A Nuclear Receptor Corepressor Modulates Transcriptional Activity of Antagonist-Occupied Steroid Hormone Receptor
Xun Zhang1,
M. Jeyakumar,
Sergei Petukhov and
Milan K. Bagchi
The Population Council and The Rockefeller University New York,
New York 10021
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ABSTRACT
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Synthetic steroid hormone antagonists are
clinically important compounds that regulate physiological responses to
steroid hormones. The antagonists bind to the hormone receptors, which
are ligand-inducible transcription factors, and modulate their
gene-regulatory activities. In most instances, a steroid receptor, such
as progesterone receptor (PR) or estrogen receptor (ER), is
transcriptionally inactive when complexed with an antagonist and
competitively inhibits transactivation of a target steroid-responsive
gene by the cognate hormone-occupied receptor. In certain cellular and
promoter contexts, however, antagonist-occupied PR or ER acquires
paradoxical agonist-like activity. The cellular mechanisms that
determine the switch from the negative to the positive mode of
transcriptional regulation by an antagonist-bound steroid receptor are
unknown. We now provide strong evidence supporting the existence of a
cellular inhibitory cofactor that interacts with the B form of human PR
(PR-B) complexed with the antiprogestin RU486 to maintain it in a
transcriptionally inactive state. In the presence of unliganded thyroid
hormone receptor (TR) or ER complexed with the antiestrogen
4-hydroxytamoxifen, which presumably sequesters a limiting pool of the
inhibitory cofactor, RU486-PR-B functions as a transcriptional
activator of a progesterone-responsive gene even in the absence of
hormone agonist. In contrast, hormone-occupied TR or ER fails to induce
transactivation by RU486-PR-B. Recent studies revealed that a
transcriptional corepressor, NCoR (nuclear receptor corepressor),
interacts with unliganded TR but not with liganded TR. Interestingly,
coexpression of NCoR efficiently suppresses the partial agonistic
activity of antagonist-occupied PR-B but fails to affect
transactivation by agonist-bound PR-B. We further demonstrate that
RU486-PR-B interacts physically with NCoR in vitro. These
novel observations suggest that the inhibitory cofactor that associates
with RU486-PR-B and represses its transcriptional activity is either
identical or structurally related to the corepressor NCoR. We propose
that cellular mechanisms that determine the switch from the
antagonistic to the agonistic activity of RU486-PR-B involve removal of
the corepressor from the antagonist-bound receptor so that it can
effect partial but significant gene activation.
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INTRODUCTION
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The nuclear hormone receptors belonging to the
steroid/thyroid/retinoid receptor superfamily are ligand-inducible
transcription factors (1). These receptors modulate transcription of
specific cellular genes, either positively or negatively, by
interacting with specific hormone response elements (HREs) located near
the target promoters (for reviews see Refs. 24). Recent studies in
many laboratories revealed that nuclear receptors repress or enhance
transcription by interacting with multiple coregulatory factors, which
function as signaling intermediates between the receptors and the RNA
polymerase II transcription machinery (for a review see Ref.5).
Certain nuclear receptors, such as thyroid hormone receptor (TR) and
retinoic acid receptor (RAR), display transcriptional silencing
activity in the hormone-free state (6, 7, 8). We and others have
demonstrated that unliganded TR or RAR associates with a negative
coregulatory factor, termed corepressor (9, 10, 11, 12, 13, 14, 15). The receptor recruits
the corepressor to the HRE, and the receptor-corepressor complex
actively represses target gene transcription by impairing the activity
of the basal transcription machinery (11, 12, 13, 14, 15). The binding of hormone
to TR or RAR results in the dissociation of the corepressor from the
receptor, leading to the reversal of transcriptional repression
(11, 12, 13, 14, 15). The hormone-bound corepressor-free receptor then acts in
unison with a positive coregulatory factor, termed coactivator, to
promote gene activation (16, 17, 18, 19).
Recently the cDNAs encoding two distinct but structurally related
candidate corepressors, NCoR (nuclear receptor corepressor) and SMRT
(silencing mediator for retinoic acid receptor and thyroid hormone
receptor), were isolated by yeast two-hybrid cloning (12, 14, 15). NCoR
is a 270-kDa nuclear protein that binds to the TR or RAR component of
DNA-bound TR-retinoid X receptor (RXR) or RAR-RXR heterodimers in the
absence of T3 or all-trans-retinoic acid but
fails to interact with the ligand-occupied receptors (12, 13).
Domain-mapping studies indicate that the transcriptional repression and
nuclear receptor interaction domains of the corepressor are physically
discrete: N-terminal amino acids 254312 of the molecule contain a
major repression function, while the extreme C-terminal 400 amino acids
harbor the receptor-interaction region (12, 20). Additional repression
domains involving amino acids 752-1016 and 18291940 have also been
reported (20a). SMRT, which encodes a 168-kDa polypeptide, also
interacts with the ligand-binding domain (LBD) of unliganded TR or RAR.
Like NCoR, association of SMRT with the receptor-DNA complexes is
destabilized by ligand (14, 15).
