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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 2–4). 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 254–312 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 1829–1940 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).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1Go, 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. 1Go, lanes 3, 5, and 7; Fig. 2Go, 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. 1Go, 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.

 
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. 2Go, 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 168–260 containing the D domain (positions 170–238) and the N-terminal region (positions 238–260) 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 145–260 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 203–230 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 230–260.

We therefore examined whether the peptide 145–260 of TRß containing the corepressor-binding region can induce transactivation by antiprogestin-bound PR-B. As shown in Fig. 3Go, when we coexpressed the entire LBD fragment (145–456) 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 145–256 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 (145–456) (5 µg), lanes 5 and 6); pCI-TR (145–260) (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.

 
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. 4Go, 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 25–30% 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.

 
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. 5Go, 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. 5Go, 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. 5Go, 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. 5Go, 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.

 
We then analyzed the effect of overexpression of NCoR on transcriptional activity of agonist-bound PR-B. As shown in Fig. 6Go, 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.

 
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 2057–2453 of NCoR that harbor a nuclear receptor-interaction domain (12, 20). The results of this experiment are shown in Fig. 7AGo. 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 2057–2453 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 2–6 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.

 
We also assessed the ability of the carboxy terminus 2057–2453 of NCoR to interact with ER. As shown in Fig. 7BGo, 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).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 O’Malley 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. 8Go). Rather, it is a receptor in which the transactivation functions remain masked by the corepressor (Fig. 8BGo). 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. 8CGo). 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. 8AGo). 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 25–30% of that induced by agonist-bound receptor (Figs. 4Go and 5Go). 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. 6AGo) 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. 6BGo, 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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 24–48 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 12–14 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. 6Go), CV1 cells were transiently transfected with the indicated DNAs using Lipofectamine (Life Technologies, Grand Island, NY) according to the manufacturer’s guidelines.

In Vitro Protein-Binding Assays
A recombinant polypeptide, GST-NCoRC', containing the C-terminal amino acids 2057–2453 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. Back

This work was supported by NIH Grant R01DK-50257–02 (to M.K.B) and an NIH Center Grant P50 HD-13541–18. 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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