From the Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts 01605
Received for publication, June 26, 2002, and in revised form, November 4, 2002
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ABSTRACT |
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Androgen receptor (AR) is a hormone-regulated
transcription factor that mediates a wide array of biological processes
including sexual differentiation, spermatogenesis, and prostate cancer
progression. The transcriptional activity of AR and other
members of the nuclear receptor superfamily are modulated by
coregulatory proteins. In this study, we have investigated the
regulation of AR transcriptional activity by the silencing mediator for
retinoid and thyroid hormone receptors (SMRT). We found that AR
possesses an intrinsic transcriptional repression activity, and AR
interacts directly with SMRT. One interacting surface on AR is mapped
to the ligand-binding domain, and the presence of a DNA binding/hinge
region enhances this interaction. The binding surface on SMRT is mapped
to the C-terminal ID2 region, and mutation in the ID2 corepressor motif
inhibits the interaction. Overexpression of SMRT inhibits
dihydrotestosterone-dependent transactivation by AR and
further suppresses the antiandrogen flutamide-mediated inhibition of AR
activity. We provide evidence to suggest that the mechanisms of
SMRT-mediated inhibition of AR activity involves inhibition of AR N/C
interaction and competition with the p160 coactivator. Our data
establish a significant role of SMRT in modulating AR
transcriptional activity.
Androgen receptor (AR)1
is a member of the nuclear receptor (NR) superfamily that mediates the
biological effects of the male sex hormone androgens in a wide range of
physiological processes, including sexual differentiation and
maturation, spermatogenesis, and gonadotropin regulation (1). AR is
also involved in the development and progression of prostate cancer,
which is one of the most frequently diagnosed cancers in males. Indeed,
somatic mutations of the AR gene have been found in prostate
tumors, which may contribute to androgen-independent growth of the
cancer cell (2). Treatment with antiandrogens can partially or
completely arrest prostate cancer cell proliferation. Similar to other
steroid receptors the unliganded AR is held in an inactive conformation by association with heat shock proteins in the cytoplasm. The heat
shock protein complex assists in maintaining an AR conformation that is
active to ligand binding. Upon ligand binding, the heat shock protein
complex dissociates from the receptor (3), allowing AR to homodimerize
and translocate to the nucleus, where it recognizes and binds specific
promoter elements to activate gene expression (4).
Like other members of the NR superfamily, AR contains an N-terminal
AF-1 transactivation domain (or A/B domain), a centrally located
DNA-binding domain (DBD or C domain), a hinge region (or D domain), and
a ligand-binding domain (LBD or E domain). The E domain is also
involved in receptor dimerization and contains ligand-dependent AF-2 function. The A/B domain is the most
variable among NRs, and in the case of AR, it contains two separate
transactivation domains that are required for full ligand-inducible
transcription activity (5, 6). The A/B region also contains a
polyglutamine tract whose expansion may influence AR transcription
activity and cause infertility and Kennedy's disease (7, 8). In
addition, mutations in the AF-1 function have also been identified in
patients suffering from androgen insensitivity or oligospermia (9-11), indicating the importance of this region in AR function. Interestingly, the A/B domain has been also shown to interact directly with the E
domain in a ligand-dependent manner (12, 13). This process is known as N/C interaction, and it is required for full transcription potential of AR. In addition to ligand binding, transcriptional coactivators such as the CREB-binding protein (CBP), the p160 coactivators (14-17), and the proto-oncogene c-Jun (18) have been
reported to enhance AR activity by promoting N/C interaction. Two
LXXLL-related sequences in the A/B domain have been found responsible for mediating the N/C interaction (19). In addition to
binding ligand and mediating N/C interaction, the E domain also
mediates AR homodimerization and interactions with heat shock proteins
(20, 21) and transcriptional coregulatory proteins (4). X-ray
crystallographic studies indicate that the AR LBD adopts a similar
structural fold as other NRs (22-24), suggesting that the regulatory
mechanisms for AR activity may be conserved among NRs.
In the past few years, it has become clear that the transcriptional
activity of AR, as well as other members of the NR superfamily, are
modulated by cellular coregulatory proteins known as coactivators and
corepressors (1, 4, 25-27). The silencing mediator for retinoid and
thyroid hormone receptors (SMRT) and the related nuclear receptor
corepressor (N-CoR) were originally isolated as RAR- and TR-interacting
proteins that form complexes with the unliganded receptors to
facilitate transcriptional repression of gene expression (28, 29). SMRT
and N-CoR were later found to interact with antagonist-bound
progesterone receptor (PR) and estrogen receptor (ER) to repress
transcription (30-32), and they also serve as corepressors for several
additional members of the NR superfamily, including RevErb (33),
chicken ovalbumin upstream promoter (COUP)-transcription factor I (34),
peroxisome proliferator-activated receptor (PPAR) Despite the established function of SMRT and N-CoR in regulating
transcriptional repression activity of several NRs, the role SMRT and
N-CoR in regulating AR transcriptional activity is less clear.
Recently, it has been shown that AR can interact with N-CoR in HEK293
cells in the presence of DHT (40). Interestingly, this interaction is
enhanced substantially by an acetylation site mutation at the hinge/D
region of AR that affected transactivation function of AR without
affecting its transrepression activity (40). In this study, we
demonstrate that the N-CoR-related corepressor SMRT interacts directly
with both the unliganded and liganded AR in vivo and
in vitro. We demonstrate that this interaction is mediated
through the C-terminal NR-interaction domain (ID2) of SMRT. The minimal
interacting surface on AR is mapped to the LBD/E domain, while the
presence of DBD/hinge region of AR enhances this interaction
substantially. We show that SMRT inhibits ligand-dependent transcriptional activation by AR and further suppresses
antiandrogen-mediated inhibition of AR activity. Finally, we provide
evidence to suggest that the mechanisms of SMRT-mediated inhibition of
AR activity are through disruption of AR N/C interaction and/or
competition with the p160 coactivators. These data establish a function
role for the corepressor SMRT to regulate the transcriptional activity of AR.
