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. 2010 Mar;30(6):1434-45.
doi: 10.1128/MCB.01002-09. Epub 2010 Jan 11.

Estrogen receptors recruit SMRT and N-CoR corepressors through newly recognized contacts between the corepressor N terminus and the receptor DNA binding domain

Natalia Varlakhanova  1 , Chelsea Snyder, Soumia Jose, Johnnie B Hahm, Martin L Privalsky
Affiliations

Estrogen receptors recruit SMRT and N-CoR corepressors through newly recognized contacts between the corepressor N terminus and the receptor DNA binding domain

Natalia Varlakhanova et al. Mol Cell Biol. 2010 Mar.

Abstract

Estrogen receptors (ERs) are hormone-regulated transcription factors that regulate key aspects of reproduction and development. ERs are unusual in that they do not typically repress transcription in the absence of hormone but instead possess otherwise cryptic repressive functions that are revealed upon binding to certain hormone antagonists. The roles of corepressors in the control of these aspects of ER function are complex and incompletely understood. We report here that ERs recruit SMRT through an unusual mode of interaction involving multiple contact surfaces. Two surfaces of SMRT, located at the N- and C-terminal domains, contribute to the recruitment of the corepressor to ERs in vitro and are crucial for the corepressor modulation of ER transcriptional activity in cells. These corepressor surfaces contact the DNA binding domain of the receptor, rather than the hormone binding domain previously elucidated for other corepressor/nuclear receptor interactions, and are modulated by the ER's recognition of cognate DNA binding sites. Several additional nuclear receptors, and at least one other corepressor, N-CoR, share aspects of this novel mode of corepressor recruitment. Our results highlight a molecular mechanism that helps explain several previously paradoxical aspects of ER-mediated transcriptional antagonism, which may have a broader significance for an understanding of target gene repression by other nuclear receptors.

