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Roles of three domains of CCAR1 in transcription activation by nuclear receptors

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Roles of three domains of CCAR1 in transcription activation by nuclear receptors

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ROLES OF THREE DOMAINS OF CCAR1 IN TRANSCRIPTION
ACTIVATION BY NUCLEAR RECEPTORS

by

In Kyoung Mah

A Thesis Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(BIOCHEMISTRY AND MOLECULAR BIOLOGY)

May 2008

Copyright 2008 In Kyoung Mah

ii
Table of Contents

List of Figures iii

Abstract iv

Introduction 1

Materials and Methods 8

Results 10

Discussion 30

Bibliography 35

iii
List of Figures

Figure 1: Mechanism of steroid hormone and coactivator complex and schematic 6
drawing of CCAR1 structure.

Figure 2: Mapping the domains of CCAR1 required for stimulation of ER. 7

Figure 3: CCAR1 interacts with ER LBD in vitro. 11

Figure 4: Domain requirement for CCAR1 and TRAP220 interaction. 12

Figure 5: Contribution of the R rich region of CCAR1 to coactivator function. 15

Figure 6: Coactivator activity of CCAR1 and its deletion mutants. 17

Figure 7: CCAR1 cooperates with CoCoA. 21

Figure 8: Synergistic effects of five coactivators. 22

Figure 9: Cooperation of CCAR1 with TRAP220. 24

Figure 10: Synergistic enhancement by multiple coactivators. 26

Figure 11: Cooperation of CCAR1 with TRAP220 in GR activity. 29

Figure 12: CCAR1 might work differently in different assay systems. 34

iv
Abstract

Nuclear receptors are ligand dependent transcription factors that activate target genes
involved in diverse physiological processes such as metabolism, development, reproduction
through the recruitment of multiple coactivators.
Cell cycle and apoptosis regulator 1 (CCAR1) is a nuclear receptor coactivator and helps
to enhance the transcription activation by nuclear receptors. CCAR1 is also associated with
the mediator complex, which recruits RNA polymerase II to the target gene promoters.
One of three domains of CCAR1 interacts with estrogen receptor. Deletions of three
domains diminish the coactivator function of CCAR1. One domain is required for cooperation
with CoCoA. In addition, three domains are required for collaboration with TRAP220 in
estrogen receptor mediated transcription. One of three domains of CCAR1 is not necessary
for the multiple coactivator complexes activity. Two domains of CCAR1 play important roles in
TRAP220 associated GR dependent activity. Thus, three domains of CCAR1 might work
differently in different conditions.

1
Introduction

Nuclear receptors (NRs) are ligand responsive transcription factors which activate target
genes involved in diverse physiological processes such as metabolism, development,
reproduction, and tissue homeostasis (Mckenna et al., 1999). Members of the nuclear
receptor family include steroid hormone receptors such as the estrogen receptor (ER), the
androgen receptor (AR), and the glucocorticoid receptor (GR); receptors for non steroid
hormones such as thyroid hormone and retinoic acid; and orphan receptors for which ligands
have not been identified (Perissi and Rosenfeld, 2005).
Ligands of nuclear receptors are small and hydrophobic, and therefore enter the target
cells by simple diffusion (Sykiotis and Papavassiliou, 2002). Ligand bound NRs undergo a
conformational change which causes a series of events. There are several well known steps
of nuclear receptor signaling. For steroid hormone receptors, ligand binding triggers the
dissociation of heat shock proteins, allowing receptor dimerization, nuclear translocation, and
binding to specific sequence of DNA known as hormone response elements (HRE) (Mckenna
et al., 1999, Sykiotis and Papavassiliou, 2002). Then, the steroid receptors mediate
transcription through the recruitment of multiple coactivators and corresponding complexes to
the promoter of the target genes (Perissi and Rosenfeld, 2005). Unliganded non steroid
hormone receptors are bound to corepressor proteins which mediate repression, but ligand
binding to NRs causes a dissociation of corepressors and recruitment of coactivators which
result in recruiting additional proteins such as RNA polymerase II, and basal transcriptional
machinery. (Perissi and Rosenfeld, 2005) Through the balance between coactivators and
corepressors, transcription activation by non steroid hormone receptors is regulated (Lonard
and O’Malley, 2006).
Nuclear receptors consist of a highly conserved central DNA binding domain, C-terminal
ligand binding domain, and less conserved N-terminal region. Most NRs have two activation
domains, the N terminal activation function, AF-1, and C-terminal activation function AF-2

2
(Mckenna et al., 1999). AF-1 operates autonomously and in ligand-indepenent manners, while
AF-2 works in ligand- dependent ways (Aranda and Pascual, 2001).
Coactivators help to enhance the transcription activity through remodeling the chromatin
structure and assembling RNA polymerase II and basal transcriptional machinery (Fig.1A).
The coactivator complexes make up a signal transduction pathway that transports signals
from activated NRs to downstream transcriptional machinery (Stallcup et al., 2003). A number
of coactivators of NRs have been identified, including the member of the p160 coactivator
family, p300 and CBP protein.
The three members of the p160 coactivator family are SRC-1, GRIP1/TIF2 and
p/CIP/RAC3/ACTR/A1B1/TRAM/SRC-3 (Mckenna et al., 1999). The p160 coactivators have
common structures that include a central region containing LXXLL NR box motifs (where L is
Leucine and X is any amino acid), a C-terminal region including two transcriptional activation
domains - AD1 and AD2, and an N-terminal basic helix-loop-helix Per-Arnt-Sim (bHLH-PAS)
domain (Mckenna et al., 1999). AD1 of the p160 coactivators mediates the interaction with
histone acetyltransferases p300 and CBP, while AD2 recruits the histone methyltransferases
CARM1 and PRMT1 (Chen et al, 1999, Torchia et al., 1997). These enzymatic secondary
coactivators contribute to chromatin remodeling and transcription activation through histone
modification (Chen et al., 1999). The p160 coactivators interact with NRs through their LXXLL
motif (Perissi and Rosenfeld, 2005), and provide additional contacts to secondary
coactivators. Also N-terminal bHLH-PAS domain of p160 coactivators acts as an additional
transcriptional activation domain, AD3. This AD3 domain recruits numerous additional
coactivators including CoCoA and GAC63 (Kim et al, 2003, Chen et al 2005). Thus, the p160
coactivators play important roles in transcriptional activation by acting as a linker between
NRs and downstream secondary coactivators (Chen et al., 2005). The interaction among
coactivator complexes is regulated by posttranslational modifications such as methylation,
acetylation, and phosphorylation which alter the function of p160 coactivators (Lee et al.,
2005). Coactivators and corepressors are recruited to target promoters dynamically and