Plasmids and Reagents--
The human AR cDNA was kindly
provided by Dr. Chawnshang Chang (University of Rochester Medical
Center, Rochester, NY). The Gal4 DBD (G4) fusions of AR fragments
(G4-AR A/B, G4-AR D/E, and G4-AR E) were kindly provided by Dr. Lirim
Shemshedini (University of Toledo, Toledo, Ohio). The G4s-AR and G4-AR
were made by inserting full-length human AR cDNA into the pCMX-Gal
vector at the XhoI and Asp718 sites,
respectively. The AR full-length, 1-500, 1-660, 501-660, 501-919,
and 661-919 fragments were amplified by PCR and subcloned into the
pCMX-HA vector. The human SMRTe (41), GST-cSMRT, GST-SMRT-ID1, and
GST-SMRT-ID2 have been previously described (42). The ID1 and ID2
corepressor motif mutants (ID1mt, ID2mt) were made by site-directed
mutagenesis in the context of GST-SMRT-ID1 and -ID2, respectively. The
oligonucleotides used for the site-directed mutagenesis were
5'-GCACATCAGTGAGGCCGCCACACAGGACTACACC-3' and
5'-GGTGTAGTCCTGTGTGGCGGCCTCACTGATGTGC-3' for ID1mt, and
5'-CATGGGGCTGGAGGCCGCAGCTAGAAAGGCACTCATG-3' and
5'-CATGAGTGCCTTTCTAGCTGCGGCCTCCAGCCCCATG-3' for ID2mt. The pSG5-TIF2 vector (43) was kindly provided by Hinrich Gronemeyer (INSERM, Strasbourg, France). The androgen dihydrotestosterone (DHT)
and the antiandrogen flutamide were purchased from Sigma.
Transient Transfection--
Human kidney 293 cells were grown in
phenol red-free Dulbecco's modified Eagle's medium media supplemented
with 10% charcoal-stripped fetal bovine serum (Invitrogen). One day
prior to transfection, cells were seeded in 12-well plates at 10,000 cells per well. A DNA mixture containing the NR expression vector, the
control vector pCMX- Co-immunoprecipitation Assay--
Co-immunoprecipitation was
conducted according to standard procedure using protein A-agarose beads
(Santa Cruz Biotechnology). Whole cell extracts were prepared from
LNCaP human prostate cancer cells after treatment with 100 nM DHT, 100 nM flutamide, or solvent alone for
24 h. The cells were lysed in cell lysis buffer (0.4 M
NaCl, 0.2 mM EGTA, 10% glycerol, 1% Nonidet P-40), and
cell extracts were pre-cleared by incubating with protein A-agarose beads for 1 h at room temperature. The affinity-purified IP
antibodies were conjugated with protein A-agarose beads in cell lysis
buffer for 2 h at room temperature. The antibody protein A-agarose
was collected by brief centrifugation and incubated with cell extracts (100 µg) overnight at 4 °C. The precipitates were collected by centrifugation and washed extensively with phosphate-buffered saline
containing 0.1% Nonidet P-40. The final precipitate was dissolved in
SDS protein sample buffer and analyzed by SDS-PAGE and Western
blotting. Western blotting was conducted using the ECL reagents
according the manufacturer's recommendation (Amersham Biosciences).
The anti-AR polyclonal antibody was purchased from Santa Cruz
Biotechnology. The anti-SMRT monoclonal antibody was a gift from
GeneTex (San Antonio, TX).
GST Pull-down Assay--
Approximately 5 µg of purified GST
fusion protein was incubated with 5 µl of in vitro
translated 35S-labeled receptors with moderate shaking at
4 °C overnight in binding buffer (20 mM HEPES, pH 7.7, 75 mM KCl, 0.1 mM EDTA, 2.5 mM
MgCl2, 0.05% Nonidet P-40, 1 mM
dithiothreitol, and 1 µg/ml bovine serum albumin). The bound protein
was washed five times with the binding buffer, and the beads were
collected by mild centrifugation at 3,000 rpm. The bound protein was
eluted into SDS sample buffer, subjected to SDS-PAGE, and detected by autoradiography.
Indirect Immunofluorescence Microscopy--
HeLa cells were
grown on coverslips in 12-well plates 24 h prior to transfection.
Immunostaining was performed as previously described (45). Briefly,
cells were washed twice with phosphate-buffered saline and fixed in
methanol/acetone (1:1) for 1 min on dry ice. Cells were stained
sequentially with primary antibodies and rhodamine or
fluorescein-conjugated secondary antibodies. Cell nuclei were counterstained with DAPI (4',6-diamidino-2-phenylindole dihydrochloride hydrate) (Sigma). The coverslips were mounted with a ProLong Antifade kit (Molecular Probes) and imaged on an inverted epifluorescence microscope (Axiovert 200, Zeiss) equipped with a cooled CCD camera (Axiocam) and the Axiovision software (Zeiss).
