5'TG3' Interacting Factor Interacts with Sin3A and Represses AR-Mediated Transcription
Manju Sharma and
Zijie Sun
Departments of Surgery and Genetics, Stanford University School of
Medicine, Stanford, California 94305
Address all correspondence and requests for reprints to: Zijie Sun, Ph.D., Departments of Surgery and Genetics, R135, Edwards Building, Stanford University School of Medicine, Stanford, California 94305-5328. E-mail: zsun{at}stanford.edu
 |
ABSTRACT
|
---|
Like other nuclear receptors, the AR exerts its transcriptional
function by binding to cis elements upstream of
promoters and interacting with other transcriptional factors
(e.g. activators, repressors, and modulators). Among
them, histone acetyltransferases (HATs) and histone deacetylases
(HDACs) play critical roles in altering the acetylation state of core
histones, thereby regulating nuclear hormone receptor-mediated
transcription. The nuclear receptor corepressor can repress the TR and
RAR in the absence of ligand through either a Sin3A-dependent or
-independent manner by recruiting HDACs. AR and some other steroid
hormone receptors cannot silence transcription through a similar
mechanism in that they are located in the cytoplasm as complexes with
heat-shock proteins before exposure to ligand. It has been shown that
AR can bind to p160/SRC, cAMP response element-binding protein-binding
protein (CBP)/P300 and other coactivators to increase the
AR-mediated transcription. However, the molecular mechanism for turning
AR from transcriptionally active into silent states is unknown. In this
study, we demonstrated that the transcription repressor, 5'TG3'
interacting factor (TGIF), selectively represses AR-mediated
transcription from several AR-responsive promoters. The repression
is mediated through binding of TGIF to the DNA binding domain of AR and
is trichostatin sensitive. We also identified a direct protein-protein
interaction between TGIF and a transcription corepressor, Sin3A, which
suggests a novel pathway for TGIF recruiting HDAC1 to the repression
complex. These results provide fresh insight into understanding the
mechanism for repressing AR-, and perhaps other steroid hormone
receptor-, mediated transcriptions.
 |
INTRODUCTION
|
---|
RECENT STUDIES HAVE demonstrated that the
acetylation state of core histones plays a critical role in regulating
eukaryotic gene transcription (1, 2). Histone
acetyltransferases (HATs) alter nucleosomal structure by acetylation of
histone amino-terminal tails, which increases accessibility of
promoters to the transcriptional machinery (3). The
recruitment of HAT activity by a sequence-specific DNA-binding protein
may be a general feature of transcriptional activation
(1). In contrast, histone deacetylases (HDACs) play the
opposite role in this process (2). HDACs facilitate
transcriptional repression on specific promoters by binding to
transcriptional repressors, leading to a more compact nucleosomal
structure that results in decreased transcription factor accessibility
to the promoter.
In recent years, numerous studies have shown that nuclear hormone
receptors mediate specific gene transcription by directly or indirectly
interacting with other transcriptional cofactors (4).
Several nuclear receptor coactivators possessing intrinsic HAT
activities have been identified in recent years, including cAMP
response element-binding protein-binding protein (CBP)/P300
(5), the p160/SRC family (6, 7), and
pCAF/GCN5 (8, 9). In the presence of ligand, these
coactivators bind to the nuclear receptors and acetylate histone in
chromatin to facilitate nuclear receptor-mediated, ligand-dependent
transcription. The TR, the RAR, and other nuclear hormone receptors can
function as potent transcription repressors in the absence of ligand
(4). Recent studies on the mechanisms led to the
identification of two related proteins, known as nuclear receptor
corepressor (NCoR) and silencing mediator for RAR and TR (SMRT)
(10, 11, 12). These two proteins mediated the repressive
effect of unliganded nuclear receptors through Sin3A-dependent or
-independent mechanisms to recruit HDACs in a multisubunit repressor
complex leading to a more compact, transcriptionally repressed
chromatin structure. Based on these observations, it has been suggested
that recruitment of corepressors to the nuclear receptors may be a
conversion point from transcription activation to repression.
AR belongs to the nuclear hormone receptor superfamily and plays a
critical role in promoting normal and tumoral cell growth (13, 14). However, an important feature of the AR and some other
steroid hormone receptors that distinguishes them from TR and RAR is
that they are compartmentalized to the cytoplasm as complexes with
heat-shock proteins (HSPs) before exposure to ligand (15, 16). Upon binding to ligand, AR dissociates from the HSPs and
translocates into the nucleus, where it facilitates androgen-regulated
transcription (17, 18). It has been shown that AR can bind
to p160/SRC, CBP/P300, and other coactivators to increase AR-mediated
transcription (19, 20). However, the molecular mechanism
for converting AR from transcriptionally active into silent states is
unknown. For the ER
, it has been suggested that the ubiquitin
proteasome pathway is involved in down-regulation by protein
degradation (21). However, the mechanism by which the
nucleosome is repacked into an inactive state, and whether HDACs are
involved in transcriptional repression of AR and other steroid hormone
receptors, is still unclear.
5'TG3' interacting factor (TGIF) is a homeodomain transcription
repressor that was previously shown to bind to the RXR response element
and inhibit RXR-mediated transcription by competitive DNA binding to an
overlapping site (22). Recently, it has been demonstrated
that TGIF interacts with Smad2, the mediator of TGF-ß signaling, and
represses TGF-ß-activated transcription (23). Repression
of Smad2-mediated transcription by TGIF is mediated through recruitment
of HDACs (23, 24). Identification of a protein-protein
interaction between TGIF and HDACs suggested a molecular mechanism by
which Smad2 switched transcriptional activation to repression by
recruiting TGIF to compete with p300/CBP binding.
In this study, we demonstrated that TGIF selectively represses
AR-mediated transcription on the androgen-induced promoters. The
repression is mediated through binding of TGIF to the DNA binding
domain (DBD) of AR and is trichostatin (TSA), a HDAC inhibitor,
sensitive. The results provide the first line of evidence showing that
AR-mediated transcription is regulated via the HDAC pathway. We also
identified a protein-protein interaction between TGIF and a
transcriptional corepressor, Sin3A. Interestingly, the region of TGIF
that interacts with Sin3A is also responsible for the binding to HDAC1
(24). Using several biochemical approaches, we further
demonstrated that Sin3A directly interacts with TGIF, indicating that
it may bridge both TGIF and HDAC1 in a protein complex. This study
suggests a novel mechanism for TGIF-mediated transcriptional
repression.
 |
RESULTS
|
---|
TGIF Represses Ligand-Dependent Activation of the AR
An earlier report showed that TGIF negatively regulated
RXR-mediated transcription (22). TGIF is expressed in a
number of human adult tissues including prostate and testis
(22). We hypothesized that TGIF might also function to
regulate other steroid hormone pathways, including androgen-dependent
transcription. Plasmids capable of expressing AR, TGIF, and a
luciferase reporter plasmid regulated by the androgen responsive
elements (AREs) in the mouse mammary tumor virus (MMTV) long terminal
repeat (MMTVpA3-Luc) vector were transfected into CV-1 cells. An
approximately 30-fold induction of AR-mediated transcriptional activity
was observed in the presence of 10 nM DHT (Fig. 1A
). Cotransfection of TGIF expression
construct showed a dosage-dependent repression of ligand-dependent AR
activation. In contrast, the positive control, the known AR-coactivator
ARA70 (25), brought about a doubling in DHT-dependent
transcription. Thus, this result suggests that TGIF repressed
transcription from an AR-dependent promoter.

