doi: 10.1128/MCB.20.9.3102-3115.2000.
The opposing transcriptional activities of the two isoforms of the human progesterone receptor are due to differential cofactor binding
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- PMID:10757795
- PMCID: PMC85605
- DOI: 10.1128/MCB.20.9.3102-3115.2000
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The opposing transcriptional activities of the two isoforms of the human progesterone receptor are due to differential cofactor binding
P H Giangrande et al. Mol Cell Biol.2000 May.
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Abstract
The human progesterone receptor (PR) exists as two functionally distinct isoforms, hPRA and hPRB. hPRB functions as a transcriptional activator in most cell and promoter contexts, while hPRA is transcriptionally inactive and functions as a strong ligand-dependent transdominant repressor of steroid hormone receptor transcriptional activity. Although the precise mechanism of hPRA-mediated transrepression is not fully understood, an inhibitory domain (ID) within human PR, which is necessary for transrepression by hPRA, has been identified. Interestingly, although ID is present within both hPR isoforms, it is functionally active only in the context of hPRA, suggesting that the two receptors adopt distinct conformations within the cell which allow hPRA to interact with a set of cofactors that are different from those recognized by hPRB. In support of this hypothesis, we identified, using phage display technology, hPRA-selective peptides which differentially modulate hPRA and hPRB transcriptional activity. Furthermore, using a combination of in vitro and in vivo methodologies, we demonstrate that the two receptors exhibit different cofactor interactions. Specifically, it was determined that hPRA has a higher affinity for the corepressor SMRT than hPRB and that this interaction is facilitated by ID. Interestingly, inhibition of SMRT activity, by either a dominant negative mutant (C'SMRT) or histone deacetylase inhibitors, reverses hPRA-mediated transrepression but does not convert hPRA to a transcriptional activator. Together, these data indicate that the ability of hPRA to transrepress steroid hormone receptor transcriptional activity and its inability to activate progesterone-responsive promoters occur by distinct mechanisms. To this effect, we observed that hPRA, unlike hPRB, was unable to efficiently recruit the transcriptional coactivators GRIP1 and SRC-1 upon agonist binding. Thus, although both receptors contain sequences within their ligand-binding domains known to be required for coactivator binding, the ability of PR to interact with cofactors in a productive manner is regulated by sequences contained within the amino terminus of the receptors. We propose, therefore, that hPRA is transcriptionally inactive due to its inability to efficiently recruit coactivators. Furthermore, our experiments indicate that hPRA interacts efficiently with the corepressor SMRT and that this activity permits it to function as a transdominant repressor.
Figures
hPRA and hPRB repress SHR transcriptional activity by distinct mechanisms. HeLa cells were transiently transfected with 1.3 μg of 3XERE-TATA-LUC, 50 ng of pBKC-βgal, either 103 ng of pBKC-hPRA or 112 ng of pBKC-hPRB, and increasing concentrations of pRST7-ERα (100 ng, 600 ng, or 1,200 ng) or 100 ng of pBKC-TUP1 (CONTROL) plus 1,200 ng of pRST7-ERα. Variable amounts of pBSII-KS were used for a total of 3 μg of DNA. Transcriptional activity of the 3XERE-TATA-LUC reporter was measured 24 h after the addition of 10−8 M 17-β-estradiol alone or in the presence of increasing concentrations of R5020 (A) or RU486 (B). A control was done in the absence of ligands (not shown). The data are presented as percent activation where 100% represents a measure of 17-β-estradiol dependent transactivation by hER in the absence of hPRA or hPRB (n = 2). The average coefficient of variation at each point was <15%. NR, no progestins.