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Figures

FIG. 1.
FIG. 1.
Effect of SMRTα overexpression on transcriptional activity of ERα. A pMMTV-2xERE-luciferase reporter was cotransfected into CV1 cells together with 10 ng of pSG5.1-SMRTα (full length) or an equivalent empty vector and with 10 ng of pSG5.1-ERα or an equivalent empty vector. Five nanograms of a constitutive pCMV-lacZ reporter was included in each transfection mixture as an internal control. After 24 h, the cells were transferred into medium containing ethanol vehicle only, 1 μM 17-β-estradiol (E2), 1 μM 4-hydrotamoxifen (Tam), or both 1 μM 17-β-estradiol and 1 μM 4-hydrotamoxifen, as indicated. The level of expression of the luciferase reporter was determined 24 h later, normalized relative to the β-galactosidase control, and is presented as relative luciferase activity. The means and standard deviations for two experiments are presented.
FIG. 2.
FIG. 2.
Interaction of SMRTα with ERα through an N-terminal corepressor domain. (A) Schematic of SMRTα. The transcriptional repression and nuclear receptor interaction domains are indicated, as are binding sites for additional proteins in the corepressor complex and the S1 and S2 CoRNR box motifs. Codons are numbered from the N terminus. HDAC, histone deacetylase. (B) Mapping of the ERα interaction domain in SMRTα. GST-ERα or GST alone was immobilized on glutathione-agarose and incubated with the 35S-labeled SMRTα protein constructs shown. SMRTα proteins remaining bound to the GST proteins after washing were resolved by SDS-PAGE gel/PhosphorImager analysis. A representative electrophoretogram is presented for each SMRT construct, showing 10% of the input, the SMRT protein bound to GST only, and the SMRTα protein bound to GST-ERα; a quantification of the binding relative to the input is presented below. (C) Reciprocal mapping of the SMRTα interaction domain in ERα. GST only or a GST-SMRTα(1-289) construct was immobilized on glutathione beads and incubated with full-length 35S-labeled ERα or the ERα subdomains indicated below the panel. The amount of ERα construct remaining bound to the GST proteins after washing was quantified by SDS-PAGE gel/PhosphorImager analysis relative to the input (formula image100%). The means and standard errors for three experiments are shown. All assays were performed in the absence of hormone ligand. (D) Coimmunoprecipitation of SMRT proteins and ERα from cells. Hemagglutinin (HA)-SMRTα, HA-SMRTα(1-289), HA-SMRTα(1210-2470), or the empty HA tag expression vector (HA) was transfected into HeLa cells together with an ERα expression vector. The cells were lysed, the lysates were immunoprecipitated with either nonimmune (“Normal IgG”) or anti-HA antibodies, and the immunoprecipitates were resolved by SDS-PAGE and were visualized using anti-ERα sera.
FIG. 3.
FIG. 3.
Interaction of both N- and C-terminal domains of SMRTα with the DNA binding “C” domain of ERα. (A) Schematic of SMRTα and ERα. Labels are as described in the legend of Fig. 2. (B) Mapping of the ERα and SMRTα interaction sites. GST fusions of ERα or the ERα subdomains indicated were immobilized on glutathione-agarose and incubated with the 35S-labeled SMRTα constructs shown. The amount of each SMRTα construct remaining bound to the GST proteins after washing was quantified by SDS-PAGE gel/PhosphorImager analysis relative to the input. The means and standard deviations for two or more experiments are shown. All assays were performed in the absence of hormone ligand.
FIG. 4.
FIG. 4.
Contributions of CoRNR box motifs to the SMRTα interaction with ERα. (A) CoRNR motifs in SMRT and N-CoR. CoRNR motifs present in the cRID domain of SMRT include the S1 and S2 motifs in SMRTα and the S3 motif present in the alternatively spliced SMRTγ variant. Mutations are indicated below each wild-type (wt) sequence. The codon numbering systems shown are for N-CoR and for the SMRTγ variant. (B) Requirement for the nRID CoRNR motif in the SMRTα interaction with ERα. GST only or a GST-SMRTα(1-289) construct containing the wild-type or mutant CoRNR motif indicated was assayed for the ability to bind to full-length 35S-labeled ERα using the protocol described in the legend of Fig. 2C. The means and standard deviations for two or more experiments are shown. (C) Presence of a functional nRID in N-CoR. GST only or GST-ERα was immobilized on glutathione beads and incubated with a 35S-labeled N-CoR(1-500) protein construct. The amount of N-CoR(1-500) remaining bound to the GST proteins after washing was quantified by SDS-PAGE gel/PhosphorImager analysis relative to the input. The means and standard deviations for two or more experiments are shown. (D) Requirement for the S1 CoRNR motif in the interaction of the SMRT cRID with ERα. GST only or GST-SMRTα constructs containing the wild-type or mutant cRID CoRNR motifs indicated were immobilized on glutathione beads and incubated with full-length 35S-labeled ERα or with the 35S-labeled ERα subdomains indicated. The amount of ERα construct remaining bound to the GST proteins was determined as described above (B). The means and standard errors for two or more experiments are shown. All assays were performed in the absence of hormone ligand.