View larger version (17K):
[in this window]
[in a new window]
|
Figure 1. TGIF Represses AR-Mediated Transactivation
A, CV1 cells were transiently transfected with 100 ng of pMMTV-Luc, 25
ng of pSV40-ß-gal, 10 ng of pSV-hAR, and, as indicated, different
amounts of pcDNA3-Flag-TGIF plasmids or 20 ng of pSG5-ARA70.
White bars represent the absence of DHT, and
black bars represent the addition of 10 nM
DHT. Relative luciferase activity is reported and represented as
mean ± SD (luciferase light units/ß-gal). B, The
transient transfections were repeated in PC3 cells with a luciferase
reporter driven by MMTV promoter or the 7-kb promoter fragment of human
PSA. One hundred nanograms of luciferase reporters, 25 ng of
pCMV-ß-gal, 10 ng of pCMV-hAR, and 10 ng of pcDNA3-Flag-TGIF plasmids
per well were used in the experiments. C, LNCaP cells were transfected
with 100 ng of pPSA-Luc, 25 ng of pCMV-ß-gal, and, where indicated,
10 or 20 ng of pcDNA3-Flag-TGIF constructs. No AR expression vector was
used in this experiment.
|
|
To explore the biological significance of the TGIF-mediated repression
further, we looked for a repression effect by TGIF in a prostate cancer
cell line, PC3. As shown in Fig. 1B
, TGIF reduced AR-mediated
transcription on an MMTV-luc reporter by approximately 50% at TGIF/AR
plasmid ratios of 1:1. The human prostate-specific antigen
(PSA) gene is an AR-regulated target gene that has
been widely used as a prostate-specific tumor marker (26, 27). To determine whether the repression of AR activity by TGIF
could be reproduced on a natural AR-dependent promoter, transient
transfections were repeated in PC3 cells with a luciferase reporter
gene driven by the 7-kb PSA promoter (28). TGIF plasmid
reduced AR-mediated transcription from the PSA promoter by 70% (Fig. 1B
).
We next tested repression of AR-dependent transcription by TGIF in a
physiologically relevant cellular context. Transient transfections were
repeated in LNCaP, a human prostate cancer cell line that
expresses endogenous AR protein. Repression of endogenous AR activity
by TGIF is measured with a cotransfected PSA promoter. As seen in Fig. 1C
, endogenous AR induces the PSA promoter approximately 25-fold in the
presence of DHT. Overexpression of TGIF showed a dosage-dependent
repression of AR activity. This result demonstrates that TGIF can
repress endogenous AR protein-mediated transcription from the PSA
promoter.
To ensure that the TGIF-mediated repression did not reflect toxic or
other nonspecific effects of the cotransfected plasmids, luciferase
expression in all experiments was normalized using ß-galactosidase
(ß-gal) production from a cotransfected plasmid. We also examined the
intracellular steady-state levels of AR protein in the above
transfectants and found them to be similar, indicating that the
TGIF-mediated repression was not due to reduced levels of expression of
AR (data not shown).
TGIF Selectively Represses AR-Mediated Transcription
TGIF is expressed in most human tissues, and it also
functions to repress transcription mediated by other activators such as
Smad2, a transducer of TGFß signals. To assess whether TGIF represses
other steroid hormone receptor activities, we tested GR and PRß along
with AR, using similar experimental conditions. The MMTV promoter
(MMTVpA3-Luc) containing the steroid hormone responsive elements for
the respective receptors (29, 30) were cotransfected with
AR, PRß, or GR expression vector into CV-1 cells. With the
appropriate ligand, the three receptors showed ligand-dependent
transcription from the MMTVpA3-Luc reporter vector (Fig. 2A
). As we observed previously, TGIF
showed a dosage-dependent repression of AR activity. However, TGIF only
slightly repressed GR-mediated transcription when 10 ng of the plasmid
were used, and has no repression of PRß activity (Fig. 2A
).
Based on these results, we conclude that TGIF mediated a specific
transcriptional repression on AR under identical experimental
conditions.

View larger version (25K):
[in this window]
[in a new window]
|
Figure 2. TGIF Specifically Represses AR-Mediated
Transcription
A, CV-1 cells were transiently transfected with 100 ng of pMMTV-Luc, 25
ng of pSV40-ß-gal, 10 ng of pSV-hAR, pSV-hGR, or pSV-hPRß, and as
indicated, 5 or 10 ng of pcDNA3-Flag-TGIF. The corresponding ligands
for each receptor were used in the experiments, including 10 nM DHT
(AR), 10 nM dexamethasone (GR), and 10 nM progesterone
(PR). White bars represent the absence of ligands,
whereas black bars represent the addition of ligands. B,
For each sample, 25 ng pSV-ß-gal, 100 ng luciferase reporter vectors
containing the different hormone response elements for each receptors
(pARE-luc, pVDRE-luc, pERE-luc, and pTRE-luc), and 10 ng of the
corresponding receptor expression constructs were transfected into CV-1
cells, with or without 10 ng of pcDNA3-Flag-TGIF. The specific ligands
to each receptor were added in the following concentrations: 10
nM DHT, 100 nM ß-E2 (ER), 10 nM
T3 (TR), and 10 nM 1 ,25-dihydroxyvitamin
D3 (VDR).
|
|
The specificity of TGIF repression was further investigated with other
nuclear receptors. As shown in Fig. 2B
, expression of AR produced about
a 10-fold induction on a luciferase reporter driven by duplicated AREs
in the presence of DHT. Induction was reduced by 70% by cotransfection
of TGIF, which is consistent with the results shown in Fig. 1
for both
the MMTV and PSA promoters. In contrast, there was no repression by
TGIF of ER-, TR-, and VDR-controlled promoters driven by the
corresponding responsive elements (Fig. 2B
). Taken together, our
results suggest that TGIF selectively represses AR-mediated
transcription.