3
sequentially (Metivier et al., 2006). These dynamic actions are driven partially by
posttranslational modification of coactivators and corepressors (Mckenna and O’Malley, 2000).
Mediator (TRAP/SMCC/DRIP/CRSP/NAT/ARC/PC2) is a coactivator complex which
consists of 30 subunits including MED10 (NUT2), MED20 (TRFP) and MED14 (TRAP170)
(Malik and Roeder, 2000). Although the majority of the subunits are tightly associated and
seem to constitute the core of the complex, some individual subunits including MED1
(TRAP220) display variable association with the complex (Malik et al., 2004). Some subunits
of mediator complex associate with RNA polymerase II holoenzyme (Sato et al, 2004).
Mediator complexes interact with various transcription activators such as NRs, NF- ĸB, p53
and VP16 (Blazek et al., 2005), and with the general transcriptional machinery (Malik and
Roeder, 2000). Thus, Mediator might function as a bridge between transcriptional activators,
such as NRs and RNA polymerase II (Stumpf et al., 2006). Mediator interacts with AF2
activation domain of NRs through the TRAP220 subunit in a ligand dependent manner. This
interaction is mediated via two LXXLL motifs of TRAP220 (Malik et al., 2004). For example,
ER binds mediator complex through the TRAP220 subunit, and this mediator complex
increases the ER dependent transcription activity in vitro (Kang et al., 2002). TRAP220 can
also serve as interacting subunit for other transcription activators including p53 (Wada et al.,
2004). TRAP220 might also play an oncogenic role in steroid hormone-dependent cancer
progression, because of overexpression of TRAP220 in several ER–positive breast and
ovarian cancer cell lines (Zhu, et al., 1999). Although the role of mediator in RNA polymerase
II transcription is not fully understood, by acting as a bridge between transcription activators
and the general transcription factors which are associated with RNA polymerase II, the
mediator might provide additional chances to fine tune the regulatory signals derived from
DNA binding factors to transfer to RNA polymerase II machinery (Malik and Roeder, 2005).
The coiled-coil coactivator (CoCoA) functions as a coactivator for NRs (Kim et al, 2003),
or Aryl hydrocarbon receptor (AHR) (Kim and stallcup, 2004). It also participates in
transcription activity of target genes by the LEF/TCF transcription factor through the wnt/ β-
catenin pathway (Yang et al., 2006). CoCoA consists of central coiled coil domain flanked by

4
N-terminal and C-terminal activation domains (ADs) (Kim et al, 2003). The central coiled-coil
domain binds the bHLH-PAS domain of p160 coactivator, but not directly to NRs (Kim et al,
2003). Therefore, CoCoA functions as a secondary coactivator for NRs and the activity of
CoCoA is highly dependent on the presence of p160 coactivators (Kim et al., 2003). In
addition, CoCoA cooperates synergically with GRIP1, CARM1, and p300 to make greater the
transcription activity by ER (Kim et al., 2003). The strong C-terminal AD of CoCoA is essential
for the coactivator function of CoCoA with NR and AHR (Kim et al, 2003, Kim and Stallcup,
2004). Approximately 91 C-terminal amino acids containing acidic, serine/proline-rich and
phenylalanine-rich subdomains form the minimal AD. Also C-terminal AD of CoCoA interacts
with p300 which is important for coactivator function of CoCoA (Kim et al., 2006). The N-
terminal AD of CoCoA is necessary and sufficient to interact with β-catenin, thereby the
interaction between CoCoA and β-catenin synergistically activates LEF mediated transcription.
Also the N-terminal AD interacts with p300, which is important for synergistic activation
between CoCoA and p300 in LEF mediated transcription (Yang et al., 2006). Therefore,
CoCoA acts as a coactivator for NR and LEF/TCF transcription factors through differential
utilization of the different activation domains (Yang et al., 2006).
Our lab identified Cell cycle and apoptosis regulator 1 (CCAR1) as a CoCoA C-terminal
AD binding partner using a biochemical approach. CCAR1 was initially cloned by a functional
knockout genetic approach and was identified as a novel mediator of apoptosis signaling by
retinoids (Rishi et al., 2003). Furthermore, CCAR1 is a key transducer of the epidermal
growth factor receptor (EGFR) dependent apoptosis signaling pathway (Rish et al., 2006).
Moreover, CCAR1 is suppressor of human breast cancer growth and its expression is
reduced in tumors (Zhang et al., 2007). CCAR1 is also associated with Mediator complexes
(Sato et al., 2004). The mouse CCAR1 gene encodes 1146 amino acids including a 35 amino
acid SAP domain (nuclear scaffold attachment factors A and B (SAF-A and –B), Acinus, and
protein inhibitor of activated STAT (PIAS)), an N-terminal Q rich region, and an arginine rich
region (JH Kim, unpublished data) (Fig.1B). SAP domain is involved in chromosomal
organization and contributes to the regulation of transcription, DNA repair, and RNA

5
processing (Hashill et al., 2004). By fusing CCAR1 and its fragments to GAL4 DBD, we found
that the N-terminal Q rich region is an autonomous transcriptional AD. By transient
transfection assay using an AD deletion mutant and SAP deletion mutant, we found that AD
and SAP domains play important roles in the coactivator function of CCAR1 (JH Kim,
unpublished data) (Fig.2). CCAR1 binds ER and p53 and facilitates the recruitment of
mediator to the promoter of the target genes. CCAR1 enhances the transcriptional activity of
ER and other nuclear receptors (JH Kim, Unpublished data). Importantly, CCAR1 is
associated with TRAP220 containing mediator complexes (JH Kim, Unpublished data). The
recent studies suggest an alternative mechanism for mediator recruitment to PPAR target
promoters by intermediate cofactors that are functionally associated with TRAP220 (Ge et al.,
2008). Therefore, CCAR1 probably functions as a bridge between p160 coactivator and the
mediator complexes.
The objectives of this thesis are 1) to determine which region of CCAR1 interacts with
estrogen receptor or TRAP220, 2) to find out the roles of three domains of CCAR1 in
transcriptional activation by nuclear receptors, 3) to investigate the contribution of the three
domains of CCAR1 when it cooperates with CoCoA or TRAP220, and 4) to examine the
contribution of the Arginine rich region of CCAR1 when CCAR1 cooperates with multiple
coactivators.
In this thesis, I first present that the Arginine-rich region of CCAR1 binds ER ligand
binding domain (LBD) and plays important roles in the ER activity in the transcription. I show
the contribution of three important domains (CCAR1 AD, SAP, and R domain) for CCAR1
activity in the interaction with NRs, CoCoA, and TRAP220. Moreover, I present that CCAR1
and multiple coactivators synergistically enhance the ER mediated transcription, but the R
rich domain is not required for the synergistic action among the coactivator complexes.

