preferentially bind to histone H3 mono-methylated and di-methylated at
lysine4. Finally, it is shown that a competition for methylated histones between the
wildtype Tip60 and the exogenously expressed Tip60 chromodomain fragment also
compromises its recruitment to certain EREs of ER target genes. Thus, the stable
recruitment and association of Tip60 at the EREs is dependent upon the normal function
of its N-terminal chromodomain and C-terminal NR box.
1
CHAPTER 1: INTRODUCTION
Within the nuclear receptor (NR) superfamily, steroid receptors, which include
androgen, estrogen, progesterone, and glucocorticoid receptors, are responsible for
regulating diverse biological processes such as sexual differentiation, reproduction, and
metabolism (Alberts, 2008) (Lonard and O'Malley, 2006). Though these steroid receptors
carry out different functions and recognize different ligands, they share many similar
structural traits. The steroid receptors are comprised of a ligand-independent
transcriptional activation function (AF-1) in the NH
2
-terminal region, a central DNA
binding domain which contains two highly conserved zinc finger motifs which aid in
targeting the receptor to its specific hormone response elements, and a ligand binding
domain and a ligand-dependent activation function (AF-2) in the COOH-terminal region
(Fig. 1A) (Chawla et al., 2001).
The steroid receptors reside in the cytosol bound to heat shock proteins that
render the receptors inactive. As the lipophilic steroid hormone diffuses through the
plasma membrane and into the cell, it will bind to and activate its cognate receptor. Once
bound to ligand, the receptor undergoes several transformations. First, the receptor will
dissociate from its complex of heat shock proteins. Second, two nuclear receptor-ligand
complexes will homo-dimerize. Third, the homodimer will translocate into the nucleus
and bind to the hormone response elements that are associated with the target genes that
are regulated by that specific hormone (McKenna et al., 1999). At this point, the nuclear
2
receptor-DNA complex must recruit coactivators that act through chromosomal histone
modifications
3
Fig. 1 Schematic diagrams of steroid receptors and Tip60
(A) The steroid receptors are comprised of a ligand-independent transcriptional activation
function (AF-1) in the NH
2
-terminal region, a central DNA binding domain which
contains two highly conserved zinc finger motifs which aid in targeting the receptor to its
specific hormone response elements, and a ligand binding domain (LBD) and a ligand-
dependent activation function (AF-2) in the COOH-terminal region.
(B) Tip60 is comprised of an NH
2
-terminal chromodomain, a central zinc finger and
conserved histone acetyltransferase (HAT) domain, and a COOH-terminal NR box.
4
and/or coactivators that recruit the basal transcription machinery in order to form the
preinitiation complex (Roeder, 2005). These series of events lead to the transcription of
target genes.
Tip60 is a 60 kD protein belonging to the MYST family (Sterner and Berger,
2000). Tip60 is comprised of an NH
2
-terminal chromodomain, a central zinc finger and
conserved histone acetyltransferase (HAT) domain, and a COOH-terminal NR box (Fig.
1B). Tip60 was initially discovered as a Tat-interacting protein in studies of HIV
(Kamine et al., 1996). Tip60 was also shown to have HAT activity. Specifically, Tip60
was shown to acetylate H2A, H3 and H4 (Yamamoto and Horikoshi, 1997).
Subsequently, Tip60 was identified to be a key player in processes such as DNA repair
(Sun et al., 2010) and apoptosis (Sheridan et al., 2001).
Most interestingly, Tip60 was also identified as an androgen receptor coactivator.
It was shown to interact with the androgen receptor specifically with the ligand-binding
domain of androgen receptor both in vivo and in vitro. In addition, Tip60 was shown to
enhance androgen receptor transactivation levels comparable to those achieved with
coactivators such as CBP, p300 and SRC-1 (Brady et al., 1998). Moreover, Tip60 was
shown to preferentially interact with AR, ERα, ERβ, and GR but not with TR, VDR, or
RXR (Gaughan et al., 2001).
As previously stated, Tip60 has an NH
2
-terminal chromodomain. The function of
this chromodomain has been unknown. However, chromodomains present in some other
proteins are known to mediate the protein’s interaction with either methylated histone
lysines or RNA molecules (Sapountzi et al., 2006). Very recently, the chromodomain of
5
Tip60 was discovered to recognize and bind to tri-methylated H3K9 in the process of
DNA repair (Sun et al., 2009).
The COOH-terminal NR box of Tip60 is of much interest as well. The NR box
harbors a short leucine rich sequence termed the LXXLL motif. This motif is found in
many different coactivators, such as CBP, SRC-1, and GRIP1. Structurally, the LXXLL
motif is a α-helical structure whereby the leucine residues line up on one side of the helix
to create a hydrophobic interface. Furthermore, studies have shown that the leucines of
the LXXLL motif are crucial for the coactivators’ interaction with the AF-2 domain of
the nuclear receptors (Darimont et al., 1998) (Heery et al., 1997).
Tip60 quickly became a gene of interest as it was shown to be a coactivator that
was recruited early on after steroid hormone induction (Metivier et al., 2003). In order to
examine the recruitment of Tip60 to ER target genes, we examined the function of the
chromodomain and the NR box of Tip60. It was previously shown that Tip60 interacts
with the estrogen receptor, though the mechanism of this interaction was unknown.