In contrast to TR and RAR, unoccupied steroid hormone receptors such as
progesterone receptor (PR) and ER exist in a non-DNA-binding complex
associated with several heat shock proteins (21, 22). Hormone binding
triggers the release of these heat shock proteins and allows the
hormone-receptor complex to interact with the target HRE. The hormone-
and DNA-bound receptor then interacts with the basal transcription
machinery to mediate gene activation. It has been reported that NCoR
does not interact with unoccupied steroid receptors such as ER, PR, or
glucocorticoid receptor (GR) (12). However, interaction of an agonist
or antagonist-bound steroid receptor with a corepressor, and the
functional consequence of such an event, has not been explored
previously.
Steroid receptors mediate negative transcriptional regulation when
bound to synthetic hormone antagonists (23, 24, 25). Upon binding most
antagonists, a steroid receptor sheds the heat shock proteins and
interacts with the HRE (26, 27, 28). The antagonist-occupied receptor,
however, fails to activate transcription efficiently, presumably due to
a failure to interact with the basal transcription machinery in a
productive fashion. In certain cellular and promoter contexts, however,
PR complexed with the antiprogestin, RU486, or ER complexed with the
antiestrogen, 4-hydroxytamoxifen, exhibits partial but significant
agonistic activity (23, 24, 29, 30, 31, 32). The molecular basis of this mixed
agonist/antagonist activity remains unclear. The cell type dependency
of this activity, however, raised the possibility that additional
cellular cofactors might critically influence the gene-regulatory
activity of the antagonist-occupied receptor. The present study
provides strong evidence that transcriptional activity of
antagonist-bound PR or ER in a target cell is indeed modulated by a
nuclear receptor corepressor protein(s).
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RESULTS
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Coexpression of Unliganded TRß Induces Transactivation by
RU486-Bound PR-B
Conceptually, if a limiting cofactor is critical for the
inhibitory action of an antagonist-bound steroid receptor, then the
inhibitory effect could be relieved by the ectopic expression of a
competing nuclear receptor that binds and sequesters the cofactor. We
and others have previously employed such squelching experiments to
define the requirement of corepressors in transcriptional repression by
hormone-free TR or RAR (9, 10, 11). To investigate whether coexpression of
unliganded TR, which binds to a cellular corepressor, can perturb the
inhibitory pathway of RU486-occupied PR, we performed transient
transfection experiments using CV1 cells. As shown in Fig. 1
, expression of PR-B effected only
minimal transactivation of a glucocorticoid/progesterone response
element (GRE/PRE)-containing promoter GRE/PRE2E1bTATA (33)
in the presence of RU486 (lane 1). Cotransfection of increasing amounts
of a vector expressing human TRß in the unliganded form did not
produce any effect on the basal level of transcription from the
promoter in the absence of RU486 (lanes 2, 4, and 6). Coexpression of
unliganded TRß however, triggered a marked transactivation of
the GRE/PRE-linked promoter by RU486-bound PR-B. The promoter
activation was clearly TRß dose-dependent (Fig. 1
, lanes 3, 5, and 7;
Fig. 2
, lanes 3 and 7) but occurred in
the absence of a DNA response element for TR in the target promoter. In
the presence of excess of TRß, we observed up to a 10-fold
enhancement of RU486-dependent transactivation by PR-B (Fig. 1
, compare
lanes 6 and 7; panel B, compare lanes 5 and 7). We also noted that
unliganded TRß failed to induce RU486-PR-B-mediated transactivation
of a control promoter, E1bTATA, which exactly resembled the test
promoter except for a lack of GRE/PRE sequences (data not shown). These
data indicated that RU486-PR-B is required to bind to the GRE/PRE in
the promoter to function as an activator. Taken together, our results
are consistent with the scenario that transcriptionally inactive,
antagonist-bound PR is complexed with a cellular inhibitory
coregulator. Unliganded TRß, which functions as a transcriptional
repressor, also interacts with this cofactor. Sequestration of a
limiting pool of the inhibitory coregulator by excess TR relieves
transcriptional inhibition by antagonist-bound PR-B and allows the
events leading to the activation of the GRE/PRE-linked promoter to
proceed.

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Figure 1. Coexpression of Unliganded TRß Induces
Transcriptional Activity of RU486-PR-B
Increasing amounts (1, 2.5, and 5 µg) of the expression vector
pCI-hTRß, together with 5 µg hPR-B expression vector, 10 µg
GRE/PRE2E1b-CAT as reporter plasmid, and 2 µg pSV ß-gal
as an internal control, were transiently transfected into CV1 cells as
described in Materials and Methods section. Cells were
treated with RU486 (10-8 M) or vehicle as
indicated for 24 h before harvesting. ß-Galactosidase and CAT
assays were performed as described previously (50). CAT activity was
normalized to ß-galactosidase activity. The percentage of
chloramphenicol conversion to the mono- and diacetylated forms in lane
1 (0.25 ± 0.02), lane 2 (0.11 ± 0.01), lane 3 (0.55 ±
0.13), lane 4 (0.10 ± 0.02), lane 5 (0.80 ± 0.01), lane 6
(0.15 ± 0.01), and lane 7 (1.52 ± 0.20) was determined as
described previously (50) and represents the mean ±
SEM of six independent experiments. Relative fold
activation represents the ratio of CAT activity in lysates of hormone-
or antihormone-treated cells compared with that in untreated cells for
each plasmid combination.