6

(A)
HRE
S R
p160
AD3
AD2
AD1
CoCoA
C A R M 1
p300
Nucleosome
Methyl
Acetyl
TFIIB
TBP
Pol II
Complex
TATA
SAM
AcCoA
TRAP/DRIP
HRE
S R S R
p160
AD3
AD2
AD1
CoCoA
C A R M 1
p300
Nucleosome
Methyl
Acetyl
TFIIB
TBP
Pol II
Complex
TATA
SAM
AcCoA
TRAP/DRIP

(B)
151-400
361-630
670-900
3287
290 381 633 667
670 894 992 1054
Q-rich
R-rich SAP
Acidic domain PABP
1 1146
Coiled-coil
792 819
Coiled-coil
1029 1111
LSVLL
746 750
151-400
361-630
670-900
3287
290 381 633 667
670 894 992 1054
Q-rich
R-rich SAP
Acidic domain PABP
1 1146
Coiled-coil
792 819
Coiled-coil
1029 1111
LSVLL
746 750
3287
290 381 633 667
670 894 992 1054
Q-rich
R-rich SAP
Acidic domain PABP
1 1146
Coiled-coil
792 819
Coiled-coil
1029 1111
LSVLL
746 750

Figure 1. Mechanism of steroid hormone and coactivator complex and
schematic drawing of CCAR1 structure.

(A) Hormone activated steroid hormone receptors bind to a specific hormone-responsive
enhance element (HRE) in DNA and recruit various coactivator complexes, which help to
remodel chromatin structure and recruit and activate RNA pol II complex and its associated
basal transcription machinery.
(B) Location of the Q-rich region, the basic (R-rich) region, SAP domain, poly A binding
protein (PABP) homology region, and the predicted coiled coil domains are indicated.

7

0 500 1000 1500
E2 (-)
E2 (+)
CC AR1
CC AR1 290 -1146
CCAR1 651-655 ∆
CC AR1 L64 1P/R 642P
CC AR1 R 662P/L663P
CC AR1 L74 9,75 0A
CC AR1 Y189F
1
2
4
5
6
7
8
9
3
Luciferase activity
(10 RLU)
3

Figure 2. Mapping the domains of CCAR1 required for stimulation of ER.

CV-1 cells were transiently transfected with MMTV (ERE)-LUC (200 ng), pHE0 (2 ng),
pSG5.HAb CCAR1 (600 and 900 ng), and pSG5.HAb-CCAR1 mutants (900 ng). This data
was kindly provided by Jeong hoon Kim.

8
Materials and Methods

Plasmids.

The full length CCAR1 cDNA was amplified from mouse 17 day embryo cDNA library
(Clontech) and cloned into pSG5.HAb (gift form Martin A. Privalsky, University of California at
Davis), which expresses N-terminal hemagglutinin (HA) epitope tag. PCR amplified cDNA
encoding CCAR1 290-410 was inserted into XhoI and EcoRI sites of pSG5. HA (Chen et al.,
1999), and pGEX-4T-1 (Amersham Biosciences). pGEX-4T-1 CCAR1 290-410 encodes
glutathione S-transferase (GST) fused to CCAR1 290-410. To create a CCAR1 mutant lacking
the R domain, PCR amplified cDNA encoding CCAR1 1-288 was inserted into BamHI and
SmaI sites of pSG5.HAb, and PCR amplified cDNA encoding CCAR1 411-1146 was inserted
into SmaI and XhoI sites of pSG5.HAb CCAR1 1-288 vector. Other plasmids encoding
CCAR1 fragments were created by PCR amplification and cloned into pSG5.HA. TRAP220
AB fragment from pC1N4-hTRAP220 (gift from Robert G. Roeder, Rockfeller University) was
cloned into pGEX-5X-1 (Amersham Biosciences). The full length TRAP220 was cloned into
pSG5.HAb vector. The plasmids for transient transfection are described as follows: pSG5.HA
CoCoA, pSG5.HA GRIP1, pSG5.HA CARM1, pSG5. HE0, MMTV (ERE)-LUC, 2ERE-TATA-
LUC, 2ERE-TK-LUC (Kim et al., 2003), MMTV-LUC (Chen et al., 1999), pCMV-p300 (Lee et
al., 2002), and hGR. pGEX-ER-LBD was gift from Geoffrey L. Greene (University of Chicago).

Cell Culture.

CV-1, COS7, and MCF-7 cells were maintained in the Dulbecco’s modifed Eagle’s
medium (DMEM) (Invitrogen) with 10% fetal bovine serum, penicillin, and streptomycin.

9
Transient Transfection.

For luciferase assays, CV-1 cells were plated at 1x10
5
cells/well into 12-well plates and
transiently transfected by TargeFect F1 reagent (Targeting Systems). Total amount of DNA
plasmids in each well was equalized by addition of pSG5.HA empty vector. Two hours after
transfection, cells were incubated in phenol red free DMEM containing 5% charcoal treated
fetal bovine serum, penicillin, and streptomycin, and 20 nM Na-HEPES with or without 100
nM E2 or 100 nM DEX. Two days after transfection, cell lysates were analyzed for luciferase
activity using Promega Luciferase assay kit. The data shown here are the mean and variation
of duplicate sets. Results shown are from one experiment which is representative of two
independent experiments. For checking expression of CCAR1 or CCAR1 deletion mutants,
COS7 cells were transiently transfected in 12 well plates using TargeFect F-2 reagent
(Targeting Systems) for two days before harvest. Cells were extracted in 200 ul NP40 buffer
(50 mM Tris buffer pH 8.0,150 mM NaCl, 0.1% NP40) containing protease inhibitor cocktail
tablets (Roche). Cell extracts were classified by centrifugation at Top speed of microfuge for
10 minutes, and the supernatants were analyzed by immunoblot with anti-HA antibody (Roche
Applied Science).

GST pull down assay.

HA epitope-tagged CCAR1, CCAR1 fragments, and ER were synthesized in vitro by
using TNT-Quick Coupled Transcription/Translation system (Promega) according to
manufacturer’s protocol. GST fusion proteins were expressed in E. coli BL21 and bound to
glutathione-Sepharose beads (Amersham Pharmacia). In vitro translated products were
incubated with GST fusion protein bound to beads in NETN buffer (20 mM Tris buffer pH 7.6,
1 mM EDTA, 200 mM NaCl, and 0.01% NP40). After precipitation using centrifugation, beads
were washed using 200 mM, 250 mM, or 300 mM NaCl NETN buffer. Bound proteins were
analyzed by immunoblot with anti-HA antibody or anti-ER α antibody.