However, since the LXXLL motif of other coactivators was shown to be involved in the
interaction between the coactivator and its respective nuclear receptor, we hypothesize
that the LXXLL motif of Tip60, too, is involved in the interaction with the estrogen
receptor. We show here that by abolishing the hydrophobic interface of the LXXLL motif
forms, the motif is indeed involved in the ligand-dependent interaction both in vivo and in
vitro. Furthermore, we show that the abolishment of the LXXLL motif prevents Tip60’s
recruitment to ER target gene EREs.
6
Finally, since it is known that chromodomains interact with methylated lysines in
other chromatin regulatory proteins and since it was shown that the chromodomain of
Tip60 interacts with repressive marker H3K9, we hypothesize that in the case of Tip60’s
role as a coactivator, the chromodomain can recognize and bind to active histone markers
such as H3K4. Indeed, in this study, we show that the chromodomain of Tip60 binds to
mono-methylated and di-methylated H3K4 in vitro. Furthermore, we show that by
compromising the interaction that the chromodomain makes with the methylated histone,
we compromise the recruitment of Tip60 to ER target gene estrogen response elements
(EREs).
7
CHAPTER 2: MATERIALS AND METHODS
Plasmids
The following glutathione S-transferase (GST) fused plasmids pGEX4T1 and pGEX4T1-
ER (LBD) and the expression vectors for the luciferase assay experiment, the murine
mammary tumor virus-estrogen response element (MMTV-ERE), pHE0, and BRG1 were
described previously (Jeong et al., 2009). The pTriEx4-His-Tip60 chromodomain
plasmid that encodes the Tip60 chromodomain fragment has been described in (Jeong et
al., submitted). The expression plasmid pcDNA-FLAG-Tip60 was kindly donated by Dr.
Anastasia Kralli (Scripp). Point mutations in Tip60 for the LXXAA mutation were
introduced by site-directed mutagenesis using the QuikChange II site-directed
mutagenesis kit (Stratagene, Santa Clara, CA).
Cell Culture
MCF7, COS-7, and SW-13 cells were maintained in the Dulbecco’s modified Eagle’s
medium (DMEM) from Invitrogen (Carlsbad, CA). The media was supplemented with
penicillin and streptomycin and 10% fetal bovine serum.
8
Reporter gene assay
0.75 x 10
5
SW13 cells were plated per well in a 24-well plate. The cells were transiently
transfected using Lipofectamine2000 reagent (Invitrogen) according to the
manufacturer’s protocol. Total DNA in every well was adjusted to be the same amount
by adding the appropriate amount of empty expression vectors. After transfection, the
cells were incubated for 48 hours in DMEM containing 5% charcoal-treated fetal bovine
serum and 100 nM estradiol. Luciferase assays on the cell extracts were performed with
Promega Luciferase Assay Kit.
Protein Interaction and Immunoblot analyses
For co-immunoprecipitation, 1.5 x 10
6
COS-7 cells were plated per 10 cm dish. The cells
were transfected with pHE0, encoding ERα, and pcDNA-FLAG-Tip60 or pcDNA-
FLAG-Tip60 (LXXAA) using Lipofectamine2000 reagent according to the
manufacturer’s protocol. After transfection, the cells were incubated in hormone-free
media with or without 100 nM estradiol for 48 hours. The cells were harvested with 1 mL
of RIPA buffer (50 mM Tris-Cl [pH 8.0], 120 mM NaCl, 0.1% NP-40, 0.1% SDS) and
protease inhibitor cocktail tablets (Roche, South San Francisco, CA). The cells were
briefly sonicated, then centrifuged in a microcentrifuge at maximum speed for 10
minutes. The supernatant was collected, and incubated overnight with Protein A/G beads
9
(Sigma Aldrich, St. Louis, MO). It was then centrifuged, again. The supernatant was
collected and incubated overnight with normal rabbit IgG (Santa Cruz Biotechnology,
Santa Cruz, CA) or anti-FLAG antibody (Sigma Aldrich) and Protein A/G beads. The
beads were collected and washed 5 times with 0.01% NETN buffer (300 mM NaCl, 1
mM EDTA, 20 mM Tris-HCl [pH 8.0], and 0.01% Nonidet P-40) and then resolved by
SDS-polyacrylamide gel electrophoresis. Immunoblotting was performed with rabbit
anti-ERα antibody (Santa Cruz Biotechnology). ECL reagents (Amersham, Pittsburgh,
PA) were used for detection.
GST-pulldown assays were performed as previously described. (Jeong et al., 2009) The
GST fusion proteins were expressed in E. coli BL21 strain and purified by incubating
with glutathione-Sepharose beads and washing with 0.01% NETN buffer (300 mM NaCl,
1 mM EDTA, 20 mM Tris-HCl [pH 8.0], and 0.01% Nonidet P-40). pcDNA-FLAG-
Tip60 and pcDNA-FLAG-Tip60(LXXAA) proteins were in vitro transcribed and
translated with TNT-Quick-coupled reticulocyte lysate system (Promega, San Luis
Obispo, CA) according to the manufacturer’s protocol. Immunoblotting was performed
with anti-FLAG (Sigma Aldrich). ECL reagents (Amersham) were used for detection.