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Figure 2. Ligand-Bound TRß Fails to Induce Transactivation
of a PRE-Linked Reporter Gene by RU486-PR-B
The expression vector pCI-hTRß (2.5 or 5 µg) together with 5 µg
phPR-B, 10 µg GRE/PRE2E1b-CAT, and 2 µg pSVß-gal were
transiently transfected into CV1 cells. Cells were treated with RU486
(10-8 M) and/or T3
(10-8 M) as indicated for 24 h before
harvesting. The percentage of chloramphenicol conversion to the mono-
and diacetylated forms in lane 1 (0.13 ± 0.04), lane 2 (0.14
± 0.03), lane 3 (0.79 ± 0.01), lane 4 (0.17 ± 0.03), lane
5 (0.15 ± 0.03), lane 6 (0.14 ± 0.02), lane 7 (1.45 ±
0.21), and lane 8 (0.16 ± 0.02) was determined as described
previously (50) and represents the mean ± SEM of
three independent experiments.
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Ligand-Bound TRß Fails to Induce Transactivation by RU486-Bound
PR-B
Previous studies revealed that the hormonal ligand influences the
interaction between TR or RAR and a nuclear receptor corepressor such
as NCoR or SMRT (12, 13, 14, 15). Whereas the unliganded TR or RAR associates
with the corepressor to generate the transcriptionally repressive
receptor-corepressor complex, the hormone-bound receptor fails to
interact with this regulatory molecule (12, 13, 14, 15). If the inhibitory
cofactor that mediates transcriptional inhibition by RU486-PR-B is one
of these newly identified nuclear receptor corepressors, one would
predict that only unliganded TRß, which binds to the corepressor,
will have the ability to induce transactivation by RU486-PR-B.
Ligand-occupied TRß, which fails to interact with the corepressor,
will be unable to induce such transactivation. As shown in Fig. 2
, in
contrast to unliganded TR, the ligand-occupied TR did indeed fail to
induce transcriptional activation by RU486-PR-B (compare lane 3 with
lane 4, lane 7 with lane 8), indicating that a transcriptionally
repressive form of TR is essential for the removal of the inhibitory
factor from antagonist-PR-B complex. This observation prompted us to
consider the possibility that a nuclear receptor corepressor is the
inhibitory cofactor that mediates transcriptional repression by
RU486-occupied PR-B. Consistent with our observations, the existence of
a putative corepressor that mediates transcriptional repression by
RU486-occupied PR-B has been postulated recently by Xu et
al. (34).
A Peptide Containing the Corepressor-Binding Site of TRß
Efficiently Induces Transactivation by RU486-Bound PR-B
Previous studies identified a region of TRß containing amino
acids 168 to 260 as a potential binding site of a corepressor (10, 11).
Baniahmad et al. (10) have shown that a peptide from
168260 containing the D domain (positions 170238) and the
N-terminal region (positions 238260) of the E domain of TRß
efficiently competed for the silencing activity of the receptor in
transient cotransfection assays. Tong et al. (11) observed
that addition of a TRß peptide from 145260 relieved transcriptional
repression by unliganded TR in HeLa nuclear extracts indicating that
this peptide alone was sufficient to titrate out the soluble
corepressor(s) present in the nuclear extracts. Horlein et
al. (12) recently reported that a smaller region 203230 of TRß
contains a conserved sequence, termed CoR box, which is critical for
binding the corepressor NCoR. For efficient interaction with the
corepressor, however, the CoR box is thought to require additional
contributions from its N-terminal sequences 230260.
We therefore examined whether the peptide 145260 of TRß containing
the corepressor-binding region can induce transactivation by
antiprogestin-bound PR-B. As shown in Fig. 3
, when we coexpressed the entire LBD
fragment (145456) of TRß, this mutant receptor significantly
activated trancription by RU486-bound PR in the absence of
T3 (lane 5). In contrast, the ligand-bound TR LBD fragment
failed to induce this transactivation (lane 6). Interestingly,
coexpression of the TR LBD peptide 145256 induced a marked
transactivation by RU486-bound PR-B. The magnitude of this
transactivation was comparable to that induced by the full-length TR
(compare lane 7 with lane 3). The ability of this relatively short
peptide containing the corepressor-binding site to induce
transactivation by antagonist-complexed PR-B further confirmed our
hypothesis that transactivation of a progesterone-responsive
promoter by RU486-PR-B results from the titration of a
limiting amount of a corepressor by unliganded TR.

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Figure 3. A Short Peptide Containing the Corepressor Binding
Site of TRß Induces Transactivation by RU486-Bound PR-B
Upper panel, Linear structures of human TRß and its
deletion mutants. Lower panel, CV1 cells were
transiently transfected with 5 µg phPR-B, 10 µg
GRE/PRE2E1b-CAT, and 2 µg pSVß-gal together with the
following expression plasmids: pCI (10 µg), lanes 1 and 2; pCI (5
µg) and pCI-hTRß (5 µg), lanes 3 and 4; pCI (5 µg) and
pCI-TR-LBD (145456) (5 µg), lanes 5 and 6); pCI-TR (145260) (10
µg), lanes 7 and 8. Cells were treated with RU486 (10-8
M) and/or T3 (10-8 M)
as indicated for 24 h before harvesting. The relative fold
activation represents the ratio of CAT activity in lysates of
antihormone-treated cells compared with that in untreated cells for
each plasmid combination. Two independent sets of the same experiment
were performed for reproducibility, and the results of a representative
experiment are shown.