10
Results

Determination of estrogen receptor interaction domain of CCAR1.

In previous studies, full length CCAR1 interacted with GST ER-LBD in hormone
independent manner. Among CCAR1 fragments , CCAR1 151-400, 361-630, 670-900, and
631-1146 fragments bound GST ER-LBD in hormone independent manner (JH Kim,
unpublished data) (Fig.3A). To determine which region of CCAR1 among those fragments
bind to GST ER LBD in more stringent conditions, we increased the concentration of NaCl in
the NETN washing buffer from 200 mM to 250 mM, and 300 mM NaCl. In high salt conditions,
only strongly bound proteins remained bound to beads. HA-tagged CCAR1 fragments in vitro
translated were incubated with GST or GST ER LBD with or without 100 nM E2. The bound
proteins were washed in 250 mM or 300 mM NaCl NETN buffer. Only CCAR1 151-400
fragment including arginine (R) rich region effectively and specifically interacted with GST-ER-
LBD in ligand independent manner; in contrast, CCAR1 361-630 and 670-900 containing
LXXLL motif didn’t bind GST ER LBD in the above conditions (Fig.3B). Thus, 151-400 of
CCAR1 would be the major ER LBD interaction domain in vitro. To better define the region
that binds ER LBD within the highly basic 151-400 fragment, we made only R rich region
plasmid (amino acid 290-410). To see interaction between R rich region and ER, we also
used GST pull down assay. As expected, this GST CCAR1 290-410 fusion proteins weakly
bound in vitro synthesized ER α in hormone independent way (Fig.3C). Therefore, this R rich
region is important domain to interact with ER.

11

Figure 3. CCAR1 interacts with ER LBD in vitro.

(A)HA-tagged CCAR1 or its fragments synthesized in vitro were incubated with equal
amount of GST or GST- ER ligand binding domain (LBD) fusion protein bound to beads with
or without 100 nM E2 treatment. The bound proteins were eluted and analyzed by SDS PAGE
and immunoblot with antibody againt the HA epitope tag. This data was provided by JH Kim.

CCAR1 670-900
300 mM NaCl 250 mM NaCl
CCAR1 151-400
CCAR1 361-630
GST
GST-ER-LBD
E2 -+
10% INPUT
10% INPUT
GST
GST-ER-LBD
-+ E2

(B) HA-tagged CCAR1 fragments interact with ER LBD in different washing conditions.
GST pull down was performed as in Fig. 3A using HA-tagged CCAR1 fragments and GST or
GST-ER LBD bound to beads. The pelleted beads were washed with 250 mM or 300 mM
NaCl NETN washing buffer. The bound proteins were analyzed by immunoblot with anti-HA.

12

Figure 3. (continued)

(C) GST CCAR1R fusion protein interacts with ER in vitro. GST pull down assays were
performed as in Fig.3a, using pHE0, GST CCAR1 290-410 fusion protein or GST. The bound
proteins were analyzed by immunoblot with anti-ER α antibody.

Figure 4. Domain requirement for CCAR1 and TRAP220 interaction.

HA-tagged CCAR1 or CCAR1 fragments were translated in vitro and incubated with GST
or GST-TRAP220 AB fusion protein bound to beads. The bound proteins were analyzed by
immunoblot with anti-HA antibody.

13
CCAR1 interacts with TRAP220.

Earlier studies showed that TRAP220 is a subunit of mediator complex, and interacts with
NRs via LXXLL motif (Malik et al., 2004). To assess the interaction between TRAP220 and
CCAR1, we conducted GST pull down assay. In vitro synthesized HAb. CCAR1 was
incubated with GST or GST TRAP220 AB (amino acid 1-670) fusion protein. The bound
proteins were assayed by immunoblot with anti-HA antibody. We found that full length CCAR1
interacted with GST TRAP220 AB (Fig.4). To examine further the region which interacts with
TRAP220, we again performed the in vitro interaction assay using several fragments of
CCAR1. CCAR1 1-249, 151-400, 203-660 and 670-900 strongly bound GST TRAP220 AB
fusion protein (Fig.4). From these interactions, we assumed that CCAR1 202-400 region and
CCAR1 720-900 region interact with TRAP220, but further tests are needed to confirm this.

CCAR1 functions as a coactivator in ER mediated transcription.

In prior studies, wild type CCAR1 enhanced the ER mediated transcription activity in a
dose dependent manner, and also increased the basal activity of MMTV (ERE)-LUC at high
concentrations of CCAR1 in CV-1 cells (JH Kim, Unpublished data) (Fig.2). To test whether
CCAR1 R rich region also possesses coactivator activity for ER, we transfected CV-1 cells
with luciferase reporter plasmid MMTV (ERE)-LUC, ER expression vector, CCAR1 or CCAR1
R rich deletion plasmid (deletion of 290-410 from the wild type CCAR1, indicated as CCAR1
∆R). As in previous studies, E2 treatment alone slightly enhanced luciferase activity.
Overexpression of CCAR1 strongly magnified the ER dependent transcription activity in dose
dependent and E2 dependent manner. In addition, we observed increased basal activity of
MMTV (ERE)-LUC at high levels of CCAR1 (900 ng of plamids). In contrast, overexpression
of CCAR1 ∆R failed to enhance the ER ability to stimulate transcription (Fig.5A). Although
CCAR1 ∆R was less expressed than wild type CCAR1, expression levels of CCAR1 ∆R
(900ng) was similar to that of CCAR1 (600ng). When these two conditions were compared,

14
we can still see overexpression of CCAR1 ∆R failed to enhance the ER mediated
transcription. We applied another experiment to investigate the contribution of R rich region
in ER activity. We tested the dominant negative effects of CCAR1 R (amino acid 290-410) to
CCAR1 using transient transfection. Without the coexpression of R rich domain, wild type
CCAR1 increased the luciferase activity in dose dependent manner. However, the coactivator
activity of CCAR1 was strongly reduced by coexpression of R rich region in dose dependent
manner (Fig.5B). To determine whether this inhibition of ER activity is caused by R rich region
or squelching effect by too much amounts of DNA, we transfected CV-1 cells with same
amount of DNA (1100 ng) as combined amount of DNA between CCAR1 and CCAR1 R.
CCAR1 still strongly intensified the hormone dependent activity of ER(Fig.5B). Thus, R rich
region plays important roles in the coactivator function of CCAR1 in ER mediated transcription.