Repression of AR Activity by TGIF Is Mediated Through HDAC
Pathways
Although an earlier study showed that TGIF represses RXR-mediated
transcription by competing the RXR binding to its target elements
(22), the recent studies have shown that transcriptional
repression by TGIF is mainly mediated through the HDAC pathway
(23, 24). In this regard, we tested whether repression on
AR is also through the HDAC pathway using a HDAC inhibitor, TSA
(31). In transfected CV1 cells with both AR and TGIF
expression plasmids, TGIF strongly repressed AR-mediated transcription
(Fig. 3A
). However, when the transfected
cells were treated with TSA at 5 or 10 nM, the repression
was partially or fully reversed, and high luciferase activity was
observed compared with untreated cells (Fig. 3A
).

View larger version (19K):
[in this window]
[in a new window]
|
Figure 3. TGIF-Mediated Repression on AR-Mediated
Transcription Is TSA Sensitive
A, Transient transfections were performed in CV-1 cells with 100 ng of
pMMTV-Luc, 25 ng of pSV40-ß-gal, 10 ng of pSV-hAR, and 10 ng of
pcDNA3-Flag-TGIF in the absence or presence of 5 or 10 nM
TSA. B, The experiments were repeated with 10 ng of pcDNA3-I Bß1
and other plasmids as described in A. After transfection, the cells
were treated with 10 nM of TSA.
|
|
To ensure that the effect of TSA is specific to TGIF rather than a
general effect on the basic transcription machinery, we repeated the
experiment with the pcDNA3-I
Bß1 expression plasmid as a control,
which was previously shown to repress transcription mediated by the AR
and other nuclear receptors (32). As shown in Fig. 3B
, cotransfection of AR and an equal amount of the TGIF or I
Bß1
expression construct driven by a cytomegalovirus (CMV) promoter
reduced the ligand-dependent AR transcription. Addition of 10
nM TSA in the transfected cells can reverse TGIF-mediated
repression, but showed no effect on the cells transfected with
pcDNA3-I
Bß1. These results clearly indicate that TGIF-mediated
repression is TSA sensitive and that the HDAC pathway is involved in
the regulation process.
TGIF Interacts with AR Protein
Our results indicate that repression of AR activity by TGIF is
mediated through the HDAC pathway. One possible mechanism for TGIF
repressing AR on multiple AR-dependent promoters would be by a physical
interaction with the AR. Therefore, a series of glutathione
S-transferase (GST)-AR fusion proteins containing
various functional domains were generated to assess possible
interactions with TGIF (Fig. 4A
). Binding
of [35S]methionine-labeled full-length TGIF
protein to GST-AR fusion proteins was analyzed by SDS-PAGE and detected
by autoradiography. As seen in Fig. 4B
, a specific retention of TGIF
protein was observed when AR-DBD was present, indicating that TGIF was
binding to a region of the AR in or near the DBD (Fig. 4B
). Additional
GST-AR fusion proteins were generated to determine whether the binding
site was in the DBD or in the hinge regions flanking the DBD. The
GST fusion proteins incorporating the amino-terminal
(AR-5'DBD) or carboxyl-terminal hinge regions
(AR-3'DBD) did not bind to TGIF (top panel, Fig. 4C
). In contrast, the construct containing precisely the DBD (AR-C'DBD)
showed a strong interaction with TGIF when similar amounts of the GST
fusion proteins were used in the experiments (bottom panel,
Fig. 4C
). These results indicate that a physical protein-protein
interaction occurs between AR and TGIF, and that the DBD of AR is
responsible for the binding.

View larger version (28K):
[in this window]
[in a new window]
|
Figure 4. TGIF Interacts with the DBD of AR in
Vitro
A, The different portions of AR were fused to GST in pGEX-2TK vector
and shown in the figure. B, In vitro-translated,
35S-labeled full-length TGIF was incubated with various
GST-AR fusion proteins on beads, the beads were washed three times, and
proteins were resolved by SDS-PAGE gel and visualized by
autoradiography. C, Additional GST-AR fusion proteins containing the
small fragments of the DBD were made, and the pull-down experiments
were repeated (top panel). Ten microliters of the GST-AR
DBD fusion proteins used in the above protein pull-down experiments
were resolved in SDS-PAGE and stained with Coomassie blue
(bottom panel).
|
|
TGIF Interacts with a Transcriptional Corepressor, Sin3A
A previous study showed that TGIF physically interacts with HDAC1
and negatively regulates Smad2 (23, 24). The class I HDAC
proteins, including HDAC13, can target the transcription repression
complexes through the transcriptional corepressor, Sin3A (2, 33). We therefore tested the possibility of Sin3A to interact
with TGIF. Using the N-terminal fragment of Sin3A between amino acids 1
and 700 as "bait" in a yeast two-hybrid screening, we isolated 43
putative binding cDNA clones from a myeloid cDNA library
(34). Seventeen of these clones perfectly matched the
sequence encoding TGIF (22). Retransformation with GAL4
DBD alone or the bait plasmid containing either the N- or C-terminal
fragment of Sin3A and the TGIF or pVP16 plasmid, respectively, were
carried out to confirm the interaction. Cotransformants containing both
the pGBT9-Sin3A-N and a plasmid in which the VP16 activation domain was
fused to TGIF showed a strong interaction as evidenced by high
expression of ß-gal reporter gene (Fig. 5B
). These results verify that Sin3A
specifically interacts with TGIF in yeast cells.

View larger version (19K):
[in this window]
[in a new window]
|
Figure 5. TGIF Interacts with Sin3A in Vitro
and in Vivo
A, Schematic representation of the full length of Sin3A protein.
Numbers correspond to amino acid residues. B, Yeast
strain PJ-69 4A was cotransformed with the plasmids containing GAL4-DBD
(pGBT9) and VP16-AD (pVP16). Yeast colonies from the SD-Leu-Trp
plates were cultured in SD-Leu-Trp medium. ß-Gal activities were
measured using the Galacto-light Plus kit (Tropix Inc., Bedford, MA)
and normalized by cell density (OD600). The data represent
the mean ± SD of three independent colonies. C,
Coimmunoprecipitation of TGIF and Sin3A proteins was performed in the
transfected CV-1 cells, and total cell lysates were analyzed by Western
blot to check the levels of expressed proteins (top and middle
panels). Equal amounts of cell lysates were immunoprecipitated
with normal mouse IgG or anti-Flag monoclonal antibody (F) at 4 C. The
immune complex bound to protein-A Sepharose beads was then resolved by
SDS-PAGE and analyzed by Western blot using anti-Sin3A polyclonal
antibody (bottom panel). D, In
vitro-translated Sin3A protein (35S-labeled) was
incubated at 4 C for 2 h with an equal amounts of various GST-TGIF
fusion proteins coupled to Sepharose beads (see Materials and
Methods). The beads were washed, and proteins were resolved on
10% SDS-PAGE gel and analyzed by autoradiography.