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Coexpression of Estrogen Receptor (ER) Complexed with
4-Hydroxytamoxifen Induces Partial Transactivation of a
Progesterone-Responsive Gene by RU486-PR-B
We wondered next whether ER complexed with an antiestrogen such as
4-hydroxytamoxifen also targets the same inhibitory cofactor that is
critical for transcriptional inhibition by antagonist-bound PR. If such
a scenario is true, one would expect that antagonist-bound ER would
have the ability to sequester this inhibitory factor from RU486-PR-B
and activate RU486-dependent transcription by PR-B. As shown in Fig. 4
, expression of PR-B in CV1 cells
triggered marked transactivation of a progesterone response element
(PRE)-linked minimal promoter in a progesterone-dependent manner (lanes
1 and 2) but failed to induce efficient transactivation when RU486 was
present (lanes 3 and 4). Cotransfection of CV1 cells with ER expression
vector either in the absence (lane 8) or in the presence of estrogen
(lane 12) did not affect the transcriptionally inactive state of
RU486-PR-B. Coexpression of ER in the presence of 4-hydroxytamoxifen,
however, led to a dramatic induction of transactivation of the
progesterone-responsive promoter by the antiprogestin-complexed PR-B
(compare lane 10 with either lane 6 or 8). The magnitude of the
transactivation by RU486-PR-B in the presence of tamoxifen-occupied ER
was estimated to be about 2530% of that induced by
progesterone-bound PR-B (compare lanes 2 and 10). Our results therefore
suggested that a common inhibitory cofactor mediates transcriptional
repression by either progesterone or estrogen receptor complexed with
the cognate hormone antagonist.

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Figure 4. Antagonist-Bound Estrogen Receptor Induces
Transactivation of a Progesterone-Responsive Gene by RU486-PR-B
CV1 cells were transiently transfected with 5 µg phPR-B, 10 µg
GRE/PRE2E1b-CAT, and 2 µg pSVß-gal with or without 2
µg hER expression vector. Ligands, progesterone, RU486,
17ß-estradiol, and 4-hydroxytamoxifen were added to a final
concentration of (10-8 M) as indicated. The
percentage of chloramphenicol conversion to the mono- and diacetylated
forms was as follows: lane 1 (0.37 ± 0.03), lane 2 (36 ±
1.50), lane 3 (1.70 ± 0.40), lane 4 (1.50 ± 0.30), lane 5
(0.9 ± 0.14), lane 6 (1.55 ± 0.26), lane 7 (0.42 ±
0.30), lane 8 (1.85 ± 0.33), lane 9 (1.05 ± 0.35), lane 10
(12 ± 1.10). The relative fold activation represents the ratio of
CAT activity in lysates of hormone- or antihormone-treated cells
compared with that in untreated cells for each plasmid combination. The
experiment was repeated at least five times.
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Corepressor NCoR Suppresses RU486-Dependent Transactivation by PR-B
but Has No Effect on Progesterone-Mediated Transactivation
If the transactivation of a progesterone-responsive promoter by
RU486-PR-B results from the sequestration of a limiting amount of a
nuclear receptor corepressor by unliganded TR or
4-hydroxytamoxifen-bound ER, one would predict that expression of an
excess of the corepressor would suppress such transactivation. To
investigate whether a corepressor is a target of competition between
antagonist-complexed steroid receptors, we examined the effects of
coexpression of the nuclear receptor corepressor, NCoR (12), on the
agonistic activity of RU486-PR-B that is displayed in the presence of
antagonist-bound ER. Consistent with our previous observation,
overexpression of ER complexed with 4-hydroxytamoxifen (Fig. 5
, lane 6) strongly induced activation of
transcription by RU486-PR-B. Cotransfection of increasing amounts of a
plasmid expressing NCoR led to a progessive decline in the agonistic
activity of RU486-bound PR-B (Fig. 5
, lanes 8, 10, and 12). In control
experiments, cotransfection of a similar plasmid in which the cDNA for
NCoR is cloned in a reverse orientation did not significantly affect
transactivation by RU486-PR-B (Fig. 5
, lanes 14 and 16), demonstrating
that the inhibition of transcription was indeed mediated by NCoR. As
much as 80% of the agonistic activity of RU486-PR-B was suppressed in
the presence of 1 µg of NCoR expression plasmid (Fig. 5
, compare
lanes 6 and 12). Our studies clearly demonstrated that overexpression
of a nuclear receptor corepressor can mask the transactivation function
of RU486-PR-B to render it transcriptionally inactive. This finding
strongly suggested that NCoR or a structurally related corepressor is
the cellular cofactor that mediates transcriptional repression by
antagonist-complexed steroid receptors.

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Figure 5. Corepressor NCoR Suppresses Transactivation of a
Progesterone-Responsive Gene by RU486-PR-B
CV1 cells were transiently transfected with 2 µg phPR-B, 10 µg
GRE/PRE2E1b-CAT, and 2 µg pSVß-gal with or without 2
µg hER expression vector. Increasing amounts (0.25, 0.5, 1, and 2
µg) of the expression vector pCI-NCoR, or pCI-Rev.NCoR, were
transfected as indicated. pCI-NCoR and pCI-Rev.NCoR were constructed by
inserting the murine NCoR cDNA in the correct and reverse orientations,
respectively, into vector pCI under the control of human
cytomegalovirus immediate-early promoter (Promega Corp.). Ligands,
progesterone, RU486, and 4-hydroxytamoxifen were added as indicated.