Roles of three domains, AD, SAP, and R rich domains, in the coactivator
function of CCAR1.

In earlier studies, deletion or point mutations of the N-terminal AD or SAP domain
abolished the coactivator activity of CCAR1 (Fig.2). Cotransfection of these two domains with
CCAR1 strongly reduced the coactivator function of CCAR1 (JH Kim, unpublished data). We
wanted to confirm the roles of two other domains in coactivator function, so we conducted
transient transfection assays. CV-1 cells were transfected with two different luciferase reporter
plasmids (MMTV (ERE)-LUC, 2ERE-TATA-LUC), expression vectors encoding ER, and
CCAR1 or CCAR1 ∆AD (amino acid 290-1146), or ∆SAP (deletion of 651-655 from the wild
type CCAR1, indicated CCAR1 ∆SAP), or ∆R. As expected, overexpression of wild type
CCAR1, but not CCAR1 ∆R enhanced the ability of ER to activate both luciferase reporter
genes (Fig.6 lane 3-4 and lane 7-8). Also, both CCAR1 ∆AD and ∆SAP failed to stimulate
thecoactivator function in ER mediated transcription (Fig.6 lane 5-6 and 9-10). Two deletion
mutants were expressed at wild type levels, but CCAR1 ∆R was less expressed than wild
type CCAR1 (Fig. 6 lower panel). The expression levels of CCAR1 ∆R (600ng) was similar to

15

0
10
20
30
40
50
60
70
Luciferase Activity(10
3
RLU)
E2-
E2+

FL. CCAR1
CCAR1 R ∆

FL CCAR1 300 450 600 900 1100
CCAR1 R ∆ 300 450 600 900 1100

Figure 5. Contribution of the R rich region of CCAR1 to coactivator
function.

(A) (Upper panel): CV-1 cells were transfected with MMTV (ERE)-LUC reporter plasmids
(200 ng), pHE0 encoding ER (2 ng), pSG5.HA CCAR1 (300, 600, or 900 ng) or pSG5.HA
CCAR1 ∆R (300, 600, or 900 ng) as indicated. Cells were incubated 48 hours after
transfection and were assayed for luciferase activity. The luciferase activity results shown are
representative of two independent experiments.
(Lower panel): Expression level of CCAR1 and CCAR1 ∆R. Different amounts (300, 450,
600, 900, or 1100 ng) of plasmids encoding HAb-tagged CCAR1 or CCAR1 del R were
transfected into CO7 cells. Cell lysates were collected two days after transfection and were
analyzed by immunoblot using anti-HA antibody.

16

E2 - + + + + + + + + + + + + + + +
FL.CCAR1 + + + + + + + ++
CCAR1 R
0
10
20
30
40
50
60
70
80
90
10 0
Luciferase A ctivity(10
3
RLU)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

Figure 5. (continued)

(B) Dominant negative effects of CCAR1 R rich region. Transient transfection using
pSG5.HAb CCAR1 (+, 900 ng, ++, 1100 ng) and variable amounts of pSG5.HA R (290-410)
(5, 25, 50,100, 150, and 200 ng) were performed as in Fig.5A. Luciferase activity results
shown are representative of two independent experiments.

17

Figure 6. Coactivator activity of CCAR1 and its deletion mutants.

(A) (Upper panel): Transient transfection using two different luciferase reporter plasmids
(2ERE-TATA-LUC, MMTV (ERE)-LUC) (200 ng), pHE0 encoding ER (2 ng), variable amounts
(+,300 ng and ++, 600 ng) of pSG5.HAb CCAR1 and its deletion mutants ( CCAR1 del AD,
del SAP, and del R) were performed as in Fig 5A. Luciferase activity data shown are
representative of two independent experiments.
(Lower panel): Expression level of CCAR1 and CCAR1 deletion mutants.
Different amounts (300, 450, 600 ng) of plasmids encoding HAb-tagged CCAR1 or CCAR1
deletion mutants were transfected into CO7 cells. Cell lysates were collected two days after
transfection and were anaylzd by immunoblot using anti-HA antibody.
(B) CV-1 cells were transfected with 2ERE-TATA-LUC (200 ng), pHE0 (2 ng), pSG5.HAb
CCAR1 or its deletion mutants (+, 600 ng, ++, 900 ng, +++, 1100 ng); CCAR1 ∆AD, ∆SAP,
and ∆R, as indicated.

18

(A)

FL.CCAR1
CCAR1 R ∆
CCAR1 SAP ∆
CCAR1 AD ∆
FL.CCAR1
CCAR1 R ∆
CCAR1 SAP ∆
CCAR1 AD ∆

19

(B)
E2 - + + + + + + + + + +
FL. CCAR1 + ++
CCAR1 AD ∆ + ++
CCAR1 R ∆
+ ++ CCAR1 SAP ∆
+ ++ +++
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
12 34 5 6 7 8 9 10 11
Luciferase Activity(10
3
RLU)

20
that of CCAR1 (450ng), so we performed another transient transfection assay to correct
differences of the expression levels using increased amounts of CCAR1 ∆R. CCAR1 ∆R in
high levels still failed to enhance the ER ability to stimulate the transcription (Fig. 6B). Thus,
these three domains are required for the coactivator function of CCAR1 for ER.

CCAR1 cooperates with CoCoA.

CCAR1 is the binding partner of CoCoA AD and cooperated with CoCoA to enhance the
activity for ER (JH Kim, Unpublished data). To address the contribution of these three
domains in cooperation with CoCoA, CV-1 cells were transiently transfected with pHE0 and
2ERE-TATA-LUC reporter. For the cooperation effect, we used low levels of CCAR1 or
CCAR1 deletion mutants. Expression of CoCoA or CCAR1 alone increased the ER activity,
but cotransfection with CoCoA and CCAR1 strongly augmented luciferase activity (Fig.7 lane
3 and 4 versus 5). However, CCAR1 ∆AD failed to cooperate with CoCoA to stimulate ER
mediated transcription (Fig.7 lane 5 versus lane 7). Both SAP and R deletion mutants showed
partial cooperation with CoCoA (Fig.7 lane 5 versus lane 9 and 11). We also observed that
high concentration of CCAR1 ∆R still cooperated partially with CoCoA (Data not shown).
These results showed that AD domain plays essential roles in collaboration with CoCoA.

Synergistic effect with other coactivators.