FIG. 5.
FIG. 5.
Effect of hormone ligand on ERα binding to SMRTα. (A) Effect of hormone on binding of full-length (FL) ERα to full-length SMRTα. The experiment described in the legend of Fig. 2B was repeated in the presence of ethanol carrier alone (−), 1 μM estradiol (E2), or 1 μM tamoxifen (Tam). (B) Effect of hormone on the interaction of different domains of ERα and SMRTα. The experiment described in the legend of Fig. 2C was repeated for the GST-SMRTα and 35S-ERα domains indicated, in the presence of ethanol carrier alone (−), 1 μM estradiol (E2), or 1 μM tamoxifen (Tam). Representative electrophoretograms are shown.
FIG. 6.
FIG. 6.
Inhibition of the SMRT interaction with ERα by estrogen response elements (EREs). (A) Binding of the SMRTα nRID to ERα. A GST-SMRTα(1-289) construct was incubated with 35S-radiolabeled ERα in the presence of ethanol carrier (none), 1 μM 17-β-estradiol (E2), or 1 μM 4-hydrotamoxifen (Tam). Binding reaction mixtures also included 125 nM oligonucleotides representing the estrogen response element from the vitellogenin promoter (Vit-ERE), the estrogen response element from the pS2 gene promoter (pS2-ERE), or the binding element for an irrelevant transcription factor (RBP-Jk). The amount of ERα remaining bound to the GST protein after washing was quantified by SDS-PAGE gel/PhosphorImager analysis relative to the input. The means and standard errors for two or more experiments are shown. (B) Binding of the SMRTα cRID to ERα. The ability of a GST-SMRT(2050-2470) fusion to bind to 35S-labeled ERα in the presence of different hormone ligands and DNA binding sites was determined by the same methodology as that described above (A). (C) Binding of the SMRTα cRID to TRα. The ability of a GST-SMRT(2050-2470) fusion to bind to 35S-labeled TRα-1 in the presence of different DNA binding sites was determined as described above (A). Incubations were performed in the presence of ethanol carrier. In addition to the DNA oligonucleotides listed in panel B, a thyroid hormone response element from the F2 lysozyme promoter (cLys-F2) was also included. (D) Binding of full-length SMRTα to ERα. The ability of a GST-ERα (full-length) fusion to bind 35S-radiolabeled SMRTα(1-2470) in the presence of different hormone ligands and DNA binding sites was determined by the same methodology as that described above (A).
FIG. 7.
FIG. 7.
Requirement for both nRID and cRID in SMRTα repression of ERα function in cells. (A) Loss of SMRTα repression of ERα due to N- or C-terminal corepressor deletions. The transfection experiment described in the legend to Fig. 1 was repeated using full-length SMRTα(1-2470), a SMRTα protein lacking the nRID domain [SMRTα(310-2470)], a SMRTα protein lacking the cRID domain [SMRTα(1-1968)], or an empty expression vector (none). The cells were incubated with ethanol carrier only, 1 μM estradiol (E2),1 μM tamoxifen (Tam), or both E2 and Tam; harvested; and assayed for relative activity as described in the legend to Fig. 1. (B) Reduced SMRTα repression of ERα due to amino acid substitutions in the nRID CoRNR box. The protocol described above (A) was repeated by using wild-type SMRTα(1-2470) or SMRTα(1-2470) bearing the AAAA or ISEVI nRID substitution described in the legend to Fig. 4A. The relative luciferase activity in the absence of SMRT was defined as 100. (C) Loss of SMRTα repression of TRα due to a C-terminal but not an N-terminal corepressor deletion. The transfection experiment described above (A) was repeated by using a TRα1 expression vector, a TRE-luciferase reporter, and an ethanol carrier (none). Reporter activity in the absence of any additional constructs was defined as “1.” (D) Dominant negative activity of the SMRTα nRID and cRID. The transfection experiment described above (A) was repeated using full-length (FL) SMRTα, SMRTα(1-427), SMRTα(2050-2470), full-length SMRTα plus SMRTα(1-427), or full-length SMRTα plus SMRTα(2050-2470), as indicated. The means and standard errors for two or more experiments are shown.
FIG. 8.
FIG. 8.
Interaction of the SMRTα nRID with a panel of nuclear receptors. A GST-SMRTα(1-289) construct was immobilized on glutathione-agarose beads and incubated with the 35S-labeled nuclear receptors indicated, in the absence or presence of cognate ligand (estradiol, tamoxifen, all-trans-retinoic acid, rosiglitazone, chenodeoxycholate, or gluggulsterone). The amount of each nuclear receptor remaining bound to the GST proteins after extensive washing was quantified by SDS-PAGE gel/PhosphorImager analysis and is presented relative to the input (formula image100%). The means and standard deviations for two experiments are shown.
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