RNA Interference and qRT-PCR
Small interfering RNA experiments were carried out as previously described (Jeong et
al., 2009): MCF7 cells were transfected with appropriate small interfering RNA using
10
Lipofectamine2000 (Invitrogen) according to the manufacturer’s protocol. The sequences
of the siRNA used are as follows:
Table 1: Sequences of siRNA.
nonspecific siRNA (siNS), 5’-UUCUCCGAACGUGUCACGUdTdT-3’ (sense)
nonspecific siRNA (siNS), 5’-ACGUGACACGUUCGGAGAAdTdT-3’ (antisense)
siTip60, 5’ – GCUCCAUUACAUUGACUUCdTdT – 3’ (sense)
siTip60, 5’ – GAAGUCAAUGUAAUGGACGdTdT – 3’ (antisense)
The cells were cultured for 72 hours in phenol red-free Dulbecco’s modified Eagle’s
medium supplemented with 5% charcoal-dextran stripped fetal bovine serum. The cells
were then treated with 10 nM estradiol for 8 and 24 hours. Total RNA was isolated with
Trizol (Invitrogen), followed by reverse transcription using iScript cDNA synthesis kit
(Bio-rad). qPCR analysis was done with the following primers:
Table 2: Sequences of primers used for qRT-PCR.
pS2, 5’–GAACAAGGTGATCTGCG-3’ (forward)
pS2, 5’-TGGTATTAGGATAGAAGCACCA-3’ (reverse)
GREB1, 5’–CAAAGAATAACCTGTTGGCCCTGC-3’ (forward)
GREB1, 5’–GACATGCCTGCGCTCTCATACTTA-3’ (reverse)
cathepsin D, 5’–GTACATGATCCCCTGTGAGAAGGT-3’ (forward)
cathepsin D, 5’–GGGACAGCTTGTAGCCTTTGC-3’ (reverse)
CYCLIN D1, 5’–AAGCTCAAGTGGAACCT-3’ (forward)
CYCLIN D1, 5’–AGGAAGTTGTTGGGGC-3’ (reverse)
progesterone receptor, 5’–GTGCCTATCCTGCCTCTCAATC-3’ (forward)
11
Table 2: Sequences of primers used for qRT-PCR continued.
progesterone receptor, 5’-CCCGCCGTCGTAACTTTCG-3’ (reverse)
CXCL12, 5’–TCAGCCTGAGCTACAGATGC-3’ (forward)
CXCL12, 5’–CTTTAGCTTCGGGTCAATGC-3’ (reverse)
PKIB, 5’–CCAATTTTGCATCTTCAGCA-3’ (forward)
PKIB, 5’–GGCTTTTCCAATTGGTCTTG-3’ (reverse)
SGK3, 5’–TGAGGCCAGGAGTGAGTCTT-3’ (forward)
SGK3, 5’–TATCATCTGGTCCAGCCACA-3’ (reverse)
GAPDH, 5’–TCTGGTAAAGTGGATATTGTTG-3’ (forward)
GAPDH, 5’–GATGGTGATGGGATTTCC-3’ (reverse)
18S, 5’–GAGGATGAGGTGGAACGTGT-3’ (forward)
18S, 5’–TCTTCAGTCGCTCCAGGTCT-3’ (reverse)
Relative expression levels were normalized to 18S mRNA levels.
Chromatin Immunoprecipitation Assay
Procedure followed the published protocol (Jeong et al., 2009): MCF7 cells were
transfected with appropriate expression plasmids and cultured for 2 days in phenol red-
free Dulbeccos’ modified Eagle’s medium supplemented with 5% charcoal-dextran
stripped fetal bovine serum. The cells were then treated with 100 nM estradiol for various
times. They were then cross-linked using formaldehyde. Then the chromatin in the cell
extracts was sheared by sonication. The sonicated cell extracts were then incubated
12
overnight at 4
o
C with appropriate antibodies. The cross-linking was reversed through
heating. The DNA was then purified by phenol-chloroform extraction and ethanol
purification. The purified DNA was dissolved in 50 µL of RNAse-free H
2
O. It was then
analyzed by qPCR with the following primers:
Table 3: Sequences for primers used for ChIP.