Since CoCoA, p300, and CARM1 bind to separate activation domains of GRIP1, and
synergistically increased the ER mediated luciferase activity (Kim et al., 2003), we next tested
synergic activity of multiple coactivators with CCAR1. Since low levels of ER facilitate the
synergistiv interaction among multiple coactivcators (Kim et al., 2003), we employed low ER
concentrations. CV-1 cells were transfected with reporter plasmid, 2ERE-TATA-LUC,
combined with plamids encoding GRIP1, CARM1 (C1), p300, CoCoA, CCAR1 or CCAR1 ∆R.
The combination of GRIP1, CARM1, p300, and CoCoA slightly increased the luciferase

21

0
0.5
1
1.5
2
2.5
3
Luciferase Activity(10
3
RLU)
1 2 3 4 5 6 7 8 9 10 11
E2 - + + + + + + + + + +
CoCoA + + + + +
FL.CCAR1 + +
+ +
CCAR1 R ∆ + +
CCAR1 SAP ∆ + +
2ERE-TATA-LUC
CCAR1 AD ∆

Figure 7. CCAR1 cooperates with CoCoA.

CV-1 cells were transfected with 2ERE-TATA-LUC (200 ng), pHE0 (2 ng), pSG5.HAb
CCAR1 or its deletion mutants (450 ng); CCAR1 ∆AD, ∆SAP, and ∆R, and pSG5.HA CoCoA
(400 ng), as indicated. Results shown are representative of two independent experiments.

22

1 2 3 4 5 6 7 8 9 10
E2 - + + + + + + + + +
GRIP1 + + + + + + +
C1+p300 + + + + + +
CoCoA + + + + +
CCAR1 + + + +
CCAR1 R ∆ +
0
1
2
3
4
5
6
7
8
9
10
L u c iferase A c tivity (1 0
3
RL U )

Figure 8. Synergistic effects of five coactivators.

CV-1 cells were transfected with 2ERE-TATA-LUC reporter plasmid (200 ng), pHE0 (0.2
ng), pSG5.HA GRIP1 (200 ng), pSG5.HA CARM1 (200 ng), pCMV-p300 (200 ng), pSG5.HA
CoCoA (200 ng), pSG5.HAb CCAR1 (200 ng), or pSG5.HAb CCAR1 ∆R (200 ng), as
indicated. Transfected cells were grown in medium with or without 100 nM E2. These results
shown are representative of two independent experiments.

23
activity (Fig.8 lane 1-5), while the combination of GRIP1, CARM1, p300, CoCoA, and CCAR1
caused a dramatic synergy of ER mediated transcription activity ( lane 6), which was fully
dependent on the coexpression of GRIP1 (lane 8). Interestingly, cotransfection of CCAR1 ∆R
with other coactivators enhanced the reporter activity even more, compared with CCAR1
(Lane 6 versus lane 7). Thus, it seems that the R rich domain is not necessary for the
synergistic enhancement of ER dependent reporter gene activity.

Cooperation of CCAR1 with TRAP220.

In previous studies, CCAR1 facilitated the recruitment of mediator complex to the
promoter of the nuclear receptor target genes (JH Kim, Unpublished data). Also, TRAP220 in
the mediator complex is an NR interacting subunit and enhanced NR activity (Malik et al.,
2004). We examined the collaboration between CCAR1 or CCAR1 deletion mutants and
TRAP220. We transfected CV-1 cells with 2ERE-TATA-LUC, pHE0, TRAP220, and CCAR1 or
CCAR1 deletion mutants ( ∆AD, ∆SAP, and ∆R). Expression of TRAP220 alone slightly
increased the luciferase activity, and expression of CCAR1 alone enhanced the ER activity in
a dose dependent manner (Fig.9 lane 2-4). Coexpression of CCAR1 with TRAP220
synergistically increased 2ERE-TATA-LUC dependent licuferase activity in a dose dependent
manner (lane 5 and 6). In contrast, cotranfected CCAR1 deletion mutants with TRAP220
failed to enhance the luciferase activity (Lane 7-12). Thus, these three domains are required
for its cooperation with TRAP220 in ER medicated transctiption.

Synergistic enhancement by CCAR1, TRAP220, and multiple
coactivators.

We next tested the coopetation activity between CCAR1 and TRAP220 in combination
with several coactivators in low ER conditions. GRIP1 and CoCoA together three times

24

TRAP220 + + + + + + + + +
CCAR1 + ++ + ++
CCAR1 AD ∆ + ++
CCAR1 SAP ∆ + ++
CCAR1 R ∆ + ++
0
5
10
15
20
25
30
Luciferase A ctivity(10
3
RL U)
E2-
E2+
1 2 3 4 5 6 7 8 9 10 11 12

Figure 9. Cooperation of CCAR1 with TRAP220.

CV-1 cells in 12-well plates were transfected with 2ERE-TATA-LUC (200ng), pHE0 (2ng),
pSG5.HAbTRAP220 (400 ng), pSG5.HAB CCAR1 (+, 600 ng, ++, 900 ng) or its deletion
mutants; CCAR1 ∆AD, ∆SAP, and ∆R (+, 600 ng, ++, 900 ng), as indicated. Transfected cells
were grown in medium with or without 100 nM E2. These results shown are representative of
two independent experiments.

25
enhanced the ER mediated transcription activity (Fig.10A lane 4), and GRIP1, CoCoA, and
TRAP220 further increased the reporter activity (Lane 5). CCAR1 along with the three other
coactivators showed additive effect (lane 6) which is entirely reliant on the presence of GRIP1
(lane 7 and 12-14). We also examined the synergistic effects between TRAP220 and five
other coactivators. As expected, five coactivators synergistically magnified the luciferase
activity (Fig.10C lane 1-6). However, cotransfection of five coactivators and TRAP220 didn’t
further enhance the ER dependent transcription activity (Lane 7). Curiously, CCAR1 ∆R with
other coactivators even more stimulated the ER dependent transcription, compared with
CCAR1 (Fig.10B). Therefore, CCAR1 cooperated with TRAP220, but this interaction is not
enough to stimulate synergistic effect with multiple coactivators.

Cooperation between TRAP220 and CCAR1 in GR mediated
transcription.