35S-labeled Sin3A is marked in the figure.
|
|
To determine whether the interaction between Sin3A and TGIF occurs
in vivo, we examined the protein-protein interaction by
coimmunoprecipitation analysis. A Flag-tagged TGIF expression plasmid
was transfected into CV1 cells. Both endogenous Sin3A and Flag-tagged
TGIF proteins were detected in the transfected cells (Fig. 5C
, top and middle panels). Whole-cell lysates were
immunoprecipitated with normal mouse IgG or an anti-Flag monoclonal
antibody. To increase the specificity of protein complexes precipitated
by the Flag antibody, immunoprecipitates were eluted with the Flag
peptide. Eluted protein complexes were then analyzed by Western blot
using a Sin3A N-terminal antibody. As shown in the bottom
panel of Fig. 5C
, endogenous Sin3A protein was detected in
anti-Flag immunoprecipitates from cells transfected with the Flag-TGIF
plasmid (lane 4), but not in untransfected cells (lane 2) or when
normal mouse IgG was used (lanes 1 and 3). These results demonstrate
that interaction between the full-length Sin3A and TGIF proteins occurs
in vivo.
To precisely map the interaction region of TGIF protein with Sin3A, GST
pull-down experiments were carried out with a series of GST-TGIF fusion
proteins (Fig. 5D
). [35S]Methionine-labeled
full-length Sin3A protein bound to GST-TGIF fusion proteins was
analyzed by SDS-PAGE and detected by autoradiography. As shown in Fig. 5D
, a strong retention of Sin3A protein was specifically observed for
the samples with GST-TGIF protein containing amino acids 108192.
These results are consistent with those of the yeast two-hybrid and
coimmunoprecipitation experiments and suggest that the region between
108 and 192 amino acid residues within TGIF is required for the
interaction with Sin3A.
TGIF Directly Interacts with Sin3A
Previous studies showed that Sin3A functions as a corepressor
through recruitment of HDAC factors to transcriptional complexes
(10, 11, 12). An interaction between TGIF and HDAC1 was
observed previously in coimmunprecipitation experiments using the
overexpressed proteins (24). In this study, we have
identified a protein-protein interaction between Sin3A and TGIF.
Importantly, the interacting region of TGIF for both Sin3A and HDAC1 is
mapped to amino acids 108192 (24). Therefore, it is
possible that the TGIF-HDAC1 interaction identified previously may be
mediated through Sin3A protein. To test this model, we first examined
the interaction between TGIF and HDAC1 or Sin3A using in
vitro protein pull-down experiments. The truncated GST-TGIF
proteins between amino acids 108 and 192 and the control constructs
were incubated with in vitro-translated HDAC1 or Sin3A
proteins. As observed previously, the GST-TGIF protein containing amino
acids 108192 showed a strong retention of in
vitro-translated Sin3A protein (right panel, Fig. 6A
). In contrast, in the identical
experiment setting, no binding was detected between the GST-TGIF
proteins and HDAC1 (left panel, Fig. 6A
). The results from
these experiments suggest that TGIF directly binds Sin3A but not
HDAC1.

View larger version (22K):
[in this window]
[in a new window]
|
Figure 6. TGIF-Recruiting HDAC1 Is Sin3A-Dependent
A, In vitro-translated, 35S-labeled
full-length Sin3A or HDAC1 were incubated with equal amounts of the GST
proteins on beads. The beads were washed three times, and proteins were
resolved by SDS-PAGE gel and visualized by autoradiography. B, Both
overexpressed Sin3A and Flag-tagged HDAC1 proteins were
coimmunoprecipitated with anti-Sin3A and -Flag antibodies and were then
analyzed by SDS-PAGE and transferred to two identical nitrocellulose
membranes. One membrane was analyzed by Western blotting for detection
of the protein expression (left panel). The other blot
was renatured and then hybridized with a 32P-labeled TGIF
protein fragment between amino acids 108192. C, CV-1 cells were
transiently cotransfected with Flag-tagged HDAC1 and T7-tagged TGIF.
Equal amounts of cell lysate were immunoprecipitated with normal mouse
IgG or anti-Flag monoclonal antibody at 4 C. The precipitated fractions
with either normal IgG or flag antibody (IP) were then resolved by
SDS-PAGE and analyzed by Western blot (WT) using anti-Flag antibody
(top panel), anti-Sin3A antibody (middle
panel), or anti-T7 antibody (bottom panel).
|
|
Although the results from the GST pull-down experiments suggest that
TGIF may directly interact with Sin3A, it still remains possible that
the interaction may be mediated indirectly by another protein in the
programmed cell lysates that were used to synthesize Sin3A and HDAC1
proteins. For this reason, we performed a Far Western blotting assay, a
potentially more specific method, to further confirm the direct
interaction that we observed above. Equal amounts of both full-length
Sin3A and HDAC1 proteins were precipitated with anti-Sin3A and -Flag
(for HDAC1) antibodies from the whole-cell lysates and were run on
SDS-PAGE and transferred to a nitrocellulose membrane (Fig. 6B
). Blots
were renatured for 48 h and probed with the
32P-labeled TGIF protein fragment (amino acids
108192). As shown in Fig. 6B
, a specific Sin3A band was labeled with
the TGIF probe but not HDAC1. These data confirmed the above-described
GST pull-down results and indicate that Sin3A protein directly
interacts with TGIF.
To further test our hypothesis that Sin3A bridges TGIF and HDAC1
proteins in the same protein complex, we cotransfected both Flag-tagged
HDAC1 and T7-tagged TGIF expression constructs into CV-1 cells. CV-1
cells were chosen because they contain detectable endogenous levels of
Sin3A protein. A Flag-tag antibody was used for coimmunoprecipitations,
and then the coimmunprecipitates with both normal IgG and Flag antibody
were analyzed by Western blots with Flag, T7, and Sin3A antibodies. As
shown in Fig. 6C
, both Sin3A and TGIF proteins were detected in the
complex with HDAC1. These results provide an additional line of
evidence showing that Sin3A mediates the interaction between HDAC1 and
TGIF proteins in the cells.
 |
DISCUSSION
|
---|
Transcriptional activation by AR and other nuclear hormone
receptors is a dynamic and complex process that requires recruitment of
other transcriptional cofactors to assemble a transactivation complex
on the target promoter. It is likely that the acetylation state of core
histones plays an important role in the regulation of transcription
(5, 35), and selective recruitment of histone acetylases
and HDACs can convert transcriptionally active states into silent
states. A number of HATs, including p300/CBP, PCAF, SRC-1, and
ACTR, which possess intrinsic HAT activity, have been identified
as transcriptional coactivators of nuclear hormone receptors.