Transcriptional Repression by AR and Interaction with SMRT in
Vivo--
To determine if AR contains intrinsic transcriptional
repression activity, we first analyzed the transcriptional activity of
a series of Gal4 DBD (G4)-AR fusion proteins by transient transfection assay in human HEK293 cells. The full-length AR was expressed as G4
(amino acids 1-147) or G4s (amino acids 1-74) fusion proteins, and
the AR A/B, D/E, and E fragment were expressed as G4 fusions (Fig.
1A). The ability of these
G4-AR fusion proteins to regulate transcription of a
Gal4-dependent MH100-Luc reporter was determined in HEK293
cells (Fig. 1B). In this assay, the G4s-AR and G4-AR exhibit
5.2- and 3.5-fold repression activities, respectively. The G4-AR A/B
shows a strong transcriptional activity as expected from the AF-1
function. Similar to the full-length G4-AR, the G4-AR D/E and G4-AR E
also show 2.5- and 4-fold repression activities, respectively. These
data suggest that AR may contain an intrinsic transcriptional
repression function.
The ability of AR to repress basal transcription led us to test whether
AR might interact with the nuclear receptor corepressor SMRT. To do so,
we first determined if the G4-AR fusion proteins could interact with a
VP-cSMRT fusion protein in a mammalian two-hybrid assay (Fig.
1C). The VP-cSMRT contains the viral VP16 activation domain
fused with the SMRT C-terminal NR-interacting domains (IDs). Co-transfection of G4-AR with VP-cSMRT moderately activates the reporter gene 2.2-fold above the G4-AR and VP control. Similarly, VP-cSMRT enhances G4-AR A/B activity about 4-fold above background. It
also enhances G4-AR D/E activity 7-fold and G4-AR E activity 8.5-fold,
respectively. In contrast, VP-cSMRT has no effect on G4 alone (not
shown). When cells were treated with DHT, there were no effects on
these two-hybrid interactions. These results suggest that AR may
interact with SMRT in mammalian cells and that these interactions may
occur even in the presence of AR ligand.
To confirm the association of AR and SMRT in vivo, we
conducted co-immunoprecipitation assays using LNCaP human prostate
cancer cell lysate. The cells were treated with either DHT or flutamide for 24 h before cell lysate preparation. Western blot analysis showed that the SMRT protein levels were similar in different cell
lysate (Fig. 1D, top). The endogenous AR proteins
were then immunoprecipitated by anti-AR polyclonal antibodies that were conjugated with protein A-agarose beads. The precipitates were resolved
by SDS-PAGE followed by Western blotting for AR and SMRT. We found that
the anti-AR antibodies could efficiently pull down endogenous AR, while
control IgG did not bring down AR (Fig. 1D). We found that
DHT treatment of LNCap cells enhanced the amount of AR protein in the
lysate, while flutamide treatment slightly reduced AR protein level.
When the precipitates were analyzed for SMRT proteins, we detected
full-length SMRT in all complexes precipitated with the anti-AR
antibodies, but not with the control IgG (Fig. 1D). DHT
treatment enhanced slightly the co-precipitated SMRT level, while
flutamide treatment increased the co-precipitated SMRT to a higher
level. These data suggest that AR and SMRT may form a stable complex
in vivo and that flutamide may enhance the formation of this complex.
Physical Interactions between AR and SMRT in Vitro--
To
determine if the in vivo association between AR and SMRT are
results of direct protein-protein interactions, we analyzed the AR-SMRT
interactions in vitro by GST pull-down assays. A series of
AR deletion mutants containing various domains (Fig.
2A) were expressed and labeled
with [35S]methionine in reticulocyte lysate. These AR
proteins were tested for binding with GST-cSMRT, GST-SMRT-ID1,
GST-SMRT-ID2, and ID motif mutants (Fig. 2B). In this assay,
we found strong interactions of full-length AR with GST-cSMRT and
SMRT-ID2 but not with GST alone or GST-SMRT-ID1 (Fig. 2C).
This suggests that SMRT may bind to AR in vitro through the
ID2 domain. We next tested the effects of androgens on this interaction
and found that, unlike the ligand-dependent dissociation of
SMRT from RAR and TR (28, 42, 44), DHT (D) and flutamide
(F) had no effects on the binding of AR to SMRT in this
in vitro binding assay (Fig. 2D).
The mechanisms of AR-SMRT interaction were further investigated by
analyzing the interactions of different AR fragments with SMRT. In the
experiment shown in Fig. 2E, GST-cSMRT did not bind to the
AR-(1-500) fragment (A/B domain), but it bound weakly to the
AR-(1-660) fragment (A/B/C/D domain). It also bound strongly to the
AR-(501-919) fragment (C/D/E) and moderately to the AR-(661-919) fragment (E domain). The AR-(1-500) fragment also did not interact with GST-SMRT-ID1, SMRT-ID2, or ID motif mutants (Fig. 2F).
The AR-(501-660) fragment that contains only the DBD and hinge regions (C/D domain) did not bind to GST-SMRT-ID1 or SMRT-ID2 in the absence or
presence of DHT (Fig. 2G), suggesting both the A/B and C/D domains alone are not sufficient to bind SMRT in vitro. In
contrast, the AR-(661-919) fragment (E domain) alone was sufficient to
bind GST-cSMRT (Fig. 2E) and SMRT-ID2 (Fig. 2H).
Interestingly, the AR-(501-919) fragment (C/D/E domain) bound both
GST-cSMRT (Fig. 2E) and SMRT-ID2 (Fig. 2I) much
more strongly, suggesting a role for the C/D domain in regulating
AR-SMRT interaction. The interaction of AR-(501-919) with SMRT-ID2 was
not affected by DHT treatment in this in vitro assay (Fig.