pS2 ERE1, 5’–CCGGCCATCTCTCACTATGAA-3’ (forward)
pS2 ERE1, 5’–CCTTCCCGCCAGGGTAAATAC-3’ (reverse)
pS2 ERE2, 5’–CCTCCCCAGCTCACGTTGT-3’ (forward)
pS2 ERE2, 5’–GGGTTGCATTTAAGGGACCTT-3’ (reverse)
pS2 ERE3, 5’–GTCGTTGCCAGCGTTTCC-3’ (forward)
pS2 ERE3, 5’-CTTCTCCACGCCCTGTAAATTT-3’ (reverse)
GREB1 ERE1, 5’–GTGGCAACTGGGTCATTCTGA-3’ (forward)
GREB1 ERE1, 5’-CGACCCACAGAAATGAAAAGG-3’ (reverse)
GREB1 ERE2, 5’–GCCACCTCTGCAGGATTGTA-3’ (forward)
GREB1 ERE2, 5’–CAAAACAGAGCAAGGCCAAA-3’ (reverse)
GREB1 ERE3, 5’–TGTGCTCAGTGACCCTTGTG-3’ (forward)
GREB1 ERE3, 5’-CTGCCCCAACAACTGAAAGA-3’ (reverse)
GREB1 ENH2 5’-TCTGTGGAGTGCCTGAAGTG-3’ (forward)
GREB 1 ENH2 5’–GCCAATGCTTTGCCATTATT-3’ (reverse)
GREB1 ENH3 5’–GAAGGGCAGAGCTGATAACG-3’ (forward)
GREB1 ENH3 5’–GACCCAGTTGCCACACTTTT-3’ (reverse)
GREB1 ENH5 5’–GCCCAGGAGACAGGTTGTAA-3’ (forward)
13
Table 3: Sequences used for ChIP continued.
GREB1 ENH5 5’–TATGACTCTTGGCCCTGTCC-3’ (reverse)
CATD ERE1, 5’–GGAGCGGAGGGTCCATTC-3’ (forward)
CATD ERE1, 5’–TCCAGACATCCTCTCTGGAA-3’ (reverse)
CATD ERE2, 5’–CCTCCTCAACTGCTCTTGCA-3’ (forward)
CATD ERE2, 5’–GCGGCTGAGATGCTGAGTCA-3’ (reverse)
CATD ENH1, 5’–GCCACAGGCAGCTTTAGTTC-3’ (forward)
CATD ENH1, 5’–CATTCACAGCCTCCACCTTT-3’ (reverse)
CATD ENH2, 5’–GCTAACATGTTGCCTGCTCA-3’ (forward)
CATD ENH2, 5’–TTCATATCTACCGCCCAAGG-3’ (reverse)
14
CHAPTER 3: RESULTS SECTION
Tip60 occupies estrogen responsive elements and enhancer regions of various target
genes in a cyclical manner.
It was previously reported that Tip60 is recruited to the pS2 ERE1 in a cyclical
manner (Metivier et al., 2003). However, the recruitment pattern of Tip60 to other ER
target genes is not known. Therefore, the occupancy of endogenous Tip60 at other
estrogen responsive elements of different ER target genes was analyzed using MCF7
cells at time points between 0 and 60 minutes after adding E2.
Consistent with previous data, Tip60 shows a cyclical recruitment to the estrogen
responsive element 1 of the pS2 gene (Metivier et al., 2003) at 20 minutes and 40
minutes. Sure enough, Tip60 was also recruited to the ERE 2 and 3 of the pS2 gene with
peaks at 20 and 40 minutes (Fig. 2A). Likewise, Tip60 is recruited to the GREB1 ERE1,
ERE2, and ERE3 regions and the distal, enhancer 2 and enhancer 3 regions in a cyclical
fashion. However, the percent occupancy of Tip60 at the GREB1 ERE2 region is very
low. This suggests that there is a regional specificity of Tip60 recruitment (Fig. 2B).
Correspondingly, Tip60 is recruited to the cathepsin D ERE2 and Enh2 regions in a
cyclical style. However, the occupancy of Tip60 at the ERE1 region is low (Fig. 2C),
which further supports the theory that Tip60 is preferentially recruited to specific regions.
15
Fig. 2 Tip60 is recruited to the estrogen responsive elements and enhancer regions of
various ER target genes.
Chromatin immunoprecipitation assays were performed with untransfected MCF7 cells in
150-mm dishes treated with 100 nM E2 for the indicated times. After
immunoprecipitation of cross-linked chromatin fragments with antibodies against the
endogenous Tip60, the amount present at (A) pS2 ERE1, ERE2, and ERE3, (B) GREB1
ERE1, ERE2, ERE3, ENH2, ENH3, ENH5, and (C) cathepsin D ERE1, ERE2, ENH1,
ENH2 were determined by qPCR. The data are plotted as the percentage of total input
before immunoprecipitation. Results shown are the mean and range of variation of
duplicated PCR reactions. The standard variation was too small to be illustrated in some
graphs.
16
Fig. 2 Tip60 is recruited to the estrogen responsive elements and enhancer regions of
various ER target genes continued.
(A)
17
Fig. 2 Tip60 is recruited to the estrogen responsive elements and enhancer regions of
various ER target genes continued.
(B)
18
Fig. 2 Tip60 is recruited to the estrogen responsive elements and enhancer regions of
various ER target genes continued.
(C)
19
Knockdown of endogenous Tip60 attenuates the expression of ER target genes.
It was previously reported that in transient reporter gene assays, Tip60 is a
coactivator for androgen receptor. In addition, Tip60 was shown to interact with the
estrogen receptor (Brady et al., 1999; Gaughan et al., 2001). This suggests a theory that
Tip60 may also be a coactivator for the estrogen receptor. Indeed, Tip60 is shown to
occupy the promoter and enhancer regions of ER target genes in a hormone dependent
manner (Fig. 2). Therefore, the effect of Tip60 on the expression of endogenous ER
target genes in MCF7 cells was examined.