Interaction of hPRA-selective peptides with hPRA and hPRB in vivo. (A) Schematic of the mammalian two-hybrid assay. (B) Sequences of peptides which were fused to the Gal4DBD and used in the mammalian two-hybrid assay. These peptides were isolated by affinity selection from an X7LXXLLX7 phage display library using R5020-activated hPRA. (C) HepG2 cells were transiently transfected with 1,000 ng of 5XGal4-TATA-LUC reporter, 200 ng of pBKC-βgal, either 400 ng of VP16-hPRA, VP16-hPRB, or VP16 alone, and either 400 ng of a vector encoding Gal4DBD alone or Gal4DBD fused to a peptide; 1,000 ng of pBSII-KS was used to bring the total amount of DNA per triplicate up to 3 μg. At 24 h after transfection, cells were treated with no hormone or with 10−7 M R5020 for 24 h. Transcriptional activity was assayed on the 5XGal4-TATA-LUC reporter and represents an indirect measure of the binding of the fusion proteins. Transfections were normalized for efficiency using an internal β-galactosidase control plasmid (pBKC-βgal). The data are represented as fold induction over the no-peptide response for each condition tested, which was set to 1.0. Each data point represents the average of triplicate determinations of the transcriptional activity under the given experimental conditions (n = 3).
hPRA-interacting peptides differentially modulate hPRA and hPRB transcriptional activities. HeLa cells were transiently transfected with 1,500 ng of 3XPRE-TATA-LUC reporter, 50 ng of pBKC-βgal, either 50 ng of pBKC-hPRA or 50 ng of pBKC-hPRB, and increasing amounts of pM-LX-H10 (from 0 to 1,350 ng). Various amounts of pM vector were used for a total of 3,000 ng of DNA per triplicate. Transcriptional activity was assayed following the addition of 10−7 M R5020. Transfections were normalized for efficiency as mentioned above. The data are represented as percent hPR transcriptional activity where 100% represents hPR transcriptional activity in the absence of peptide. Each data point represents the average of triplicate determinations from two separate experiments.
The ID facilitates hPRA's interaction with the corepressor SMRT. (A) Schematic of the mammalian two-hybrid assay. The receptor-interacting domain of SMRT (C'SMRT; aa 981 to 1495) was fused to the Gal4DBD (aa 1 to 147). hPRA, hPRB, or ΔhPRA was fused onto VP16 (VP16 acidic activation domain; aa 411 to 455). The fusion constructs were cotransfected into HeLa cells along with a reporter plasmid containing five copies of a Gal4-responsive element (Gal4-RE) upstream of the luciferase gene. (B) HeLa cells were transiently transfected with 0.5 μg of 5XGal4-TATA-LUC, 50 ng of pBKC-βgal, 1 μg of pCMX-Gal4-C'SMRT (Gal4-C'SMRT), 1 μg of either pVP16-hPRB, pVP16-hPRA, or pVP16-ΔhPRA, and 0.45 μg of pBSII-KS. Transcriptional activity was assayed on the 5XGal4-TATA-LUC reporter and represents an indirect measure of the binding of the fusion proteins. Transcriptional activity was measured following the addition of increasing concentrations of R5020, RU486, or ZK98299. Transfections were normalized for efficiency using an internal β-galactosidase control plasmid (pBKC-βgal). The data are represented as fold induction over the no-hormone (No L) response, which was set to 1.0. Each data point represents the average of triplicate determinations of the transcriptional activity under the given experimental conditions (n = 2).
hPRA has a higher affinity for the corepressor SMRT than hPRB. For GST pull-down assays, the fusion protein GST-C'SMRT, containing the carboxyl terminus of SMRT fused to GST, was immobilized on glutathione beads and incubated at 4°C for 24 h with either in vitro-translated hPRA or hPRB, in the presence of either vehicle (NH), R5020, or RU486 (A) or baculovirus-purified receptors bound to RU486 (B). An equimolar amount of GST was used as a negative control for each condition tested. Following incubation at 4°C, the unbound proteins were removed with five washes of NENT-B buffer. The bound receptors were subsequently visualized by Western analysis using a polyclonal antibody against PR. (C) Equal amounts of BSA and either baculovirus-purified hPRA or hPRB bound to RU486 were immobilized onto 96-well plates and incubated in the presence of increasing concentrations of bacterially purified GST-C'SMRT. Following incubation at 4°C for 24 h, the unbound fusion protein was removed by washing five times with NENT-B buffer. The amount of bound GST-C'SMRT was determined by ELISA. The response was measured at 405 nm after 30 min of incubation with ABTS plus 0.05% H2O2. The OD405 readings for hPRA and hPRB were normalized by subtracting those obtained with the BSA control and subsequently setting the highest reading value to OD405 = 1. The data were fitted to a two-site binding curve, and the values for each curve are reported. Hill-1 and Hill-2 for hPRA = 3.66 and 0.33, respectively; Hill-1 and Hill-2 for hPRB = 2.49 and 0.43 respectively.ymax-1 andymax-2 for hPRA = 0.272 and 0.098 respectively;ymax-1 andymax-2 for hPRB = 0.274 and 0.052, respectively.