2J). Finally, although the AR-(1-500) and -(501-660)
fragments both showed no interaction with SMRT, the AR-(1-660)
fragment that combines the A/B and C/D domains displayed some binding
to GST-cSMRT (Fig. 2E) and SMRT-ID2 (Fig. 2K),
again indicating a role for the C/D domain in regulating SMRT binding.
Finally, we show that the interactions of SMRT-ID2 with AR-(661-919),
AR-(501-919) and AR-(1-660) fragments were inhibited by mutation of
the ID2 corepressor motif (Fig. 2, H, I, and
K). Together, these data indicate that SMRT may directly interact with AR in vitro. The AR-SMRT interaction may be
mediated through the SMRT-ID2 corepressor motif. The AR E domain
appears to be sufficient for SMRT binding, while the C/D domain may
play a critical role in regulating AR-SMRT interaction either directly or indirectly.
Subcellular Colocalization of AR with SMRT--
To further
demonstrate AR-SMRT interaction in mammalian cells, we cotransfected
HA-tagged AR and full-length SMRT (SMRTe) in HeLa cells and analyzed
potential subcellular colocalization by double immunofluorescence
staining. Transfection was conducted under normal serum condition
because the AR protein is unstable in the absence of ligand (46). We
found that in about 90% of cells where AR and SMRT are coexpressed,
the SMRT speckles are greatly reduced in size into microspeckled or
uniform patterns (Fig. 3, row
A), suggesting an interaction between SMRT and AR in these cells.
Strong evidence of AR-SMRT interaction is found in the remaining 10%
of transfected cells, where AR and SMRT appear simultaneously in
discrete speckles (Fig. 3, row B). These data suggest a
close association of AR and SMRT within nuclear speckles in mammalian
cells.
To confirm the association of AR and SMRT in mammalian cells, we
further analyzed the colocalization of three SMRT-binding AR fragments
with SMRT. Coexpression of SMRT with AR-(501-919) or -(661-919)
fragment results in clear colocalization at nuclear speckles (Fig. 3,
rows C and D), indicating an association of SMRT
with these two AR fragments. In fact, unlike full-length AR, the
501-919 and 661-919 fragments alone already localize in nuclear
speckles (data not shown). Interestingly, coexpression of the
AR-(1-660) fragment with SMRT results in dispersed AR and SMRT
localization pattern (Fig. 3, row E), suggesting an
association of SMRT with the AR N-terminal domain. These data suggest
that the AR N-terminal domain may be able to recruit SMRT from nuclear speckles to nucleoplasm and thus could be responsible for the disruption of SMRT speckles by full-length AR. Together, these data
suggest an association between AR and SMRT in cultured mammalian cells.
Inhibition of AR Transcription Activity by SMRT--
The
interaction between SMRT and AR prompted us to address the role of SMRT
in regulating the transcriptional activity of AR. To address this
question, we conducted transient transfection assays to determine the
effect of SMRT overexpression on AR activity using the AR-responsive
MMTV-Luc reporter in HEK293 cells (Fig. 4A). We found that
coexpression of SMRT resulted in a SMRT dose-dependent inhibition of the reporter gene expression that was activated by AR in
response to DHT treatment. Importantly, SMRT also had an additive
effect with the AR antagonist flutamide to reduce MMTV promoter
activity below basal levels. These data suggest that SMRT may inhibit
ligand-dependent transcriptional activation by AR, and it
also may act together with antagonist to further suppress AR target
gene expression.
The transcriptional activity of AR is determined by a strong
constitutive AF-1 activity and a relatively weak
ligand-dependent AF-2 function. To determine if SMRT can
affect either AF-1 or AF-2 function, we analyzed the effect of SMRT
overexpression on each of these two activation functions. In the
experiment shown in Fig. 4B, the autonomous AF-1 function
observed in the G4-AR 1-500 fusion protein was gradually inhibited by
SMRT coexpression in a dose-dependent manner, suggesting
that SMRT may inhibit the AF-1 activity of AR. Furthermore, we found
that SMRT could also inhibit the DHT-dependent AF-2
activity as well (Fig. 4C). The inhibition of AR
transcriptional activity by SMRT is specific because overexpression of
SMRT had no effect on G4 or G4-VP16 activity (Fig. 4D).
These data suggest that SMRT may inhibit the transcriptional activity
of AR.
Inhibition of AR N/C Interaction by SMRT--
The
overall transcriptional activity of AR is determined by a process known
as N/C interaction where two LXXLL-like motifs in the A/B
domain interact with the coactivator-binding pocket within the E domain
in a ligand-dependent manner (12, 13). To provide insight
into the molecular basis of SMRT-mediated inhibition on AR activity, we
tested whether SMRT could affect AR N/C interaction. As a result of the
N/C interaction, cotransfection of the AR N-terminal 1-500-amino acid
fragment with the C-terminal 501-919-amino acid fragment resulted in a
strong ligand-dependent activation of the MMTV promoter
activity (Fig. 5A).
Remarkably, co-expression of SMRT abolished this
ligand-dependent activation by 90% (a 10-fold reduction),
indicating that SMRT may inhibit AR N/C interaction. Interestingly,
cSMRT also had a similar effect, abolishing the N/C interaction by 85%
(a 6.7-fold reduction). As control, we found that expression of SMRT
and cSMRT had no significant effect on the basal expression of the
MMTV-Luc reporter (data not shown). The inhibition of AR N/C
interaction by SMRT and cSMRT were further measured at increasing
concentrations of SMRT or cSMRT to demonstrate that both SMRT and cSMRT
can inhibit AR N/C interaction in a dose-dependent manner
(Fig. 5B). These data suggest that SMRT may inhibit the transcriptional activity of AR by disrupting N/C interaction.