Transfected siRNA against Tip60 mRNA reduced the expression levels of Tip60
mRNA in the MCF7 cells compared to the transfected non-specific siRNA (siNS) (Fig.
3A). Tip60 siRNA did not affect the levels of GAPDH mRNA, as expected. Interestingly,
though, it significantly reduced the E2-dependent expression of the endogenous pS2 and
PKIB both at 8 and 24 hours. On the other hand, the hormone dependent expression of
other genes such as SGK3 and CXCL12 were not affected at all by the reduction of Tip60
mRNA. Still for other genes, such as the GREB1, cathepsin D, progesterone receptor, and
cyclinD1 genes, a temporary reduction of expression at 8 hours was observed, but by 24
hours, the expressions were rescued (Fig. 3B). However, a similar experiment done in
tandem by a colleague shows that the expressions of these genes are in fact reduced at
both 8 and 24 hours. Moreover, the latter experiment shows that the expression of
cyclinD1 was unaffected. (Jeong et al., unpublished) Clearly, more experiments must be
carried out in order to make a firm conclusion. However, for the purposes of this study
20
we will assume that the levels of mRNA expression is decreased for GREB1, cathepsin
D, and progesterone receptor at 24 hours as well as 8 hours and that the mRNA levels of
cyclinD1 is not affected at all by the reduction of Tip60. Nevertheless, it is clear that
Tip60 affects the hormonal induction of only a subset of ER target genes.
Tip60 interacts with the estrogen receptor in a ligand dependent manner.
Earlier studies employing the mammalian two-hybrid system showed that Tip60
interacts with the ligand-binding domain of estrogen receptor in a hormone dependent
manner (Gaughan et al., 2001). Here, an in vitro translated full length Tip60 was
incubated with a GST fused ligand-binding domain of ER in the presence and absence of
hormone. The GST pulldown assay shows that in the absence of hormone Tip60 interacts
with the estrogen receptor. Thus, Tip60 can specifically bind to the estrogen receptor in a
ligand independent manner. However, this binding is further enhanced by the presence of
hormone. Thus, Tip60 interacts with the ligand binding domain of the estrogen receptor
in a ligand-dependent manner in vitro (Fig. 4) as well as in vivo (Gaughan et al, 2001).
21
Fig. 3 Tip60 affects the expression of a subset of the hormonally induced ER target
genes.
MCF7 cells were transfected with 200 µM of either non-specific siRNA (siNS) or
siTip60. The cells were then grown in hormone-free media for 72 hours. After 72 hours,
the cells were treated with 100 nM E2 or vehicle for 8 and 24 hours and then harvested.
Total RNA was analyzed by qRT-PCR and normalized to the level of 18S mRNA. The
results shown are mean and range of variation of duplicate sets of PCR reactions.
(A)
22
Fig. 3 Tip60 affects the expression of a subset of the hormonally induced ER target
genes continued.
(B)
23
Fig. 4 Tip60 interacts with estrogen receptor in a hormone dependent manner.
GST pulldown assays were performed with in vitro translated FLAG-tagged Tip60 and
incubated with GST fused ER-LBD bound to glutathione-Sepharose beads in the
presence and absence of hormone. Protein bound to beads was analyzed by immunoblot
with anti-FLAG antibody.
24
The LXXLL motif of Tip60 is essential for ligand-dependent binding.
Now that the ligand-dependent interaction between Tip60 and estrogen receptor
has been established for in vivo as well as in vitro, the interaction between the two is
analyzed further. In order to determine the role of the NR box, specifically the LXXLL
motif, in the interaction between Tip60 and the estrogen receptor the leucines at position
496 and 497 of Tip60 were modified to alanines. The exchange with the less hydrophobic
amino acid destroys the hydrophobic interface the α-helix once formed with the ligand
binding domain of ER without greatly altering the overall structure of Tip60.
Once again, in vitro translated full length Tip60 and the Tip60 (LXXAA) mutant
were each incubated with GST fused ligand-binding domain of ER in the presence and
absence of hormone. While the wildtype Tip60 showed an enhanced interaction with ER-
LBD in the presence of E2, the LXXAA mutant failed to show the same enhancement
(Fig. 5A).
Next, the role of the LXXLL motif of Tip60 was observed in vivo. The FLAG-
tagged wildtype Tip60 and the FLAG-tagged LXXAA mutant Tip60 were independently
overexpressed in COS-7 cells in the presence and absence of hormone. The prepared cell
extracts were immunoprecipitated with anti-FLAG antibody. Then the
immunoprecipitated proteins were analyzed by western blot using ERα antibodies.
Again, the wildtype Tip60 showed an enhanced interaction with the ER (LBD) in the
presence of hormone. However, the LXXAA mutant failed to show an enhanced
interaction with estrogen receptor in the presence of hormone (Fig. 5B). Consequently,
25
this suggests that Tip60 interacts with the estrogen receptor through its NR box, in
particular its LXXLL motif, in a ligand-dependent manner both in vitro and in vivo.
26
Fig. 5 Tip60 interacts with estrogen receptor through its NR box in a hormone-
dependent manner both in vivo and in vitro.