C'SMRT can partially reverse hPRA-mediated repression of hER transcriptional activity. HeLa cells were transiently transfected with 1 μg of 3XERE-TATA-LUC, 50 ng of pBKC-βgal, 450 ng of pRST7-ERα, 300 ng of pBKC-hPRA, and increasing concentrations (ranging from 0 to 1.2 μg) of either Gal4-SMRT (A), Gal4-C'SMRT (B), or ΔN4, used as a control (C). Various amounts of pBKC-DBD were added to balance the amount of input Gal4DBD. Transcriptional activity of the 3XERE-TATA-LUC reporter was measured 24 h after the addition of 10−7 M 17-β-estradiol and 10−7 M RU486. A control was done in the absence of ligands (not shown). The data are presented as percent activation where 100% represents a measure of 17-β-estradiol-dependent transactivation by hER in the absence of RU486 (Cont). The average coefficient of variation at each point was <12% (n = 2).
The ability of C'SMRT to reverse hPRA-mediated transrepression is ligand dependent. HeLa cells were transiently transfected as for Fig. 4. Transcriptional activity was measured 24 h after the addition of 10−7 M 17-β-estradiol and either 10−7 M R5020, 10−7 M RU486, or 10−7 M ZK98299. A control was done in the absence of ligands (not shown). The data are presented as percent activation where 100% represents a measure of 17-β-estradiol-dependent transactivation by hER in the absence of progestins or antiprogestins (Cont) for each experimental condition. The average coefficient of variation at each point was <12%. The data from a single representative experiment are shown (n = 3).
The deacetylase inhibitor TSA can partially reverse hPRA-mediated repression of hER transcriptional activity. HeLa cells were transiently transfected with 1.5 μg of 3XERE-TATA-LUC, 50 ng of pBKC-βgal, 500 ng of pRST7-ERα, and either 481 ng of pBKC-hPRA or 467 ng of pBKC-Rev-TUP1 (not shown). Variable amounts of pBSII-KS were used for a total of 3 μg of DNA. Transcriptional activity of the 3XERE-TATA-LUC reporter was measured 24 h after the addition of 10−7 M 17-β-estradiol and 10−7 M RU486, alone or in combination with increasing concentrations of TSA (0, 10−8, 10−7, and 10−6 M). A control was done in the absence of ligands (not shown). The data are presented as percent activation where 100% represents a measure of 17-β-estradiol-dependent transactivation by hER in the absence of RU486 (CONT). The data from one representative experiment are shown (n = 2). The average coefficient of variation at each point was <10%.
Inactivation of the nuclear receptor silencer SMRT does not convert hPRA to a transcriptional activator. (A) HeLa cells were transiently transfected with 1.5 μg of 2XPRE-TK-LUC, 50 ng of pBKC-βgal, either 52 ng of pBKC-hPRB, 48 ng of pBKC-hPRA, or 46 ng of pBKC-RevTUP1, and increasing concentrations (from 0 to 1 μg) of Gal4-C'SMRT. Various amounts of pBKC-DBD were added to balance the amount of input Gal4DBD. pBSK-II was added to normalize the total DNA to 3 μg. The transcriptional activity of these vectors was assayed on a 2XPRE-TK-LUC reporter and measured after the addition of 10−7 M R5020. Transfections were normalized for efficiency as mentioned previously. R5020-mediated transcriptional activity in the presence of increasing concentrations C'SMRT was normalized to the no-ligand control for each concentration of C'SMRT used. Each data point represents the average of triplicate determinations (± standard error of the mean) from two separate experiments (n = 2). The control represents basal reporter activity in the presence of control vector and was set to 100%. (B) HeLa cells were transiently transfected with 1.5 μg of 2XPRE-TK-LUC, 50 ng of pBKC-βgal, either 50 ng of pBKC-hPRA or 48 ng of pBKC-Rev-TUP1, and various amounts of pBSK-II for a total of 3 μg. Transcriptional activity of the constructs was measured following the addition of 10−7 M R5020 alone or in combination with increasing concentrations (0, 10−8, 10−7, and 10−6 M) of the deacetylase inhibitor TSA. Transfections were normalized for efficiency as mentioned above. R5020-mediated transcriptional activity in the presence of increasing concentrations of TSA was normalized to the no-ligand control for each TSA treatment used. Each data point represents the average of triplicate determinations (± standard error of the mean) from two separate experiments (n = 2).