Transcriptional activation by TR, RAR, and other nuclear hormone
receptors has been shown to be regulated by both HATs and HDACs
(7, 36). Unbound TR, RAR, and other nuclear hormone
receptors stay in a latent state in the nucleus by association with
NCoR and SMRT (10, 11, 12). After binding to ligand, the
receptors can be switched from repressed states into activated states
by disassociation of repressors and association with transcriptional
activators. Because most steroid hormone receptors are sequestered by
HSPs in the cytoplasm, the mechanism by which transcriptional
activation is switched to repression for AR and other steroid hormone
receptors may be different from the other nuclear hormone receptors.
Recently, it has been reported that the active transcriptional complex
formed by ER
and its coactivator is down-regulated through the
ubiquitin proteasome pathway (21). In addition, a recent
report has demonstrated that upon binding an antagonist, tamoxifen,
ER
recruits both NCoR and SMRT to form a transcriptional repressor
complex (37). Although both reports have shed light on the
negative regulation of ER-mediated transcription, it is unclear whether
AR and other steroid hormone receptors are also regulated in the same
manner. Moreover, there is still a gap in our knowledge regarding how
an open, active nucleosome with an active transcriptional complex can
turn into a compact, inactive form on AR- and other steroid hormone
receptor-regulated promoters. In this report, we have identified the
transcriptional repressor, TGIF, which functions as a bona
fide repressor to specifically repress AR-mediated transcription.
The repression by TGIF is mediated through a protein complex containing
TGIF, Sin3A, and HDAC1 proteins, which provides evidence for the first
time that the HDAC pathway is involved in AR-mediated
transcription.
TGIF is a homeobox protein that contains multiple transcriptional
repression domains (24). Although it was shown earlier
that TGIF binds to RXR response element and interferes with RXR
DNA
binding, recent evidence has suggested that TGIF repression is mediated
through the HDAC pathway (23). In our transient
transfection assays, we found that TGIF substantially represses
AR-mediated transcription on several androgen-induced promoters. A
protein-protein interaction between the DBD of AR and TGIF was
demonstrated. The repression by TGIF was further determined to be TSA
sensitive. In the process of probing the mechanism of TGIF-mediated
repression, using several in vivo and in vitro
approaches, we determined that TGIF physically interacts with a
corepressor, Sin3A. Previous studies have shown that Sin3A is a
component of transcription repression complexes and functions as a
mediator between sequence-specific DNA binding repressors and HDAC
proteins. Our results suggest a potential role for Sin3A in the
TGIF-mediated repression.
Using a series of deletion mutants of both TGIF and Sin3A, we mapped
the interaction regions of the two proteins. The interaction region of
Sin3A with TGIF was mapped to the N-terminal region (Fig. 5A
), which
contains the paired amphipathic
-helix 1, 2, and 3 domains.
This region was also previously shown to interact with the
transcriptional repressors MAD and NCoR/SMRT (38, 39, 40),
suggesting that a common mechanism may apply to TGIF and these
transcriptional repressors. The binding region of TGIF with Sin3A was
mapped to the middle portion of TGIF (amino acids 108192), which is
the exact same region that interacts with HDAC1 (24).
Interestingly, in our GST pull-down experiments, we only detected the
interaction between TGIF and Sin3A, but not HDAC1, under identical
experimental conditions. One possibility is that the interaction
between TGIF and HDAC1 is not direct and is possibly mediated through
Sin3A. Using far Western blot, we confirmed that a direct interaction
between TGIF and Sin3A occurs. Taken together, these results suggest
that Sin3A is necessary for TGIF to recruit HDAC1 for the repression of
AR-mediated transcription.
More than seven HDAC proteins have been identified. Based on their
sequences, they can be divided into two classes: class I includes
HDAC13, whereas class II includes HDAC46 (36, 41, 42).
Several reports have shown that Sin3A appears to play a pivotal role in
targeting the class I HDACs to transcriptional repressor complexes
(10, 36). However, HDAC4, HDAC5, and an as yet unnamed new
member of the class II HDACs are capable of interacting directly with
the corepressors NCoR and SMRT to target various transcriptional
complexes in a Sin3A-independent manner (43). Our finding
that Sin3A allows HDAC1 to complex with TGIF is consistent with the
previous observation and suggests that TGIF may function on AR in a
Sin3A-dependent manner.
TGIF has been reported previously to be a corepressor of Smad2 protein.
In this report, we show that TGIF interacts with the AR and represses
AR-mediated transcription. However, we found that TGIF probably does
not act broadly as a nuclear hormone corepressor, because it repressed
AR-mediated transcription but not the activities of other nuclear
hormone receptors, including PRß, ER
, TR, and VDR. In the context
of the MMTV promoter, we showed that TGIF slightly inhibits GR
activity. Because the DBD of AR, which is composed of two zinc fingers,
was mapped as the interacting region with TGIF, it is likely that the
selective repression by TGIF on AR and GR may be due to the close
sequence similarities in the DBD regions of these proteins.
In this report, we demonstrated that TGIF interacts with the DBD of AR,
facilitating binding of Sin3A and HDAC1 to form a repression complex
resulting in deacetylation of the core histones. Although it is unclear
whether other cellular factors are involved in this process, it is
likely that TGIF can trigger this regulation. Repression of AR-mediated
transcription by TGIF suggests a potentially significant mechanism to
control androgen-induced cell growth, which may play a critical role in
the growth and development of normal prostate tissue, and in the
development and progression of prostate cancer.
 |
MATERIALS AND METHODS
|
---|
Cell Cultures and Transient Transfections
A monkey kidney cell line, CV-1 (44), and a human
prostate cell line, PC-3 (45), were maintained in RPMI
1640 medium supplemented with 5% FCS (HyClone Laboratories, Inc., Logan, UT). The AR-positive human
prostate cancer cell line, LNCaP (46), was cultured in T
medium (Life Technologies, Inc., Gaithersburg, MD) with
5% FCS.
Transient transfections for CV-1 were carried out by using a
LipofectAMINE transfection kit (Life Technologies, Inc.)
as described previously (47). About 200 ng of total
plasmid DNA per well was used in the transfection. The total amount of
DNA per dish was kept constant by adding pBluescript plasmid
(Stratagene, La Jolla, CA). Approximately 16 h after
transfection, the cells were washed and fed medium containing 5%
charcoal-stripped (steroid hormone-free) FCS (HyClone Laboratories, Inc.) in the presence or absence of steroid
hormones. LNCaP and PC3 cells were transfected with the LipofectAMINE
2000 reagent. Because we have observed that simian virus 40
(SV40) promoter is transcriptionally inactive in these prostate
cancer cell lines, the expression vectors driven by a CMV promoter were
used in the experiments. Luciferase activity was measured in relative
light units as previously described (48, 49). The relative
light units from individual transfections were normalized by
measurement of ß-gal activity expressed from a cotransfected plasmid
in the same samples. Individual transfection experiments were done in
triplicate and the results are reported as mean luciferase/ß-gal
(±SD) from representative experiments.