Competition with TIF2 by SMRT to Regulate AR Activity--
The
p160 family coactivator TIF2 has been shown to enhance AR
transcriptional activity through stabilization of AR N/C interaction (15, 18). Members of the p160 coactivator family and the SMRT/N-CoR corepressors are known to bind to overlapping surfaces in the AF-2
domain of the receptor (47-51). Therefore, it is reasonable to
speculate that SMRT might compete with p160 coactivator for binding to
liganded AR. We tested whether SMRT and TIF2 could compete with each
other to control the transcriptional activity of AR. Consistent with
prior findings (15, 18), we found that co-transfection of TIF2 enhanced
DHT-dependent transcriptional activity of AR by about
2-fold (Fig. 6A). As expected,
co-transfection with SMRT abolished this TIF2 coactivation effect
completely. Interestingly, this inhibition of AR activity by SMRT could
be alleviated gradually by increasing TIF2 concentration (Fig.
6A), suggesting that TIF2 may revert SMRT-mediated
inhibition on AR activity. Conversely, increasing SMRT concentration
could also reduce the enhancement of AR activity by TIF2 (Fig.
6B), suggesting that SMRT mutually competes with TIF2 to
inhibit AR transactivation. In contrast, we found that TIF2 was not
able to reverse the SMRT-mediated inhibition on AR activity in the
presence of flutamide (Fig. 6C). This is consistent with the
fact that TIF2 does not bind to antagonist-bound AR. Together, these
data suggest that the coactivator TIF2 and the corepressor SMRT may
compete with each other to regulate the transcriptional activity of
AR.
In this study, we have investigated the physical and
functional interactions between AR and the corepressor SMRT. We show that SMRT interacts with both the unliganded and liganded forms of AR
in vitro and in vivo. The AR-SMRT interaction
in vivo appears to be enhanced by agonist or antagonist
treatment. We have mapped the interaction surface to the SMRT-ID2
domain and showed that mutation of the ID2 corepressor motif disrupted
the interaction. We found that the AR LBD/E domain is sufficient to
bind SMRT, while the C/D domain also play a critical role in regulating
SMRT binding. In addition, we demonstrate an in vivo
colocalization between SMRT and AR in cultured mammalian cells. The
biological significance of these in vivo and in
vitro interactions between AR and SMRT is supported by transient
transfection assay showing that overexpression of SMRT robotically
inhibits AR-mediated transcriptional activation of target promoter.
Finally, we provide evidence to suggest that the mechanisms of
SMRT-mediated inhibition on AR transcriptional activity may be due to
either disruption of the AR N/C interaction and/or functional
competition with the p160 coactivator.
Using the Gal4 DBD fusion protein system, we show that AR contains
transcriptional repression activity. We localized such repression
activity to the E domain of the receptor (Fig. 1B). Consistent with existing knowledge, we show that the AR A/B domain contains a strong transcriptional activation function, and that this
activity is masked in the context of full-length AR. The ability of the
G4-AR fusion protein to repress basal transcription is consistent with
the interaction of AR with SMRT as first demonstrated by mammalian
two-hybrid assay (Fig. 1C). We speculate that the inability
of G4-AR A/B to repress transcription despite its interaction with SMRT
is due to the presence of a strong AF-1 function. The in
vivo interaction between AR and SMRT is further supported by co-immunoprecipitation of endogenous AR and SMRT from the LNCaP human
prostate cancer cell lysate (Fig. 1D). Interestingly, we found that the AR ligands, especially the antagonist flutamide, appear
to enhance the in vivo association of AR with SMRT. This result is consistent with a previous chromatin immunoprecipitation study showing that the AR antagonist bicalutamide could enhance the
recruitment of N-CoR and SMRT to the prostate-specific antigen (PSA) promoter in LNCap prostate cancer cells (52).
The physical interaction between AR and SMRT was demonstrated by GST
pull-down assay, which indicates that SMRT may interact with AR
directly in vitro. We have mapped the interacting surfaces on SMRT to the ID2 domain. Interestingly, the SMRT-ID1 domain has no
affinity to AR, despite the fact that this domain is the primary
interacting surface for RAR and TR (42) and that ID1 and ID2 share a
consensus LXXXIXXX(I/L) sequence that mediate direct binding of SMRT and N-CoR to several NRs (47-51). Intriguingly, the SMRT-AR interaction is inhibited by mutation of the ID2 corepressor motif, suggesting that the mechanism of SMRT binding to AR may be
conserved with other NRs (48, 49). Our data suggest that there might be
at least two SMRT binding sites on the AR protein, although the exact
binding mechanisms remain to be elucidated. The best AR fragment for
SMRT binding is the AR-(501-919) amino acid fragment that contains
both the DBD/hinge region and LBD. Since the LBD alone binds to SMRT
but DBD/hinge alone shows no interaction with SMRT, we speculate that
there may be a SMRT binding site within the LBD. The fact that the
AR-(501-919) fragment binds to SMRT much stronger that the
AR-(661-919) fragment suggest a critical role for the DBD/hinge region
of AR in regulating corepressor binding. It is possible that the
presence of the DBD/hinge domain may help to stabilize a conformation
of the LBD and A/B domain that is more compatible with SMRT binding.