hPRA interacts weakly with the NR boxes of the coactivator proteins GRIP1 and SRC-1. (A) HeLa cells were transiently transfected with 0.5 μg of 5XGal4-TATA-LUC, 50 ng of pBKC-βgal, 1 μg of either pM-GRIP1(NR) or pM-SRC-1(NR), 1 μg of either pVP16-T, pVP16-ERα, pVP16-hPRB, pVP16-hPRA, or pVP16-ΔhPRA, and 0.45 μg of pBSII-KS. Transcriptional activity of the luciferase gene was assayed on the 5XGal4-TATA-LUC reporter as in Fig. 2B. Transcriptional activity was measured following the addition of 10−7 M R5020 for PR or 10−7 M 17-β-estradiol for ERα (H); a control was done in the absence of ligands (NH). Transfections were normalized for efficiency as mentioned previously. The data are represented as fold induction over the control interaction between Gal4-GRIP1(NR) or Gal4–SRC-1(NR) and VP16-T for each ligand treatment group, which was normalized to 1.0. Each data point represents the average of triplicate determinations from three separate experiments. The average coefficient of variation at each point was <10% (n = 3). (B and C) GST pull-down assays. The fusion proteins GST-GRIP1(NR) (top) and GST-SRC-1(NR) (bottom) were immobilized on glutathione beads and incubated at 4°C for 24 h with in vitro-translated35S-hPRA or35S-hPRB in the presence of vehicle (NH), R5020, or RU486 (B) or baculovirus-purified hPRB bound to R5020 or RU486 (C). The bound baculovirus-purified receptors were analyzed by Western analysis using a polyclonal antibody against PR. An equimolar amount of GST alone was used as a negative control for each condition tested.
hPRA does not associate with the ERα transcription complex. HeLa cells were transiently transfected with 500 ng of 5XGal4-TATA-LUC, 50 ng of pBKC-βgal, 1,000 ng of pM-hPRA, and either 1,000 ng of pVP16-T (control), pVP16-hPRA, or pVP16-ERα (gray bars). Transcriptional activity was assayed on the 5XGal4-TATA-LUC reporter following the addition of 10−7 M 17-β-estradiol or 10−7 M R5020 and represents an indirect measure of the binding of the fusion proteins. Transfections were normalized for efficiency as mentioned above. The data are represented as fold induction over the control interaction between Gal4-hPRA and VP16-T in the absence of ligands, which was normalized to 1.0 (black bars). Each data point represents the average of triplicate determinations from three separate experiments.
Two distinct models are required to describe the molecular mechanism of action of hPRA. (A) Transcriptional activation. Based on the in vivo and the in vitro binding studies, we propose that hPRA interacts more efficiently with corepressors and less efficiently with coactivators than hPRB. In the presence of hormone, hPRB, but not hPRA, undergoes a favorable conformational change which allows it to displace corepressors (CoR) and recruit coactivator proteins (CoA), thus allowing hPRB to activate transcription from progesterone-responsive promoters. HD, histone deacetylase; A, hPRA; B, hPRB. (B) Transrepression. Based on our in vivo transrepression data, we propose that hPRA transrepresses ERα-mediated transcription by a transcriptional interference mechanism. In this model, ERα activates transcription by recruiting a complex of coactivator proteins (ERα CoA complex) to the regulatory region of target genes. hPRA (A), but not hPRB (B), targets and sequesters a member of the ERα CoA complex, thus preventing ERα from activating transcription. hPRA transrepression of ERα transcriptional activity is further enhanced by the recruitment by hPRA of the corepressor SMRT (CoR).
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