Plasmid Construction
The human TGIF cDNA was a kind gift of Dr. Roger Clerc
(Roche, Basel, Switzerland). GST-TGIF constructs
containing different portions of TGIF and the full-length TGIF
expression vector were generated by PCR with specific primers and
subcloned into the pGEX (Amersham Pharmacia Biotech,
Piscataway, NJ) and pcDNA3-Flag vectors, respectively. For yeast
two-hybrid screening, N-terminal (amino acids 1700) and C-terminal
segments (amino acids 7001,219) of human Sin3A were generated by PCR
and fused to the GAL4 DBD in the pGBT9 vector (CLONTECH Laboratories, Inc., Palo Alto, CA). Human HDAC1 expression
vector (pBJ5.1-HDAC1) with a carboxyl-terminal Flag epitope tag was
kindly provided by Dr. Edward Seto (University of South Florida, Tampa,
FL).
The AR expression vector, pSV-hAR, was provided by Dr. Albert Brinkmann
(Erasmus University, Rotterdam, The Netherlands). pMMTV-luc was
provided by Dr. Richard Pestell (Albert Einstein College of Medicine,
New York, NY). The pPSA-luc reporter plasmid was obtained from Dr.
Belldegrun (UCLA, Los Angeles, CA) (28).
pSV-ß-gal, an SV40 driven ß-gal reporter plasmid (Promega Corp., Madison, WI) was used in CV-1 cells as an internal
control. The pSG5-ARA70 plasmid, containing the full-length ARA70 cDNA,
and the reporter plasmid pARE-luc were the kind gifts of Dr. Chawnshang
Chang (University of Rochester, Rochester, NY)
(25). pCMV-VDR, pSV-hGR, and pVDRE-luc were provided by
Dr. David Feldman (Stanford University, Stanford, CA). A human ER
expression construct (pcDNA3-ER) and a luciferase reporter plasmid with
three estrogen responsive elements were kindly provided by Dr. Myles
Brown (Dana-Farber Cancer Institute, Boston, MA). A human TRß
expression vector and a luciferase reporter controlled by two
testosterone responsive elements were kindly provided by Dr. Anthony
Hollenberg (Beth Israel Deaconess Medical Center, Boston, MA). The
human PRß expression vector, pSV-hPRß, was a gift of Dr. Nancy L.
Weigel (Baylor College of Medicine, Houston, TX). The human I
Bß1
expression vector, pcDNA3-I
Bß1, was kindly provided by Dr. David
Moore (Baylor College of Medicine, Houston, TX).
Yeast Two-Hybrid Screen
The interaction between Sin3A and TGIF was examined in
a yeast two-hybrid assay. pGBT9-Sin3A-N (amino acids 1700),
pGBT9-Sin3A-C (amino acids 700-1219), and a myeloid cDNA library
(34) were used in the experiments with a modified yeast
strain, PJ69-4A (50). The experimental conditions for
yeast two-hybrid screenings were previously described
(49). Transformants were selected on Sabouraud dextrose
medium lacking adenine, leucine, and tryptophan. Specific interactions
were measured by the production of adenine and ß-gal. A liquid
ß-gal assay was used to quantify the interaction.
GST Pull-Down Assay
Expression and purification of GST fusion proteins were
performed as described previously (51). The full-length
human TGIF and Sin3A proteins were translated and labeled in
vitro using the TNT-coupled reticulocyte
lysate system (Promega Corp.). Equal amounts of GST-AR or
-TGIF fusion proteins coupled to glutathione Sepharose beads were
incubated with radiolabeled TGIF or Sin3A proteins at 4 C for 2 h.
The buffer used for protein binding is a modified NETN buffer (0.2%
Nonidet P-40 (NP-40), 1 mM EDTA, 20
mM Tris-Cl (pH 8.0), 100 mM
NaCl, 5% glycerol, 4 mM
MgCl2, 0.5 mM
dithiothreitol (DTT), 1 mM phenylmethylsulfonyl
fluoride, 5 µg/ml leupeptin, and 5 µg/ml aprotinin). Beads
were carefully washed four times in NETN buffer and then analyzed by
SDS-PAGE followed by autoradiography.
Immunoprecipitation and Western Blotting
The human Sin3A expression vector, pIRES1HisSin3A
(33), alone or with a Flag-tagged pcDNA3-TGIF expression
plasmid, were transfected into CV-1 cells. After incubation for 24
h, cells were harvested into a buffer containing 20 mM
HEPES (pH 8.0), 0.5% NP-40, 100 mM NaCl, 1 mM
EDTA, 5 mM MgCl2, 50 mM
NaF, 0.3 mM sodium vanadate, 1 mM DTT, 1
mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, and
5% glycerol). Whole-cell lysates were incubated with mouse normal IgG
or Flag monoclonal antibody (Sigma, St Louis, MO) at 4 C
for 2 h. Pre-equilibrated protein A-Sepharose beads were then
added and, after 1 h of incubation, collected by centrifugation
and gently washed three times with the same buffer as described above.
Specific protein complexes were eluted with 100 ng/ml of Flag peptide
in a buffer containing 10 mM HEPES (pH 8.0), 100
mM NaCl, 1 mM EDTA, and 0.1% NP-40. The eluted
samples were boiled in SDS sample buffer and resolved on a 10%
SDS-PAGE. The proteins were transferred onto a nitrocellulose membrane
and probed with a 1:500 dilution of a polyclonal antibody against the N
terminus of mSin3A (Santa Cruz Biotechnology, Santa Cruz,
CA; catalog no. sc-994). Proteins were detected using an ECL kit
(Amersham Pharmacia Biotech, Arlington Heights,
IL).
Far Western Blot Analyses
The overexpressed Sin3A and Flag-tagged HDAC1 proteins were
precipitated with specific antibodies. The precipitated Sin3A and
HDAC1-F were then resolved on an 8% SDS-PAGE and transferred onto
nitrocellulose membranes. One set of membranes was analyzed by Western
blotting with Sin3A or Flag antibodies. The other was first denatured
in 6 M guanidine hydrochloride buffer [50 mM
HEPES (pH7.5), 50 mM NaCl, 0.1 mM EDTA, 10
mM MgCl2, 1 mM DTT, 0.1
mM ZnSO4, and 10% glycerol). The
membrane was renatured by removing the guanidine hydrochloride
gradually (from 6 M to 0.5 mM) in changing the
buffer every 23 h and was then blocked in renaturation buffer with
5% fat-free milk overnight. The blocked membrane was then incubated
with
-P32-labeled TGIF (amino acids 108192) for
1014 h in the renaturation buffer with 0.25% fat-free milk. The
membrane was then washed in the same buffer three times at room
temperature. Each wash was for 30 min. The membrane was then air-dried
and exposed to x-ray film using intensifying screens.
 |
ACKNOWLEDGMENTS
|
---|
We are especially grateful for the various reagents received
from Drs. David Feldman, Nancy Weigel, Albert Brinkmann, Richard
Pestell, Myles Brown, Chawnshang Chang, Anthony Hollenberg, David
Moore, and Roger Clerc. We thank Drs. Fajun Yang and Olga Petrauskene
for invaluable technical assistance and useful discussions and Mr.