Three independent sets of the same experiment were performed, and the
results of a representative experiment are shown. Relative fold
activation represents the ratio of CAT activity in lysates of hormone-
or antihormone-treated cells compared with that in untreated cells for
each plasmid combination.
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We then analyzed the effect of overexpression of NCoR on
transcriptional activity of agonist-bound PR-B. As shown in Fig. 6
, overexpression of NCoR did not
significantly affect progesterone-dependent transactivation of a
progesterone-responsive promoter by PR-B (panel A), whereas similar
amounts of corepressor strongly suppressed low levels of agonist
activity, which is displayed by RU 486-complexed PR-B (panel B).
Interestingly, in the presence of excess NCoR, RU 486-PR-B repressed
transcription to even below basal levels (panel B, lanes 5 and 6).
Overexpression of NCoR, however, did not repress transcription from a
control SV40 promoter (data not shown). Based on these results, we
conclude that NCoR does not exert a general repressive effect on
transcription and, in the context of a progesterone-responsive
promoter, it functions as a corepressor for the antagonist-bound PR
only.

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Figure 6. The Effects of Overexpression of NCoR on
Transcriptional Activity of Agonist- and Antagonist-Bound PR-B
CV1 cells were transiently transfected with 1 µg phPR-B, 1 µg
GRE/PRE2E1b-CAT, and 1 µg pSVß-gal vector by the
Lipofectamine method as described in Materials and
Methods. Increasing amounts (0.1, 0.25, 0.5, and 1 µg) of the
expression vector pCI-NCoR were transfected as indicated. In each
reaction, the total amount of pCI vector was maintained at 1 µg by
adjusting with empty pCI plasmid. Ligands, progesterone, and RU486 were
added as indicated. Cell lysates were normalized to ß-galactosidase
activity, and CAT assays were performed as described above. The
percentage of chloramphenicol conversion to the mono- and diacetylated
forms were as follows: panel A, lane 1 (15.84), lane 2 (95.3), lane 3
(33.35), lane 4 (95.5), lane 5 (95.3), lane 6 (95.3), lane 7 (94.7);
panel B, lane 1 (8.4), lane 2 (95.7), lane 3 (18.8), lane 4 (10.9),
lane 5 (4.8), lane 6 (2.2). Three independent sets of each experiment
described in the panels A and B were performed for reproducibility, and
the results of a representative experiment are shown. The experiment in
panel A was repeated under conditions of lower percentage conversion of
chloramphenicol, and essentially similar results were obtained.
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Interactions of Steroid Receptors with NCoR
Previous studies indicated that NCoR physically associates with
nuclear receptors such as TR and RAR to generate the inhibitory
receptor-corepressor complex in the absence of the hormonal ligand
(12, 13, 14, 15). Since NCoR repressed the transcriptional activity of
RU486-PR-B, we next examined whether it exhibits any direct and
specific interaction with the antagonist-complexed receptor. We
analyzed in vitro the interaction of 35S-labeled
hPR-B with a recombinant polypeptide,
glutathione-S-transferase (GST)-NCoRC', which contains the
carboxy-terminal amino acids 20572453 of NCoR that harbor a nuclear
receptor-interaction domain (12, 20). The results of this experiment
are shown in Fig. 7A
. PR-B did not
interact with a control GST polypeptide (lane 2). Only modest binding
of NCoRC' to PR-B was observed when the receptor was either unoccupied
or occupied with progesterone (lanes 3 and 4). In contrast, a
significant enhancement (
4- to 5-fold) in the binding of NCoRC' to
PR-B occurred when the receptor was occupied with RU486 (compare lane 5
with either lane 3 or 4). These results support the scenario that NCoR
or a closely related corepressor modulates the transcriptional activity
of RU486-PR-B by directly interacting with the antagonist-bound
receptor. Our results are consistent with those of Horwitz and
co-workers (35) who recently described the isolation of a human homolog
of NCoR by using RU486-occupied ligand binding domain of hPR-B as a
bait in a yeast two-hybrid screen. Interestingly, the fragment of NCoR
initially isolated by this group contained the C-terminal sequences of
NCoR that we have used in our in vitro binding
experiments.

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Figure 7. Interactions between NCoR and Antagonist-Bound PR-B
or ER
A, Top panel: 35S-labeled hPR-B was
synthesized from a linearized expression plasmid by in
vitro transcription-coupled translation using the TNT kit from
Promega and treated with either progesterone or RU486 or solvent as
described in Materials and Methods. A recombinant
polypeptide GST-NCoRC' containing the carboxy-terminal amino acids
20572453 of NCoR fused to glutathione-S-transferase (GST) was
expressed in E. coli BL21. The fusion protein was
immobilized on glutathione affinity resin, and the resin was washed
extensively. The resin containing GST-NCoRC' (200 ng) was incubated
with 10 µl of translation mix containing 35S-labeled hPR-B
as described in Materials and Methods.
After repeated stringent washings, the resin-bound proteins were eluted
and analyzed by SDS-PAGE and fluorography. The arrow
indicates the full-length, 35S-labeled hPR-B polypeptide.
The input lane represents 20% of the total volume of reticulocyte
lysate added to each reaction. Bottom panel: The optical
densities of the 35S-labeled hPR-B signals (indicated by
arrow) in lanes 26 were determined by densitometry.