Alternatively, it is equally possible that there might be a weak
binding site within the AR DBD or hinge region that may synergize with
the other binding site for SMRT binding. Contrary to a recent report
(53), we found no interaction between AR A/B alone and SMRT in GST
pull-down assays using either GST-cSMRT or SMRT-ID2. However, the
interaction between SMRT and AR LBD is readily detectable in our assay.
It appears that SMRT utilizes the ID2 corepressor motif for interaction with AR, suggesting that the mechanisms of SMRT binding to these two
sites may be related. Since the amino acid sequences of the ID1 and ID2
corepressor motifs are similar, the mechanisms by which AR
distinguishes one motif from the other remains to be determined.
Unlike RAR and TR, whose interactions with SMRT are ligand-independent
(28, 42, 44), or the PR and ER whose interactions with SMRT are
antagonist-dependent (30, 32, 54), we found that the
interaction between full-length AR and SMRT is not affected by
androgens in the in vitro binding assay (Fig.
2D). These data suggest that SMRT might be able to interact
with both the unliganded and liganded forms of AR. The ability of SMRT
to interact with liganded AR is consistent with the reporter gene assay
demonstrating that SMRT can inhibit ligand-dependent
transcriptional activation by AR (Fig. 4). Alternatively, we have
previously shown that there are a substantial number of cells
containing cytoplasmic SMRT staining (55), implying that SMRT might be
involved in the signal transduction pathway of AR in the cytoplasm
during its ligand-induced transformation process. It is also possible
that SMRT might help to maintain an inactive AR conformation in either
the cytoplasm or nucleus.
The subcellular distribution of unliganded AR is cell
line-dependent as the AR is predominantly nuclear when
expressed in HeLa cells but mainly cytoplasmic in COS-1 cells (56). In
the presence of hormone, the AR is translocated into the nucleus and distributed in a microspeckles (57-59). Our data show that
coexpression of AR and SMRT in HeLa cells results in apparent
colocalization in the speckles (Fig. 3), thus confirming in
vivo association of AR and SMRT. Interestingly, unlike the
recruitment of RAR to the SMRT speckles (55), AR seems to disrupt the
SMRT speckles as a majority of the transfected cells contain
microspeckled SMRT staining when AR is coexpressed (Fig. 3). It is
possible that AR may be able to recruit SMRT to its nuclear sites.
Consistently, SMRT has been detected on the natural AR target promoter
by chromatin immunoprecipitation assay in the presence of antiandrogen
(52). Likely, when AR binds to the response element in the presence of
antiandrogen, it is able to recruit SMRT to the target promoter because
of lack of competition from coactivator. In contrast, in the presence
of androgen, SMRT will have to compete with coactivator, and only when
SMRT is overexpressed will it be able to show binding to the liganded receptor.
Our data suggest that SMRT can inhibit both the AF-1 and AF-2 function
of AR in a SMRT dose-dependent manner (Fig. 4). This inhibition is likely due to direct binding of SMRT as demonstrated by
in vivo and in vitro binding assays. We predict
that SMRT binding to liganded AR may block recruitment of coactivators
or bring in deacetylase activity to the liganded receptor. It is
reasonable to speculate that binding of SMRT to the liganded receptor
may be sufficient to block AR transcriptional activity. Interestingly, the inhibition of AR activity can be reverted by overexpression of the
coactivator TIF2 (Fig. 6). Because TIF2 also binds to the liganded AR,
it is conceivable that the reinstatement of AR activity by TIF2 may be
due to displacement of SMRT from the liganded AR, resulting in
subsequent recruitment of CBP/p300 or other coactivators to the
receptor. The existence of a mutual competition between TIF2 and SMRT
on regulating AR transactivation suggests that the corepressor SMRT
might inhibit transactivation by the liganded receptor. Similarly, we
also observed potent inhibition of AR activity by the SMRT-related
corepressor N-CoR.2 Because
SMRT and N-CoR share highly conserved NR interaction domains, we
believe that both SMRT and N-CoR can function independently to
negatively regulate the transcriptional activity of liganded AR.
The AR has been shown to undergo several rounds of "recycling"
before being subjected to proteasome-mediated degradation (57), indicating that a mechanism of transient AR inactivation must exist. It
has been suggested that hormone inactivation or degradation may result
in the transient deactivation of AR activity prior to receptor export
from the nucleus (57). In contrast, a recent report indicates that
HDAC1 may play a key role in the acute repression of AR transcriptional
activity (60). We suggest here that SMRT may be another key player in
the down-regulation of AR activity. The abilities of SMRT to interact
with the liganded AR, to compete with TIF2 coactivator, and to block AR
N/C interaction are all consistent with the hypothesis that SMRT is
capable of antagonizing the transcriptional activity of AR.
Interestingly, SMRT is known to form a stable complex with HDAC1 (61),
implying that SMRT and HDAC1 may act synergistically to inhibit AR
activity. However, our data also indicate that the primary mechanism
for the SMRT-mediated AR inactivation may be due to the blockage of AR
N/C interaction. This is supported by the fact that the C-terminal
fragment of SMRT, which contains only the receptor-interacting domain
but no transcriptional repression activity, is capable of blocking N/C
interaction (Fig. 5). The molecular mechanisms by which SMRT blocks AR
N/C interaction remain to be defined. However, in light of previous
observations that SMRT can function as an activating cofactor for HDAC3 to acetylate histones (62, 63) and that the AR
transcriptional activity may be dynamically regulated by acetylation
and deacetylation of AR (40, 60), it is possible that SMRT might affect
AR activity through enhancing deacetylation of AR.