Homer Abaya for administrative assistance and help in preparing
this manuscript.
 |
FOOTNOTES
|
---|
This work was supported by NIH Grant CA-70297 (to Z.S.) and American
Cancer Society Grant RPG98213 (to Z.S.).
Abbreviations: ARE, Androgen responsive element; CBP, cAMP
response element-binding protein-binding protein; CMV, cytomegalovirus;
CREB, cAMP response element-binding protein; DBD, DNA binding domain;
DTT, dithiothreitol; ß-gal, ß-galactosidase; GST,
glutathione-S-transferase; HAT, histone
acetyltransferase; HDAC, histone deacetylase; HSP, heat-shock protein;
MMTV, mouse mammary tumor virus; NCoR, nuclear receptor corepressor;
NP-40, Nonidet P-40; PSA, prostate-specific antigen; SMRT, silencing
mediator for RAR and TR; SRC, steroid receptor coactivator; SV40,
simian virus 40; TGIF, 5'TG3' interacting factor; TSA, trichostatin.
Received for publication May 14, 2001.
Accepted for publication August 3, 2001.
 |
REFERENCES
|
---|
-
Kouzarides T 1999 Histone acetylases and deacetylases in
cell proliferation. Curr Opin Genet Dev 9:4048[CrossRef][Medline]
-
Grunstein M 1997 Histone acetylation in chromatin structure
and transcription. Nature 389:349352[CrossRef][Medline]
-
Struhl K 1998 Histone acetylation and transcriptional
regulatory mechanisms. Genes Dev 12:599606[Free Full Text]
-
Xu L, Glass CK, Rosenfeld MG 1999 Coactivator and corepressor
complexes in nuclear receptor function. Curr Opin Genet Dev 9:140147[CrossRef][Medline]
-
Ogryzko VV, Schiltz RL, Russanova V, Howard BH, Nakatani Y 1996 The transcriptional coactivators p300 and CBP are histone
acetyltransferases. Cell 87:953959[Medline]
-
Hong H, Kohli K, Trivedi A, Johnson DL, Stallcup MR 1996 GRIP1, a novel mouse protein that serves as a transcriptional
coactivator in yeast for the hormone binding domains of steroid
receptors. Proc Natl Acad Sci USA 93:49484952[Abstract/Free Full Text]
-
Onate SA, Tsai SY, Tsai MJ, OMalley BW 1995 Sequence and
characterization of a coactivator for the steroid hormone receptor
superfamily. Science 270:13541357[Abstract]
-
Korzus E, Torchia J, Rose DW, Xu L, Kurokawa R, McInerney EM,
Mullen TM, Glass CK, Rosenfeld MG 1998 Transcription factor-specific
requirements for coactivators and their acetyltransferase functions.
Science 279:703707[Abstract/Free Full Text]
-
Blanco JC, Minucci S, Lu J, Yang XJ, Walker KK, Chen H, Evans
RM, Nakatani Y, Ozato K 1998 The histone acetylase PCAF is a nuclear
receptor coactivator. Genes Dev 12:16381651[Abstract/Free Full Text]
-
Chen JD and Evans RM 1995 A transcriptional co-repressor
that interacts with nuclear hormone receptors. Nature 377:454457[CrossRef][Medline]
-
Kurokawa R, Soderstrom M, Horlein A, Halachmi S, Brown M,
Rosenfeld MG, Glass CK 1995 Polarity-specific activities of
retinoic acid receptors determined by a co-repressor. Nature 377:451454[CrossRef][Medline]
-
Horlein AJ, Naar AM, Heinzel T, Torchia J, Gloss B, Kurokawa
R, Ryan A, Kamei Y, Soderstrom M, Glass CK, Rosenfeld MG 1995 Ligand-independent repression by the thyroid hormone receptor mediated
by a nuclear receptor co-repressor. Nature 377:397404[CrossRef][Medline]
-
Chang CS, Kokontis J, Liao ST 1988 Molecular cloning of human
and rat complementary DNA encoding androgen receptors. Science 240:324326[Medline]
-
Wilson EM, Simental JA, French FS, Sar M 1991 Molecular
analysis of the androgen receptor. Ann NY Acad Sci 637:5663[Medline]
-
Sanchez ER, Faber LE, Henzel WJ, Pratt WB 1990 The
5659-kilodalton protein identified in untransformed steroid receptor
complexes is a unique protein that exists in cytosol in a complex with
both the 70- and 90-kilodalton heat shock proteins. Biochemistry 29:51455152[Medline]
-
Sullivan WP, Vroman BT, Bauer VJ 1992 Isolation of steroid
receptor binding protein from chicken oviduct and production of
monoclonal antibodies. J Steroid Biochem Mol Biol 43:3741[CrossRef][Medline]
-
Jenster G 1999 The role of the androgen receptor in the
development and progression of prostate cancer. Semin Oncol 26:407421[Medline]
-
Jenster G, van der Korput HA, van Vroonhoven C, van der Kwast
TH, Trapman J, Brinkmann AO 1991 Domains of the human androgen receptor
involved in steroid binding, transcriptional activation, and
subcellular localization. Mol Endocrinol 5:13961404[Abstract]
-
Fu M, Wang C, Reutens AT, Wang J, Angeletti RH, Siconolfi-Baez
L, Ogryzko V, Avantaggiati ML, Pestell RG 2000 p300 and
p300/cAMP-response element-binding protein-associated factor acetylate
the androgen receptor at sites governing hormone-dependent
transactivation. J Biol Chem 275:2085320860[Abstract/Free Full Text]
-
Ikonen T, Palvimo JJ, Janne OA 1997 Interaction between the
amino- and carboxyl-terminal regions of the rat androgen receptor
modulates transcriptional activity and is influenced by nuclear
receptor coactivators. J Biol Chem 272:2982129828[Abstract/Free Full Text]
-
Lonard DM, Nawaz Z, Smith CL, OMalley BW 2000 The 26S
proteasome is required for estrogen receptor-
and coactivator
turnover and for efficient estrogen receptor-
transactivation. Mol
Cell 5:939948[Medline]
-
Bertolino E, Reimund B, Wildt-Perinic D, Clerc RG 1995 A novel
homeobox protein which recognizes a TGT core and functionally
interferes with a retinoid-responsive motif. J Biol Chem 270:3117831188[Abstract/Free Full Text]
-
Wotton D, Lo RS, Lee S, Massague J 1999 A Smad transcriptional
corepressor. Cell 97:2939[Medline]
-
Wotton D, Lo RS, Swaby LA, Massague J 1999 Multiple modes of
repression by the Smad transcriptional corepressor TGIF. J Biol
Chem 274:3710537110[Abstract/Free Full Text]
-
Yeh S, Chang C 1996 Cloning and characterization of a specific
coactivator, ARA70, for the androgen receptor in
human prostate cells. Proc Natl Acad Sci USA 93:55175521[Abstract/Free Full Text]
-
Dube JY, Chapdelaine P, Guerin S, Leclerc S, Rennie PS,
Matusik RJ, Tremblay RR 1995 Search for androgen response elements in
the proximal promoter of the canine prostate arginine esterase gene. J
Androl 16:304311[Abstract/Free Full Text]
-
Cleutjens KB, van Eekelen CC, van der Korput HA, Brinkman AO,
Trapman J 1996 Two androgen response regions cooperate in steroid
hormone regulated activity of the prostate-specfic antigen promoter.