Three independent sets of the same experiment were performed, and the
results of a representative experiment are shown. B,
35S-labeled hER was synthesized from a linearized
expression plasmid by in vitro transcription-coupled
translation using the TNT kit from Promega and treated with either
17ß-estradiol or 4-hydroxytamoxifen or solvent as described in
Materials and Methods. GST pull-down reactions were
performed using the recombinant polypeptide GST-NCoRC', and the bound
products were analyzed as described above.
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We also assessed the ability of the carboxy terminus 20572453 of NCoR
to interact with ER. As shown in Fig. 7B
, 35S-labeled human
ER (hER) did not bind at all to control GST polypeptide (lane 2). In
contrast, significant amounts of ER bound to NCoRC' irrespective of
whether the receptor was unoccupied or occupied with 17ß estradiol or
4-hydroxytamoxifen (compare lanes 3, 4, or 5 with lane 2). These
results indicated that under in vitro conditions, NCoR
interacted with ER in a ligand-independent manner. Similar
ligand-independent interactions between ER and the corepressor SMRT
have been recently reported by Smith et al. (36).
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DISCUSSION
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Antiprogestins and antiestrogens have emerged as powerful
pharmaceutical tools with which to counteract various physiological
effects of the steroid hormones, progesterone and estrogen. Although
RU486 is presently used for pregnancy termination and tamoxifen is the
drug of choice for the treatment of advanced hormone-dependent breast
cancer, little is known about their mechanism of action. Previous
studies indicated that PR complexed with RU486, or ER complexed with
4-hydroxytamoxifen, mimic certain of the functional responses of the
hormone-bound receptor such as the release of heat shock proteins,
nuclear localization, and dimerization (26, 27, 28). The antagonist-bound
receptor binds to the cognate DNA-response element but fails to
transactivate target promoter. An elegant set of studies by OMalley
and collaborators (37, 38) established that hormone- and
antagonist-bound steroid receptors display different conformations
within the carboxy-terminal portion of the molecule. It has been
proposed that antagonist binding induces an improper conformation in
the carboxy-terminal domain of the receptor that impairs the
ligand-dependent transactivation function, AF-2. Although
ligand-induced conformational change appears to be a critical event
leading to receptor activation or inactivation, it alone cannot explain
the mixed agonist/antagonist properties of RU486 or tamoxifen. Several
reports have documented cell type- and promoter-specific activities of
tamoxifen-bound ER or RU486-bound PR indicating the involvement of
additional regulatory factors in antihormone action (23, 24, 29, 30, 31, 32).
The findings reported in this paper support the hypothesis that steroid
receptors bound to antagonists such as RU486 and 4-hydroxytamoxifen
exist in the cell complexed with an inhibitory factor that
functionally, and perhaps structurally, resembles the nuclear receptor
corepressor NCoR. We provide evidence that NCoR physically interacts
with RU486-complexed PR-B and represses its partial agonist activity.
NCoR, however, belongs to a newly discovered family of
receptor-regulatory proteins, which also incudes SMRT. NCoR and SMRT
are highly related proteins with an overall amino acid identity of 41%
(39). Like NCoR, SMRT also associates with unliganded TR or RAR through
a conserved carboxy-terminal receptor interaction domain, and this
association is destabilized by hormonal ligand (14). Furthermore, like
NCoR, SMRT can functionally replace the corepressor of TR or RAR in
transient transfection experiments (14, 15). Therefore, based on the
accumulated data, we cannot excude the possibility that SMRT or an as
yet undiscovered member of nuclear receptor corepressor family could
function as the inhibitory cofactor that locks RU486-PR-B in a
nonfunctional state.
A steroid receptor typically possesses two distinct
transactivation functions: an amino-terminal constitutive activation
function, AF-1, and a carboxy-terminal hormone-induced activation
function, AF-2 (40, 41). PR-B harbors an additional transactivation
function, AF-3, in its unique amino- terminal segment (42). The present
study shows that in the context of the synthetic GRE/PRE-linked minimal
promoter, GRE/PRE2E1b-TATA, none of the activation
functions of the antagonist-bound, corepressor-complexed PR-B displays
any significant activity. The antagonist-receptor-corepressor complex
is transcriptionally inactive, perhaps due to its inability to undergo
functional interaction with the basal transcription machinery. It is
also conceivable that the antagonist receptor-bound corepressor
recruits a histone deacetylase to the target promoter and thereby
maintains the chromatin in an inactive state (20a, 44). In the presence
of either unliganded TR or antagonist-occupied ER, which presumably
titrates out the corepressor, RU486-PR-B acquires the ability to
transactivate the promoter.
Based on these results, we propose a working hypothesis that a steroid
receptor occupied by a mixed agonist-antagonist, such as RU486 or
4-hydroxytamoxifen, is not an intrinsic transcriptionally nonfunctional
receptor (Fig. 8
). Rather, it is a
receptor in which the transactivation functions remain masked by the
corepressor (Fig. 8B
). Removal of the corepressor by a competing
nuclear receptor unmasks at least one of the activation function(s) of
the antagonist-complexed receptor, which can then interact with the RNA
polymerase II transcription machinery by an unknown mechanism to
partially activate the target promoter (Fig. 8C
). In the proposed
model, binding of the hormone agonist to the receptor triggers
inactivation or dissociation of the corepressor, and the activation
functions of the receptor are then available for interaction with a
coactivator to achieve a fully active state (Fig. 8A
). It is important
to point out that our model is based on studies using two steroid
antagonists, RU486 and 4-hydroxytamoxifen, both of which are known to
promote receptor binding to the HRE and exhibit mixed
agonist/antagonist activity. Whether this model is also valid for a
pure antagonist such as ZK98299, which is thought to block the binding
of PR to the HRE (45), remains to be determined.