During preparation of this manuscript, Baniahmad and co-workers (53)
reported that SMRT could interact with AR in the presence of the
natural androgen DHT, the synthetic androgen R1881, or the partial
agonist cyproterone acetate (CPA). By using a modified mammalian
two-hybrid system on the complex MMTV promoter, that study demonstrated
a 4-fold interaction with VP-cSMRT in the absence of ligand and in the
presence of the full antagonists Casodex or hydroxyflutamide, which is
consistent with our findings of a ligand-independent interaction
between AR and SMRT. To address the discrepancy between the effects of
DHT on the AR-SMRT interaction, we repeated and confirmed in the same
modified mammalian two-hybrid system using MMTV-Luc reporter that DHT
indeed stimulated the reporter gene expression in the presence of
full-length AR and VP-cSMRT.2 Interestingly, this
DHT-mediated enhancement was virtually abolished when the AR N-terminal
A/B domain was removed in the same MMTV-based two-hybrid
system,2 indicating a requirement of the AF-1 function for
this enhancement effect. It is conceivable that the induction of N/C
interaction by DHT might be involved in this process; however, the
exact mechanisms remain to be determined. Collectively, these results
suggest that SMRT may play an important role in regulating the
transcriptional activity of AR and also provide a potential mechanism
for down-regulation of AR transcriptional activity. These results may
also have implications for antagonizing AR activity in prostate cancer cells.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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(35) and
(36,
37) and the orphan receptor DAX1 (38). In addition, SMRT and N-CoR have
also been implicated as corepressors for a variety of unrelated
transcription factors involved in a wide array of biological processes
(27). The promiscuity of SMRT and N-CoR on interacting with multiple
transcription factors is consistent with an essential role in embryonic
development as was revealed by studies of N-CoR-null mice, which die in
midgestation and exhibit defects in several developmental processes
(39).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Gal, the luciferase reporter, and the pGEM
carrier DNA was prepared in a final volume of 30 µl. The DNA solution was mixed dropwise with one volume of 0.5 M
CaCl2 and two volumes of 2× BBS (50 mM
N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES;
Calbiochem), 280 mM NaCl, 1.5 mM
Na2HPO4, pH 6.95). The DNA precipitates were
allowed to form at room temperature for 10 min before adding to culture
cells. Transfection was allowed to proceed for 12 h, and the
precipitates were removed by washing with phosphate-buffered saline.
Transfected cells were refed with fresh media containing vehicle alone
or vehicle plus respective ligand. Cells were harvested 24-36 h after
treatment and processed for luciferase and
-galactosidase assay as
previously described (44). The luciferase activities were determined
with a MLX microtiter plate luminometer (Dynex) using 100 µl of assay
solution (100 mM KPO4, 5 mM ATP, 10 mM MgCl2) and 100 µl of luciferin solution (10 mM D-luciferin in 100 mM
KPO4, pH 7.8). The luciferase activities were normalized to
the
-galactosidase activity.
RESULTS
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ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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View larger version (55K):
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Fig. 1.
Transcriptional regulation by AR and its
interaction with SMRT. A, schematics of the Gal4
DBD (G4)-AR and VP-cSMRT fusion proteins. The G4s (amino acids 1-74)
contains the minimal Gal4 DBD. The AR domains are depicted as
A/B, the N-terminal domain that contains the AF-1 function.
C, the DNA-binding domain; D, the hinge region;
and E, the ligand-binding domain that contains the AF-2
function. The VP-cSMRT contains the transactivation domain of VP16
(amino acids 413-490) fused to the C-terminal last 422 amino acids of
SMRT that contains two nuclear receptor-interacting domains.
B, mammalian one-hybrid assay showing transcriptional
activity of G4-AR fusion proteins. The full-length AR, the D/E, and E
domains contain moderate transcriptional repression activity. The A/B
domain contains constitutive transcriptional activation function.
C, mammalian two-hybrid assay showing interactions of AR
with SMRT in the absence or presence of DHT (100 nM). The
VP-cSMRT interacts weakly with the full-length AR but strongly with the
AR A/B, D/E, and E domains. DHT has no effect on these interactions.
D, co-immunoprecipitation of endogenous AR with SMRT in
prostate cancer cell lysate. The LNCaP human prostate cancer cell
lysate were prepared after treatment of the cells with 100 nM DHT (D), 100 nM flutamide
(F), or solvent alone ( ) for 24 h. The endogenous AR
proteins were precipitated by anti-AR polyclonal antibodies or with
control IgG (IgG) bound to protein A-agarose beads. The
immunoprecipitated complexes were resolved by SDS-PAGE and analyzed
for AR and SMRT proteins by Western blotting. Western blot analysis
indicates that there are approximately equal amounts of SMRT in the
cell lysate before immunoprecipitation (top panel). The
anti-AR immunoprecipitation followed by AR Western blot shows an
elevation of AR protein level after DHT treatment, and a reduction
after flutamide treatment. The co-immunoprecipitation data show that AR
can form a stable complex with SMRT in the absence or presence of
ligand and that AR ligands, especially, the antagonist flutamide
enhance formation of AR-SMRT complex.
View larger version (67K):
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Fig. 2.
The interacting domains between SMRT and
AR. A, schematics of AR deletion mutants and their
relative binding to SMRT. B, schematics of SMRT mutants
and their relative interactions with AR. Numbers indicate
starting and ending amino acids based on the human SMRTe sequence (41).
indicates an internal deletion of 46 amino acids following residue
2352. The ID1 and ID2 mutants (mt) were generated by
site-directed mutagenesis. C, GST pull-down assay
showing interactions of GST-cSMRT, SMRT-ID1, and SMRT-ID2 with
35S-labeled full-length AR (35S-AR).