The hormone dependent activity of GR reporter gene also was increased by CCAR1 in
CV-1 cells (JH Kim, Unpublished data). GR also cooperates with TRAP220. To investigate
whether CCAR1 or CCAR1 deletion mutants with TRAP220 affect GR dependent activity, CV-
1 cells were transfected with GR expression vector, and MMTV-LUC reporter plasmid. As in
prior studies, cotransfection of CCAR1 and GR vector increased the luciferase activity in
ligand dependent and dose dependent ways (Fig.11 lane 1, 2, 4, and 6). Futhermore, CCAR1
and TRAP220 together synergistically enhanced the GR dependent luciferase activity (lane 5
and 7). Interestingly, CCAR1 ∆SAP showed similar elevation of GR activity (lane 12-15),
compared with wild type CCAR1. Unlike CCAR1 ∆SAP, CCAR1 ∆AD and ∆R failed to enhance
the luciferase activity by GR (Lane 8-11 and 16-19). Thus, CCAR1 acts jointly with TRAP220
in GR dependent luciferase activity, and two AD and R domains are required to increase the
GR activity and cooperate with TRAP220.

26

0
5
10
15
20
25
30
35
40
45
50
Luciferase Activity(10
3
RLU)
1 2 3 4 5 6 7 8 9 10 11 12 13 14
E2 - + + + + + + + + + + + + +
GRIP1 + + + + + + + +
CoCoA + + + + + + +
TRAP220 + + + + + + +
CCAR1 + + + + + + +

Figure 10. Synergistic enhancement by multiple coactivators.

(A) Additive enhancement of ER-mediated transcription activity by four coactivators
(GRIP1, CoCoA, TRAP220, and CCAR1). Transient transfection using 2ERE-TATA –LUC
(200 ng), pHE0 (2 ng), pSG5.HA GRIP1 (200 ng), pSG5.HA CoCoA (200 ng), pSG5.HAb
TRAP220 (200 ng), and pSG5.HAb CCAR1 (200 ng) as indicated. These results shown are
representative of two independent experiments.

27

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
E2 - + + + + + + + + + + + + + +
GRIP1 + + + + + + + + +
CoCoA + + + + + + + +
TRAP220 + + + + + + + +
CCAR1 + + + + + + +
CCAR1 R ∆ +
0
5
10
15
20
25
30
35
L u c ife ra s e A c tiv ity (1 0
3
RLU)

Figure 10. (continued)

(B) Synergistic effects of four coactivators. CV-1 cells were transfected with 2ERE-TATA-
LUC reporter plasmid (200 ng), pHE0 (2 ng), pSG5.HA GRIP1 (200 ng), pSG5.HA CoCoA
(200 ng), pSG5.HAb TRAP220 (200 ng), pSG5.HAb CCAR1 (200 ng), or pSG5.HAb CCAR1
∆R (200 ng), as indicated. Transfected cells were grown in medium with or without 100 nM E2.
These results shown are representative of two independent experiments.

28

0
1
2
3
4
5
6
7
8
9
L u c ife ra s eA c tiv ity (1 0
3
RLU)
1 2 3 4 5 6 7 8 9 10 11
E2 - + + + + + + + + + +
GRIP1 + + + + + + + +
C1+p300 + + + + + + +
CoCoA + + + + + +
CCAR1 + + + + +
TRAP220 + + + + +

Figure 10. (continued)

(C) Six coactivators activity in ER mediated transcription. Transient transfection using
2ERE-TATA –LUC (200 ng), pHE0 (0.2 ng), pSG5.HA GRIP1 (200 ng), pSG5.HA CARM1
(200 ng), pCMV-p300 (200 ng), pSG5.HA CoCoA (200 ng), pSG5.HAb TRAP220 (200 ng),
and pSG5.HAb CCAR1 (200 ng) were performed as in Fig.5A. Luciferase activity results
shown are representative of two independent experiments.

29

0
10
20
30
40
50
60
70
80
90
10 0
1 2 3 4 5 6 7 8 9 1011 12 1314 15 1617 18 19
L u ciferase A ctivity(10
3
RLU)
DEX
-
+ + + + + + + + + + + + + + + + + +
TRAP220 + + + + + + + + +
FL.CCAR1 + + ++ ++
CCAR1 AD ∆ + + ++ ++
CCAR1 R ∆
+ + ++ ++ CCAR1 SAP ∆
+ + ++ ++

Figure 11. Cooperation of CCAR1 with TRAP220 in GR activity.

Transient transfection using MMTV-LUC (200 ng), hGR (2 ng), pSG5.HAbTRAP220
(400 ng), pSG5.HAb CCAR1 (+, 600 ng, ++, 900 ng) or its deletion mutants; CCAR1 ∆AD,
∆SAP and R (+, 600 ng, ++, 900 ng), Luciferase activity results shown are representative of
two independent experiments.

30
Discussion

The roles of three domains of CCAR1 in estrogen receptor mediated
transcription.

I identified the arginine rich (R) domain as an estrogen receptor interacting domain in
vitro (Fig.3C). We demonstrated that overexpression of CCAR1 ∆R failed to enhance ligand-
stimulated transcription activation by ER (Fig.3C). Also, the results showed that the R rich
region inhibited coactivator activity of CCAR1, when both of them were cotransfected into
cells (Fig.5B). Thus, the arginine rich region is important for ER stimulated transcription
activation. Also CCAR1 202-400, which includes R rich region, bound TRAP220 AB (amino
acid 1-670), which is sufficient for interaction both with the mediator complex and with NRs
(Malik et al., 2004).
In general, many coactivators bind NRs through LXXLL motifs in the coactivators (Lonard
and O’Malley, 2006). However, this R rich region doesn’t contain this LXXLL motif. Indeed, the
essential requirement of LXXLL motif for the interaction with NR LBD has been challenged by
the FXXLL motif of NSD-1 which mediates the interaction with NR LBD (Huang et al.,
1998).This R rich region also doesn’t have this FXXLL motifs. Therefore I need to further
narrow down the region to find out the minimum region, which should be different from well
known LXXLL motif.
In a previous study, the AD and SAP domains of CCAR1 were important for its
coactivator function with ER in transient transfection assays (JH Kim, Unpublished data)
(Fig.2). The transient transfection data also confirmed that AD and SAP domains of CCAR1
play important roles in ER stimulated transcription activation (Fig.6). However, I failed to
detect their interaction with ER in vitro and in vivo using coimmunoprecipitation assay. Thus,
SAP and AD domains might serve a different function, instead of binding of ER.