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|
Figure 8. Outlines of a Hypothetical Model for
Transcriptional Regulation by a Steroid Receptor Complexed with an
Agonist or a Mixed Agonist/Antagonist
The model depicts DNA-bound PR homodimers, which interact with either
coactivator or corepressor depending on the nature of the ligand bound
to the receptor. A, Binding of progesterone to PR leads to the release
of heat shock proteins, receptor dimerization, and DNA binding. The
hormone- and DNA-bound PR then transactivates the basal promoter via a
coactivator. IIA, IIB, IID, IIE, IIF, IIH, and Pol II denote the
transcription initiation factors TFIIA, TFIIB, TFIID, TFIIE, TFIIF,
TFIIH, and RNA polymerase II, respectively. B, Binding of the
antiprogestin RU486 to PR also leads to dissociation of heat shock
proteins followed by receptor dimerization and DNA binding. RU486-PR,
however, remains complexed with a corepressor and is transcriptionally
nonfunctional because of its failure to interact with the basal
transcription machinery via the coactivator. Note that RU486-PR
possesses a conformation different from that of the agonist-bound
receptor. C, When the corepressor is removed by competition with
unliganded TR or tamoxifen-bound ER, the antagonist-occupied PR
acquires partial but significant agonistic activity. It is unclear how
the RU486-PR interacts with the basal transcription machinery to
achieve this partial gene activation.
|
|
It is presently unknown which of the three AFs of the antagonist-bound
PR-B participates in the activation process as the corepressor is
removed from the receptor. Previous studies reported that in an
antagonist-complexed PR or ER, the AF-2 is inactive while the
amino-terminal constitutive transactivation functions remain active
(23, 24). It is possible that the AF-2 of the antagonist-bound receptor
remains functionally impaired even when the corepressor is removed. In
agreement with this prediction, the LBDs of antagonist-complexed ER and
PR show only weak interactions with a candidate coactivator, SRC-1,
in vitro (16, 17). We noted that the magnitude of
transactivation by RU486-PR-B in the presence of an excess of competing
receptor approached only 2530% of that induced by agonist-bound
receptor (Figs. 4
and 5
). Whether this partial activation is due to
incomplete removal of the corepressor from the antagonist-complexed PR
in competition experiments or reflects the activity of only the
amino-terminal AFs in the unmasked receptor, is unclear at present.
The lack of effect of NCoR on progesterone-dependent transactivation by
PR-B (Fig. 6A
) is somewhat surprising in light of our in
vitro experiments, which indicated weak interactions between NCoR
and progesterone-bound PR-B. Smith et al. (36) have recently
reported that under in vitro conditions, SMRT interacts with
ER irrespective of ligand. Like NCoR, SMRT also fails to affect
estrogen-dependent transactivation by ER, while it strongly represses
partial transactivation by tamoxifen-complexed ER. If interactions
between hormone-bound steroid receptor and NCoR or SMRT occur in
vivo, one would postulate that before transactivation, the
corepressor is likely to be inactivated or displaced from the
agonist-complexed receptor so that the coactivator can interact with
the receptor and mediate its positive regulatory function. We envision
that the ligand-bound receptor exists in a dynamic equilibrium with the
coactivator and the corepressor, and the ultimate direction of the
transcriptional regulation by the receptor in a given cell and promoter
context will be determined by the relative binding affinities and
cellular distributions of these coregulators.
The molecular mechanism by which the antagonist-bound,
corepressor-complexed receptor inhibits the function of the RNA
polymerase II transcription machinery at the target promoter is
unclear. Previous studies in our laboratory and elsewhere indicated
that a complex of unliganded TR or RAR, with a corepressor such as
NCoR, actively represses basal transcription by preventing the assembly
of a functional initiation complex at the target promoter (46, 47). It
is believed that unliganded TR mediates repression by targeting an
early intermediate containing the basal initiation factors TFIID and
TFIIB (46, 47). The corepressor may facilitate basal repression by TR
presumably by stabilizing inhibitory interactions between the receptor
and the basal initiation complexes. In contrast, it is unknown whether
antagonist-complexed ER or PR directly contacts any component of the
basal transcription machinery. Our results suggest that the
RU486-PR-B-NCoR complex actively represses basal transcription from a
progesterone-responsive promoter (Fig. 6B
, lanes 5 and 6). Further
studies are clearly needed to clarify the nature of the molecular
interactions between this inhibitory complex and the RNA polymerase II
transcription machinery. Recent studies indicating that a corepressor
might recruit a histone deacetylase complex to the target promoter to
augment repression on a chromatin template adds another layer of
complexity to this issue (20a, 44).
Ample evidence now exists that the antiprogestin RU486 and the
antiestrogen tamoxifen manifest a wide range of tissue-specific effects
in normal steroid-responsive tissues and anomalous agonist-like effects
in breast tumors during endocrine therapy (25, 48, 49). An important
implication of the present study is that tissue-specific differences in
the distribution of endogenous nuclear receptor corepressors may
determine the response of a target gene to a steroid hormone
antagonist. Although NCoR and SMRT are ubiquitously expressed, the
levels of expression may differ from one tissue to another. The
proposed model predicts that cellular concentrations of corepressors in
a particular tissue may dictate whether RU486 or tamoxifen acts as an
agonist or an antagonist of receptor-mediated gene transcription.