AR binds to SMRT-ID2 but not SMRT-ID1. D, GST pull-down
assay showing the effect of androgens on binding of AR to GST-cSMRT.
S, solvent (20% dimethyl sulfoxide, 80% ethanol);
D, 100 nM dihydrotestosterone; F, 1 µM flutamide. E, interactions of various
AR fragments with GST-cSMRT in the GST pull-down assay.
F-K, interactions of various AR fragments with GST-SMRT-ID1
(ID1) and GST-SMRT-ID2 (ID2) domains in the GST pull-down assay.
wt, wild type; mt, mutant.
View larger version (59K):
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Fig. 3.
Immunofluorescence colocalization of AR and
SMRT in mammalian cells. HeLa cells were transiently transfected
with plasmids encoding full-length human SMRT or HA-tagged full-length
AR or indicated AR fragments. SMRT and AR were double-labeled with a
mouse monoclonal anti-SMRT antibody (Grp5, GeneTex) and a rabbit
polyclonal anti-HA antibody (Medical & Biological Laboratory), followed
by fluorescein (green color)- or rhodamine (red
color)-conjugated secondary antibodies, respectively. The cell
nuclei were stained with DAPI and are shown at right.
Coexpression of full-length AR with SMRT results in disruption of the
SMRT speckles in most cells (row A), while in some cells
clear colocalization are observed in the speckles (row B).
The AR-(501-919) and -(661-919) alone show speckles that colocalize
with SMRT (rows C and D). The AR-(1-660)-amino
acid fragment alone shows a diffuse pattern, which appears to disrupt
the SMRT speckles (row E).
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Fig. 4.
Inhibition of AR transcriptional activation
by SMRT. A, SMRT inhibits AR-mediated
transactivation and enhances antagonism mediated by flutamide. 293 cells were transiently transfected with AR and increasing
concentrations of SMRT. The MMTV-tk-Luc reporter activity was
determined from triplicate experiments in the presence of DHT (100 nM) or flutamide (1 µM). B,
SMRT inhibits AR AF-1 activity. The G4-AR-(1-500) fusion construct was
transiently transfected into 293 cells with increasing concentrations
of SMRT and the MH100-tk-Luc reporter. The Gal4 DBD alone basal
activity is shown in D. C, SMRT inhibits the
ligand-dependent AF-2 activity of AR. The AR-(501-919)
construct was transiently transfected into HeLa cells with increasing
amounts of SMRT. The MMTV-tk-Luc reporter activity was determined by
luciferase assay. D, SMRT has no effect on the basal
promoter activity or G4-VP16-activated transcription.
View larger version (18K):
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Fig. 5.
SMRT inhibits AR N/C interaction.
A, inhibition of AR N/C interaction by SMRT and cSMRT.
The AR N-terminal domain (1-500) and C-terminal domain (501-919) were
transiently transfected into HeLa cells with SMRT or cSMRT and the
MMTV-Luc reporter. Both SMRT and cSMRT are capable of blocking AR N/C
interaction efficiently by ~10- and 6.7-fold, respectively.
B, dose-dependent inhibition of AR N/C
interaction by SMRT and cSMRT. The reporter luciferase activity in the
presence of AR-(1-500), AR-(501-919), and 100 nM DHT is
expressed as 100% N/C interaction.
View larger version (32K):
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Fig. 6.
Competition between SMRT and TIF2 on
regulating AR activity. A, TIF2 can reverse
SMRT-mediated inhibition of AR activity. 293 cells were transiently
transfected with increasing concentrations of TIF2 (in µg), and the
full-length AR (0.1 µg) and SMRT (0.5 µg) where indicated by +,
together with the MMTV-tk-Luc reporter. B, SMRT
inhibits transcriptional coactivation mediated by TIF2. Where
indicated, full-length AR (0.1 µg) and TIF2 (0.5 µg) were
transiently transfected into HeLa cells with increasing concentration
of SMRT. C, TIF2 cannot reverse flutamide- and SMRT-mediated
inhibition of AR activity. Where indicated, full-length AR (0.1 µg)
and SMRT (0.5 µg) were transiently transfected into HeLa cells with
increasing concentrations of TIF2 (in µg) in the presence of 100 nM of flutamide.
DISCUSSION
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ABSTRACT
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DISCUSSION
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ACKNOWLEDGEMENTS |
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We thank Amy (Hong-Bing) Chen for excellent technical support and David Johnson for critical reading of the article.
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FOOTNOTES |
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* This work was supported by Grants DK52542 and DK52888 from the National Institutes of Health (to J. D. C.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Research Scholar funded by the Leukemia & Lymphoma Society. To
whom correspondence should be addressed: Dept. of Pharmacology, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson
Medical School, 675 Hoes Ln., Piscataway, New Jersey 08854. E-mail:
Donchen2032@yahoo.com.
Published, JBC Papers in Press, November 18, 2002, DOI 10.1074/jbc.M206374200
2 G. Liao and J. D. Chen, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: AR, androgen receptor; SMRT, silencing mediator for retinoid and thyroid hormone receptor; DHT, dihydrotestosterone; DBD, DNA-binding domain; LBD, ligand-binding domain; NR, nuclear receptor; MMTV, mouse mammary tumor virus; RAR, retinoic acid receptor; HA, hemagglutinin; DAPI, 4',6-diamidino-2-phenylindole.
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