31
Knock out studies have defined a role for p160 coactivators in the hormone stimulated
ER dependent transcription (Shiau et al., 1998, Cavarretta et al., 2002). If wild type mouse
CCAR1 is not affected by siRNA for human CCAR1, overexpression of CCAR1 with siCCAR1
treated cells might rescue luciferase activity. Through RNA interference and rescue
experiment, we could investigate the physiological roles of the three domains of CCAR1 in
vivo. For the full recovery in the luciferase activity, siRNA resistant mutant of CCAR1 (CCAR1-
R) which included the three mismatches were made. The rescue assays will be performed
using this CCAR1-R.
CCAR1 was an interacting protein for CoCoA AD and cooperated with CoCoA (JH Kim,
Unpublished data). The results of luciferase assays showed that SAP and R domains of
CCAR1 were needed to fully cooperate with CoCoA, and the AD domain of CCAR1 was
essential for collaboration with CoCoA (Fig.7). In previous studies, CoCoA C-terminal AD is
essential for transcription activation and interacts with p300 (Kim et al., 2003, Kim et al.,
2006). For better determination of interaction between CoCoA and three domains of CCAR1,
the transient transfection assay using CoCoA AD remains to be tested.

Association of CCAR1 with TRAP220.

The mediator complex is important for transcription regulation by acting as a bridge
between DNA binding transcription factors and RNA polymerase II. TRAP220 acts as a direct
binding target for NRs (Vijayvargia et al., 2007). The results of GST pull down assay indicated
that CCAR1 202-400 region and CCAR1 720-900 region interact with TRAP220 in GST pull
down assay (Fig.4). Coexpression of CCAR1 with TRAP220 synergistically increased
licuferase activity in a dose dependent manner (Fig.9). In contrast, cotransfected CCAR1
deletion mutants with TRAP220 failed to enhance the luciferase activity (Fig.9). CCAR1 202-
400 is located between AD and R region, so this interaction between TRAP220 and CCAR1
202-400 seems to be sufficient to effect high level transcription. SAP domain doesn’t have
any interaction between TRAP220 and CCAR1 in vitro. Indeed, GST pull down assay detects

32
strong protein-protein interaction, but the interaction detected by coimmunoprecipitation is
relatively weak, so in the environment of cells, other factors may affect the interaction among
coactivators. Thus, fine domain mapping will help us to better understand the association
between three domains and TRAP220.
In GR mediated transcription activation, CCAR1 and TRAP220 enhanced luciferase
activity, and two domains, AD and R, are required to increase the GR activity and cooperate
with TRAP220 (Fig.11). This functional data is consistent with results of in vitro interaction
assays. Coactivator activity of CCAR1 without the interaction between TRAP220 and CCAR1
is almost abolished. Function of CCAR1 ∆SAP, which doesn’t contain the interaction domain,
isn’t affected by deletion of amino acid 651-655. Thus, this implies that SAP domain is not
essential for the interaction between CCAR1 and TRAP220 and coactivator function by GR.
Because all the three domains play important roles in cooperation between CCAR1 and
TRAP220 in ER dependent transcription, the three domains of CCAR1 act differently with
different nuclear receptors.

Role of CCAR1 in p160 coactivator complex.

The transient transfection assay showed that CCAR1 acted synergistically with GRIP1,
CARM1, p300, and CoCoA to increase the luciferase activity by ER (Fig.8). Also CCAR1,
GRIP1, CoCoA, and TRAP220 additionally increased the luciferase activity which is entirely
reliant to the presence of GRIP1 (Fig.10A). Interestingly, CCAR1 ∆R with other coactivators
further enhanced the reporter activity, compared with CCAR1 (Fig.10B). Thus, loss of this
domain of CCAR1 might have no significant effect on the transcriptional activity of the
complex. Because GRIP1 might bind ER and CCAR1 could interact with CoCoA in multiple
coactivator complexes, R rich region which interacts with ER is not necessary for stimulating
transcription in the multiple coactivators system, however, in single coactivator or two
coactivators condition, R rich region that bind ER LBD is important for the coactivator function
of CCAR1. Thus CCAR1 might work differently in different assay systems (Fig.12). This

33
deletion of R rich domain might stabilize the coactivator complex (Yang et al., 2006).
Alternative explanation for this difference might be because down stream molecule which acts
as a repression factor interacts with R rich region.
The luciferase assay demonstrated that TRAP220 and five other coactivators; GRIP1,
p300, CARM1, CoCoA, CCAR1 didn’t strongly enhance the ER dependent transcription
activity, compared with the five coactivators complex (Fig.10C). Therefore, this means that
CCAR1 cooperated with TRAP220, but this interaction is not enough to stimulate a synergistic
effect with four other coactivators. It seems that TRAP220 might act as a redundant factor in
this particular assay system. Thus, the mechanism of coactivator complex cooperation and
distinct contribution to the transcription activation process remains to be determined.

34

ERE
ER ER
CCAR1
CoCoA
TRAP220
Transcription activation
Single or two coactivator condition
Multiple coactivator condition
ERE
ER ER
GRIP1
AD2
p300
CARM1
AD1
CoCoA
AD3
CCAR1
Transcription activation
TRAP220
R
R
ERE
ER ER
CCAR1
CoCoA
TRAP220
Transcription activation
Single or two coactivator condition
Multiple coactivator condition
ERE
ER ER
GRIP1
AD2
p300
CARM1
AD1
CoCoA
AD3
CCAR1
Transcription activation
TRAP220
R
R

Figure 12. CCAR1 might work differently in different assay systems.

In single or two coactivators assay system, R region which interacts with estrogen
receptor is essential for the coactivator activity of CCAR1. In multiple coactivator complexes,
the interaction between CCAR1 and estrogen receptor via R rich region is not necessary for
the multiple coactivator complexes activation.

35
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Abstract (if available)

Abstract Nuclear receptors are ligand dependent transcription factors that activate target genes involved in diverse physiological processes such as metabolism, development, reproduction through the recruitment of multiple coactivators.

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Asset Metadata

Creator Mah, In Kyoung (author)

Core Title Roles of three domains of CCAR1 in transcription activation by nuclear receptors

School Keck School of Medicine

Degree Master of Science

Degree Program Biochemistry and Molecular Biology

Degree Conferral Date 2008-05

Publication Date 04/21/2008

Defense Date 03/26/2008

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

Tag coactivator,nuclear receptor,OAI-PMH Harvest

Language English

Advisor Stallcup, Michael R. (committee chair), Stellwagen, Robert H. (committee member), Tokes, Zoltan A. (committee member)

Creator Email imah@usc.edu

Permanent Link (DOI) https://doi.org/10.25549/usctheses-m1113

Unique identifier UC1221445

Identifier etd-Mah-20080421 (filename),usctheses-m40 (legacy collection record id),usctheses-c127-51656 (legacy record id),usctheses-m1113 (legacy record id)

Legacy Identifier etd-Mah-20080421.pdf

Dmrecord 51656

Document Type Thesis

Rights Mah, In Kyoung

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