J Biol Chem 271:63796388[Abstract/Free Full Text]
-
Pang S, Dannull J, Kaboo R, Xie Y, Tso CL, Michel K, deKernion
JB, Belldegrun AS 1997 Identification of a positive regulatory element
responsible for tissue-specific expression of prostate-specific
antigen. Cancer Res 57:495499[Abstract]
-
Hoeck W, Hofer P, Groner B 1992 Overexpression of the
glucocorticoid receptor represses transcription from hormone responsive
and non-responsive promoters. J Steroid Biochem Mol Biol 41:283289[CrossRef][Medline]
-
Mink S, Ponta H, Cato AC 1990 The long terminal repeat region
of the mouse mammary tumour virus contains multiple regulatory
elements. Nucleic Acids Res 18:20172024[Abstract]
-
Taunton J, Hassig CA, Schreiber SL 1996 A mammalian histone
deacetylase related to the yeast transcriptional regulator Rpd3p.
Science 272:408411[Abstract]
-
Na SY, Choi HS, Kim JW, Na DS, Lee JW 1998 Bcl3, an I
B
protein, as a novel transcription coactivator of the retinoid X
receptor. J Biol Chem 273:3093330938[Abstract/Free Full Text]
-
Hassig CA, Fleischer TC, Billin AN, Schreiber SL, Ayer DE 1997 Histone deacetylase activity is required for full transcriptional
repression by mSin3A. Cell 89:341347[Medline]
-
Lioubin MN, Algate PA, Tsai S, Carlberg K, Aebersold A,
Rohrschneider LR 1996 p150Ship, a signal transduction molecule with
inositol polyphosphate-5-phosphatase activity. Genes Dev 10:10841095[Abstract]
-
Yang XJ, Ogryzko VV, Nishikawa J, Howard BH, Nakatani Y 1996 A
p300/CBP-associated factor that competes with the adenoviral
oncoprotein E1A. Nature 382:319324[CrossRef][Medline]
-
Nagy L, Kao HY, Chakravarti D, Lin RJ, Hassig CA, Ayer DE,
Schreiber SL, Evans RM 1997 Nuclear receptor repression mediated by
a complex containing SMRT, mSin3A, and histone deacetylase. Cell 89:373380[Medline]
-
Shang Y, Hu X, DiRenzo J, Lazar MA, Brown M 2001 Cofactor
dynamics and sufficiency in estrogen receptor-regulated transcription.
Cell 103:843852[CrossRef]
-
Zhang X, Jeyakumar M, Petukhov S, Bagchi MK 1998 A nuclear
receptor corepressor modulates transcriptional activity of
antagonist-occupied steroid hormone receptor. Mol Endocrinol 12:513524[Abstract/Free Full Text]
-
Ayer DE, Laherty CD, Lawrence QA, Armstrong AP, Eisenman RN 1996 Mad proteins contain a dominant transcription repression domain.
Mol Cell Biol 16:57725781[Abstract]
-
Hurlin PJ, Foley KP, Ayer DE, Eisenman RN, Hanahan D, Arbeit
JM 1995 Regulation of Myc and Mad during epidermal differentiation and
HPV-associated tumorigenesis. Oncogene 11:24872501[Medline]
-
Hassig CA, Tong JK, Fleischer TC, Owa T, Grable PG, Ayer DE,
Schreiber SL 1998 A role for histone deacetylase activity in
HDAC1-mediated transcriptional repression. Proc Natl Acad Sci USA 95:35193524[Abstract/Free Full Text]
-
Grozinger CM, Hassig CA, Schreiber SL 1999 Three proteins
define a class of human histone deacetylases related to yeast Hda1p.
Proc Natl Acad Sci USA 96:48684873[Abstract/Free Full Text]
-
Pazin MJ Kadonaga JT 1997 Whats up and down with
histone deacetylation and transcription? Cell 89:325328[Medline]
-
Rovera G, Mehta S, Maul G 1974 Ghost monolayers in the study
of the modulation of transcription in cultures of CV1 fibroblasts. Exp
Cell Res 89:295305[Medline]
-
Isaacs JT and Kyprianou N 1987 Development of
androgen-independent tumor cells and their implication for the
treatment of prostatic cancer. Urol Res 15:133138[Medline]
-
Horoszewicz JS, Leong SS, Kawinski E, Karr JP, Rosenthal H,
Chu TM, Mirand EA, Murphy GP 1983 LNCaP model of human prostatic
carcinoma. Cancer Res 43:18091818[Abstract]
-
Yang F, Li X, Sharma M, Zarnegar M, Lim B, Sun Z 2001 Androgen
receptor specifically interacts with a novel p21-activated kinase,
PAK6. J Biol Chem 276:1534515353[Abstract/Free Full Text]
-
Sun Z, Pan J, Hope WX, Cohen SN, Balk SP 1999 Tumor
susceptibility gene 101 protein represses androgen receptor
transactivation and interacts with p300. Cancer 86:689696[CrossRef][Medline]
-
Sharma M, Zarnegar M, Li X, Lim B, Sun Z 2000 Androgen
receptor interacts with a novel MYST protein, HBO1. J Biol Chem 275:3520035208[Abstract/Free Full Text]
-
James P, Halladay J, Craig EA 1996 Genomic libraries and a
host strain designed for highly efficient two-hybrid selection in
yeast. Genetics 144:14251436[Abstract/Free Full Text]
-
Sun Z, Pan J, Balk SP 1997 Androgen receptor-associated
protein complex binds upstream of the androgen-responsive elements in
the promoters of human prostate-specific antigen and kallikrein 2 gene.
Nucleic Acids Res 25:33183325[Abstract/Free Full Text]