(A) GST pulldown assays were performed with in vitro translated FLAG-tagged Tip60 or
FLAG-tagged LXXAA mutant Tip60 and incubated with GST fused ER-LBD bound to
glutathione-Sepharose beads in the presence and absence of hormone. Protein bound to
beads was analyzed by immunoblot with anti-FLAG antibody.
(B) COS-7 cells were transfected with plasmids encoding ERα and either FLAG-tagged
Tip60 or FLAG-tagged LXXAA mutant Tip60. Immunoprecipitation was performed on
cell extracts with an antibody against the FLAG tag and then immunoblotted with anti-
ERα antibody.
27
Fig. 5 Tip60 interacts with estrogen receptor through its NR box in a hormone-
dependent manner both in vivo and in vitro continued.
(A)
(B)
28
The LXXLL motif of Tip60 is essential for stabilizing the association of Tip60 with
EREs of ER target genes.
Since the importance of the LXXLL motif in the hormone-induced interaction
between Tip60 and estrogen receptor has been established, its effect on Tip60’s
recruitment to transcription sites was studied by chromatin immunoprecipitation. FLAG-
tagged wildtype Tip60 and FLAG-tagged LXXAA mutant Tip60 were overexpressed in
MCF7 cells. The occupancy of the overexpressed proteins at the pS2 ERE1, ERE2, and
ERE3 was examined at time points between 0 and 60 minutes after the addition of
hormone (Fig. 6). As previously monitored, the transfected wildtype Tip60 showed a
cyclical recruitment to the estrogen responsive element sites compared to that of an
empty expression plasmid. Moreover, at the pS2 ERE1 and ERE3 regions, the LXXAA
mutant failed to be recruited at all and showed a similar pattern to that of the vehicle.
However, the pS2 ERE2 region saw no differentiation in apparent recruitment in cells
expressing the wildtype Tip60, the LXXAA mutant Tip60, or the exogenous Tip60! Thus
we saw no clear FLAG-Tip60 recruitment at ERE2. In conclusion, the LXXAA mutation
of Tip60 dramatically decreases the recruitment of Tip60 to the EREs of ER target genes.
The chromodomain of Tip60 recognizes methylated histones.
Chromodomains in other proteins are thought to recognize methylated histone
lysines (Sapountzi et al., 2006). In order to observe the role of the chromodomain of
29
Tip60, a His-tagged chromodomain of Tip60 was purified (Fig. 7). It was shown that the
chromodomain of Tip60 binds to H3K9me3 (Sun et al., 2009). The purified
chromodomain of His-tagged Tip60 was incubated with biotinylated histone H3,
H3K4me1, H3K4me2, and H3K4me3 peptides. The biotinylated peptides were pulled
down and the bound protein was immuno-blotted with anti-Tip60. Clearly, the
chromodomain of Tip60 preferentially interacts with mono-methylated and di-methylated
H3K4 peptides (Fig. 8).
The SWI/SNF complex is required for the recruitment of Tip60.
The SWI/SNF complex is an ATP-dependent chromatin remodeling complex
containing either BRG1 or Brm as the catalytic ATPase subunit (Trotter and Archer,
2007). The SWI/SNF complex aids in nuclear receptor mediated transcription by
remodeling chromatin and thereby facilitating the binding of transcriptional factors and
RNA polymerase (Trotter and Archer, 2008).
Here, we look at Tip60’s dependence on the SWI/SNF complex for coactivator
activity. SW13 cells, which carry all components necessary to form the SWI/SNF
complex except BRG1 and Brm, were used. Due to the lack of the core ATPase catalytic
subunit, BRG1 or Brm, the SWI/SNF complex is non-functional in these cells. The SW13
cells were transfected with MMTV-LUC reporter plasmid encoding the luciferase
reporter gene with the enhancer element specific for estrogen receptor, and plasmids
30
encoding ERα, the wildtype Tip60, and BRG1. The cells were then grown in medium
with hormone and then tested for luciferase activity.
In the absence of BRG1, no amount of Tip60 was able to activate the transcription
of the luciferase gene. However, in the presence of BRG1, Tip60 was able to enhance the
transcription of the reporter gene (Fig. 9). Strangely, there was no enhancement of
luciferase activity in the presence of BRG1, but absence of Tip60. However, very low
levels of BRG1 were transfected in order that the enhancement by Tip60 can be seen.
Thus the low level of BRG1 may account for the absence of enhancement in the absence
of Tip60. All in all, this indicates that the SWI/SNF complex is required for Tip60 to
coactivate ER-mediated transcription.
31
Fig. 6 LXXAA mutation of Tip60 compromises its recruitment to pS2 EREs.
Chromatin immunoprecipitation assays were performed with MCF7 cells transfected with
FLAG-tagged Tip60, FLAG-tagged LXXAA mutant Tip60, or empty vector and treated
with 100 nM E2 between time points 0 to 60 minutes. After immunoprecipitation of
cross-linked chromatin fragments with antibodies against the FLAG tag, the amount of
the corresponding Tip60 present at pS2 ERE1, ERE2, and ERE3 was determined by
qPCR. The pcDNA is the empty vector, which was transfected as a negative control. The
data are plotted as the percentage of total input before immunoprecipitation. Error bars
represent the range of variation of duplicate PCR reactions but were too small to be
detected at some time points.