Whereas an excess of corepressors is expected to maintain the
antagonist-bound receptor in a transcriptionally inactive state, an
absence or low levels of this cofactor in a target tissue might trigger
inappropriate agonistic activity of the receptor. Since RU486 and
tamoxifen show tremendous promise as therapeutic agents in disease
states such as hormone-dependent breast cancer and endometriosis, a
clear understanding of the molecular processes by which nuclear
receptor corepressors modulate the gene-regulatory activity of an
antagonist-complexed steroid receptor in normal and tumor cells is
of utmost importance.
 |
MATERIALS AND METHODS
|
---|
Plasmid Construction
The plasmids pCI-TRß and pCI-NCoR expressing human TRß and
murine NCoR, respectively, were constructed by inserting cDNAs encoding
these proteins into vector pCI under the control of human
cytomegalovirus immediate-early promoter (Promega Corp., Madison, WI).
The construction of human PR-B expression vector has been described
previously (37). The reporter construct GRE/PRE2E1b-CAT
contained two copies of a glucocorticoid/progesterone response element
(GRE/PRE) placed in front of a minimal promoter containing TATA box
linked to a chloramphenicol acetyl transferase (CAT) gene (33). The
constructions of GRE/PRE2 E1b-CAT and E1b-CAT were
described previously (33).
Transient Transfection Experiments
CV-1 cells were maintained in DMEM (GIBCO BRL,
Grand Island, NY) supplemented with 5% FBS (Hyclone Laboratories,
Logan, UT). Semiconfluent cells were transiently transfected using the
calcium phosphate coprecipitation procedure as described previously
(50). Briefly, 5 x 105 cells were plated on 10-cm
tissue culture dishes in phenol red-free DMEM containing 5%
charcoal-stripped serum and after 2448 h were transfected with
plasmid DNAs. Typically, cells received 10 µg of CAT reporter plasmid
and 2 µg of an internal control plasmid pSV-ßgal (Promega Corp.),
which contains the gene for ß-galactosidase enzyme. The amount of
cotransfected expression vectors containing PR-B, TRß, and NCoR
varied as indicated for each experiment. After 1214 h of exposure to
the calcium phosphate precipitate, the cells were washed with PBS and
incubated in fresh phenol red-free, serum-free medium with
10-8 M of ligands such as progesterone, RU486,
17ß-estradiol, 4-hydroxytamoxifen, T3, or solvent. Cells
were harvested after 24 h for determination of ß-galactosidase
and CAT activities as described previously (50). The amount of cell
extract used per CAT assay was determined after normalization with
respect to the ß-galactosidase activity. Quantification of the CAT
activities was performed by liquid scintillation analysis of the
acetylated [14C]chloramphenicol product and the remaining
unacetylated substrate. Each transient transfection experiment was
repeated at least three times.
In certain of the experiments (Fig. 6
), CV1 cells were transiently
transfected with the indicated DNAs using Lipofectamine (Life
Technologies, Grand Island, NY) according to the manufacturers
guidelines.
In Vitro Protein-Binding Assays
A recombinant polypeptide, GST-NCoRC', containing the C-terminal
amino acids 20572453 of murine NCoR fused to GST was expressed in
Escherichia coli BL21. 35S-labeled hPR-B or hER
was generated by in vitro transcription-coupled translation
as described previously by Allan et al. (38). An aliquot of
labeled translation mix was treated with either progesterone (1
µM), or RU486 (1 µM), or 17ß-estradiol (1
µM), or 4-hydroxytamoxifen (1 µM), or
ethanol (0.1% vol/vol) at room temperature for 15 min. Ten microliters
of in vitro-translated PR-B treated with either hormone or
antihormone or solvent was incubated with 200 ng of GST-NCoRC'
immobilized on a glutathione resin. Binding was allowed to proceed for
1 h at 4 C. The resin was washed repeatedly with a buffer
containing 40 mM Tris-HCl (pH 7.4), 60 mM NaCl,
15% glycerol, and 0.1% NP-40. The 35S-labeled PR-B or ER
that was retained on the resin was then eluted by boiling with a
denaturing buffer containing SDS, analyzed by SDS-PAGE, and visualized
by fluorography.
 |
ACKNOWLEDGMENTS
|
---|
We acknowledge the excellent technical assistance of Michael
Tanen. We thank Evan Reed for the artwork and Jean Schweis for
carefully reading the manuscript. We gratefully acknowledge Andreas
Horlein and Michael G. Rosenfeld for NCoR cDNA, Ronald M. Evans for
human TRß cDNA, Donald McDonnell for phPR-B, and John Cidlowski for
the reporter plasmid constructs GRE/PRE2E1bTATA and
E1bTATA.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Milan K. Bagchi, The Population Council and the Rockefeller University, Center for Biomedical Research, 1230 York Avenue, New York, New York 10021.
1 Present address: Division of Endocrinology, Cedars-Sinai
Medical Center, Los Angeles, California. 
This work was supported by NIH Grant R01DK-5025702 (to M.K.B) and an
NIH Center Grant P50 HD-1354118. X. Zhang is an NIH Training Grant
Fellow (Grant T32HD-07435).
Received for publication April 30, 1997.
Revision received November 20, 1997.
Accepted for publication January 11, 1998.
 |
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