32
Fig. 6 LXXAA mutation of Tip60 compromises its recruitment to pS2 EREs
continued.
33
Fig. 7 Protein Purification of His-tagged Tip60 chromodomain fragment
His-tagged Tip60 chromodomain fragment (A) was purified from E. coli using nickel
beads. The protein was eluted using high concentration of imidazole. Fractions of the
wash and elutions were immunoblotted using anti-His tag antibody for confirmation (B).
(A)
(B)
34
Fig. 8 Tip60 chromodomain preferentially interacts with mono-methylated and di-
methylated H3K4.
His-tagged Tip60 was incubated with histone H3, H3K4me1, H3K4me2, and H3K4me3
peptides. The biotinylated peptides were pulled down with streptavidin beads, and the
eluted proteins were blotted with anti-Tip60 antibody.
35
Fig. 9 The SWI/SNF complex is required for the coactivator function of Tip60 with
ERα.
The SW13 cells were transfected with murine mammary tumor virus-estrogen responsive
element (MMTV-ERE) luciferase reporter plasmid (50 ng) and expression plasmids
encoding ERα (0.02 ng), BRG1 (1 ng), and Tip60 (1 ng, 10 ng, or 100 ng). The cells
were then grown in medium with hormone for 48 hours. The cell extracts were
consequently detected for activation of luciferase activity using the luminometer.
36
The chromodomain of Tip60 is essential for stabilizing the association of Tip60 with
EREs of ER target genes.
In order to study the role of Tip60’s chromodomain and its effect on Tip60’s
recruitment to target genes, the chromodomain fragment of Tip60 was overexpressed in
MCF7 cells and the occupancy of the endogenous Tip60 at ER target gene EREs was
observed. By overexpressing the chromodomain fragment, it was believed that the
chromodomain of the endogenous Tip60 must compete with the overexpressed
chromodomain fragment.
Tip60 was immunoprecipitated with an antibody that recognizes the C-terminal
end of Tip60 to ensure that we are observing the recruitment of the endogenous Tip60,
rather than the overexpressed chromodomain fragment, to the EREs. As shown,
expression of the chromodomain fragment reduced the recruitment of endogenous Tip60
to pS2 ERE1, ERE2, ERE3 and GREB1 ERE2, ERE3 at 10 and 20 minutes.
Interestingly, Tip60’s recruitment to GREB1 ERE1 was only modestly affected (Fig.10).
However, the experiment may need to be repeated for clear conclusions.
The dominant negative effect caused by the overexpression of the chromodomain
fragment of Tip60 demonstrates that by having to compete with the chromodomain
fragment, the stable occupancy of the endogenous Tip60 at the EREs of pS2 and GREB1
were compromised.
37
Fig. 10 Dominant Negative Effect of Tip60 chromodomain fragment
Chromatin immunoprecipitation assays were performed with MCF7 cells transfected with
Tip60 chromodomain fragment. The cells were treated with 100 nM E2 between time
points 0 to 60 minutes. After immunoprecipitation of cross-linked chromatin fragments
with antibodies against the endogenous Tip60 using α-C terminal Tip60 antibody, the
amount present at pS2 ERE1, ERE2, and ERE3 and GREB1 ERE1, ERE2, ERE3 was
determined by qPCR. The data are plotted as the percentage of total input before
immunoprecipitation. Results shown are mean and range of variation of duplicate PCR
reactions and are representative of a single experiment which is representative of two
independent experiments.
(A)
38
CHAPTER 4: DISCUSSION SECTION
The COOH-terminal NR box, specifically the LXXLL motif, of Tip60 mediates the
hormone-dependent interaction with estrogen receptor.
The LXXLL motif contained in the NR box of Tip60 forms a α-helical structure
in which the leucine residues line up to form a hydrophobic interface. In many other
cofactors, such as CBP, SRC-1, and GRIP1, this LXXLL motif was shown to be the one
to mediate the interaction between the coactivator and the AF-2 domain of nuclear
receptors. Although it was previously shown that Tip60 does indeed interact with the
estrogen receptor in a ligand-dependent manner, it was unknown whether or not the
LXXLL motif of Tip60 mediated this interaction.
Here, we have shown that not only does Tip60 bind to the estrogen receptor in a
hormone-dependent manner in vitro (Fig. 4), but we have also shown that this interaction
is mediated by the LXXLL motif of Tip60 both in vitro and in vivo (Fig. 5). It is
important to note here that Tip60 seems to bind to the estrogen receptor in the absence of
hormone. Clearly, the LXXLL motif is not involved in the hormone-independent
interaction between Tip60 and the estrogen receptor. However, it would be very
interesting to see which domain/s of Tip60 are involved in the hormone-independent
interaction with the estrogen receptor. Future mapping studies could elucidate this
interaction.
39
Chromatin immunoprecipitation assays of overexpressed wildtype Tip60 and
LXXAA mutant Tip60 show that by abolishing the LXXLL motif, Tip60’s recruitment to
ER target gene EREs is compromised (Fig. 6). Overall, the interaction between the
LXXLL motif of Tip60 and the estrogen receptor α proves to be a possible, if not
important, mechanism of Tip60’s recruitment to estrogen responsive elements of ER
target genes.
The NH
2
-terminal chromodomain of Tip60 mediates the stabilization of Tip60’s
occupancy at ER target gene EREs.
The function of the NH
2
-terminal chromodomain of Tip60 is largely unknown.
Chromodomains in other proteins are believed to arbitrate the interaction between the
protein and methylated histone lysines. Very recently, the chromodomain of Tip60 was
shown to interact with tri-methylated H3K9, which is a repressive marker. Here, we show
that the chromodomain of Tip60 interacts, preferentially, with mono-methylated and di-
methylated H3K4, which is an active marker in the histone code (Fig. 8).
Moreover, by overexpressing the chromodomain fragment of Tip60 in MCF7
cells, we show a dominant negative effect caused by the competition of the endogenous
Tip60 chromodomain with the overexpressed chromodomain fragment for interaction
with the methylated histones. This competition compromises the recruitment of the
endogenous Tip60 to estrogen responsive elements of ER target genes (Fig. 10).
40
All in all, the chromodomain of Tip60 proves to provide a possible mechanism
through which it aids in the recruitment of Tip60 to ER target gene transcription sites.
The mechanism of recruitment of Tip60 to the promoters and enhancers of ER
target genes.
Tip60 interacts with the estrogen receptor through its NR box. Likewise the
chromodomain of Tip60 interacts with methylated histones. Since the compromise of
either of these two interactions can severely affect the recruitment of Tip60 to ER target
gene estrogen responsive elements and enhancer regions, it is unlikely that only one of
these interactions is responsible for Tip60’s recruitment. More likely than not, it must be
an interplay of both these interactions that promote not only Tip60’s recruitment to but
also its stabilization at the target gene transcription sites (Fig. 11).
Previous studies, in addition to the study here, show that Tip60 is recruited in a
cyclical fashion to transcription sites. For future studies, it would be very interesting to
determine which interaction is involved for which peak of recruitment or if there is an
order as to which domain of Tip60 makes the initial interaction and which domain
stabilizes Tip60’s position. To investigate the order in which these interactions come into
play, we can look at the occupancy of Tip60 at ER target gene promoters and enhancers
pre- and post- the methylation of the histones. All things considered, both interactions
from both the NR box and the chromodomain are necessary to successfully recruit Tip60
to ER target genes.
41
Fig. 11 Model of the Mechanism of Recruitment of Tip60 to ER Target Genes
Once the estrogen receptor associates with appropriate EREs, the catalytic subunit of the
SWI/SNF complex, BRG1 is recruited to remodel the chromatin structure. Shortly,
thereafter histone methyltransferases are recruited to methylate histones. Tip60’s NR box
is responsible for not only interacting with estrogen receptor but also recruiting Tip60 to
EREs. Likewise, Tip60’s chromodomain is responsible for not only interacting with
mono- and di-methylated H3K4 but also recruiting Tip60 to EREs. Thus, Tip60’s
recruitment to and occupancy at EREs is determined by an intact NR box and
chromodomain.
42
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Abstract (if available)
Abstract
The steroid receptors in the nuclear receptor superfamily are responsible for regulating many different functions including transcription. However, in order to completely and correctly activate transcription, the steroid receptors must recruit not only the general initiation factors but also other coregulatory factors, such chromatin remodelers and coactivators. Tip60, a 60 kD protein with an N-terminal chromodomain, a central intrinsic histone acetyltransferase (HAT) activity, and a C-terminal nuclear receptor binding domain (NR box) was previously reported to be a coactivator for androgen receptor, in addition to being able to bind to the estrogen receptor in vivo. Subsequently, Tip60 was identified to be recruited very early on to the pS2 estrogen response element1 (ERE1) site, hormone binding enhancer elements involved in transactivation, upon hormone treatment. Here, it is shown that the interaction between Tip60 and the estrogen receptor, both in vivo and in vitro, requires ligand binding to estrogen receptor and also interacts via the C-terminal NR box, specifically the LXXLL motif. Furthermore, the abolishment of the LXXLL motif compromises Tip60’s recruitment to certain estrogen response elements (EREs) of ER target genes. In addition, the N-terminal chromodomain is shown to preferentially bind to histone H3 mono-methylated and di-methylated at lysine4. Finally, it is shown that a competition for methylated histones between the wildtype Tip60 and the exogenously expressed Tip60 chromodomain fragment also compromises its recruitment to certain EREs of ER target genes. Thus, the stable recruitment and association of Tip60 at the EREs is dependent upon the normal function of its N-terminal chromodomain and C-terminal NR box.
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Asset Metadata
Creator
Lee, Janet M.
(author)
Core Title
The mechanism of recruitment of Tip60 to ER target genes
School
Keck School of Medicine
Degree
Master of Science
Degree Program
Biochemistry and Molecular Biology
Degree Conferral Date
2011-05
Publication Date
03/07/2011
Defense Date
02/14/2011
Publisher
University of Southern California
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coactivator,nuclear receptor,OAI-PMH Harvest,Tip60
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Rice, Judd C. (
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), Tokes, Zoltan A. (
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capricorn134@gmail.com,janetmle@usc.edu
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