Defective Release of Corepressor by Hinge Mutants of the Thyroid Hormone Receptor Found in Patients with Resistance to Thyroid Hormone

Abstract

On positive thyroid hormone response elements (pTREs), thyroid hormone receptor (TR) binding to DNA in the absence of ligand (thyroid hormone, T3) decreases transcription (silencing). Silencing is due to a family of recently described nuclear corepressor proteins (NCoR and SMRT) which bind to the CoR box in the hinge region of TR. Ligand-dependent activation of TR is associated with displacement of corepressors and recruitment of coactivating proteins. Resistance to thyroid hormone (RTH) is due to mutations in the β isoform of the thyroid hormone receptor (TR-β). To date, three RTH mutations reportedly with near-normal T3binding (A234T, R243Q, and R243W) have been described in or near the CoR box. To determine the mechanism of RTH caused by these mutants, the interaction of wild type (wt) and mutant TRs with the corepressor, NCoR, and the coactivator, SRC-1, was tested in gel-shift assays. As expected, NCoR bound wt TR in the absence of T3 and dissociated from TR with increasing T3 concentration. SRC-1 failed to bind wt TR in the absence of T3, but bound to TR with increasing avidity as T3 concentrations rose. At no T3 concentration did both NCoR and SRC-1 bind to wt TR, indicating that their binding to TR was mutually exclusive. Hinge mutants bound NCoR normally in the absence of T3; however, dissociation of NCoR and recruitment of SRC-1 was markedly impaired except at very high T3 concentrations. Importantly, hinge mutant TRs when complexed to DNA bound T3 poorly despite their near-normal T3 binding in solution. These binding studies correlated with functional assays showing defective transactivation of pTREs by hinge mutants except at high T3concentrations. Thus, we describe a novel mechanism of RTH whereby TR hinge mutants selectively affect T3 binding when complexed to DNA, and prevent NCoR dissociation from TR. Our data also suggest that solution T3 binding by RTH mutants may not accurately reflect physiologically relevant T3 binding by TR when bound to DNA. On positive thyroid hormone response elements (pTREs), thyroid hormone receptor (TR) binding to DNA in the absence of ligand (thyroid hormone, T3) decreases transcription (silencing). Silencing is due to a family of recently described nuclear corepressor proteins (NCoR and SMRT) which bind to the CoR box in the hinge region of TR. Ligand-dependent activation of TR is associated with displacement of corepressors and recruitment of coactivating proteins. Resistance to thyroid hormone (RTH) is due to mutations in the β isoform of the thyroid hormone receptor (TR-β). To date, three RTH mutations reportedly with near-normal T3binding (A234T, R243Q, and R243W) have been described in or near the CoR box. To determine the mechanism of RTH caused by these mutants, the interaction of wild type (wt) and mutant TRs with the corepressor, NCoR, and the coactivator, SRC-1, was tested in gel-shift assays. As expected, NCoR bound wt TR in the absence of T3 and dissociated from TR with increasing T3 concentration. SRC-1 failed to bind wt TR in the absence of T3, but bound to TR with increasing avidity as T3 concentrations rose. At no T3 concentration did both NCoR and SRC-1 bind to wt TR, indicating that their binding to TR was mutually exclusive. Hinge mutants bound NCoR normally in the absence of T3; however, dissociation of NCoR and recruitment of SRC-1 was markedly impaired except at very high T3 concentrations. Importantly, hinge mutant TRs when complexed to DNA bound T3 poorly despite their near-normal T3 binding in solution. These binding studies correlated with functional assays showing defective transactivation of pTREs by hinge mutants except at high T3concentrations. Thus, we describe a novel mechanism of RTH whereby TR hinge mutants selectively affect T3 binding when complexed to DNA, and prevent NCoR dissociation from TR. Our data also suggest that solution T3 binding by RTH mutants may not accurately reflect physiologically relevant T3 binding by TR when bound to DNA. thyroid hormone receptor thyroid hormone response element positive thyroid hormone response element thyroid hormone receptor beta isoform triiodothyronine nuclear corepressor nuclear corepressor inhibitor steroid receptor coactivator-1 resistance to thyroid hormone wild type direct repeat response element with 4-base pair separation chicken lysozyme response element nuclear binding domain-1 glutathione S-transferase retinoid X receptor α isoform retinoid X receptor β isoform. Thyroid hormone receptor (TR)1 is a member of the steroid/thyroid hormone receptor superfamily and regulates the expression of many target genes through its ability to bind to thyroid hormone response elements (TREs) (1Mangelsdorf D.J. Evans R.M. Cell. 1995; 83: 841-850Abstract Full Text PDF PubMed Scopus (2843) Google Scholar, 2Mangelsdorf D.J. Thummel C. Beato M. Herrlich P. Schutz G. Umeseno K. Blumberg B. Kastner P. Mark M. Chambon P. Evans R.M. Cell. 1995; 83: 835-839Abstract Full Text PDF PubMed Scopus (6110) Google Scholar). TR contains a variety of protein domains involved in DNA binding, hormone binding, receptor dimerization, and interaction with the basic transcription machinery. On positive response elements (pTREs), TR binding to DNA in the absence of ligand (thyroid hormone, T3) decreases transcription (silencing) (3Sap J. Munoz A. Schmitt H. Stunnenberg H. Vennstrom B. Nature. 1989; 340: 242-244Crossref PubMed Scopus (297) Google Scholar). 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J.K. 1996; Google was a expression in a A which the was to The binding wt and mutant TRs in solution with a binding T3 A. J. M. S. PubMed Scopus Google Scholar). a T3 binding DNA was also a gel-shift except T3 As in 1, three mutations in (A234T, R243Q, and These mutations a in and to the expression in TR binding to T3 in solution is not impaired with mutant a in T3 These results of the T3 binding of these mutant M. U. Mol. Endocrinol. 1992; Google Scholar, K. H. A. T. Y. K. K. A. 1995; PubMed Scopus Google Scholar, H. J. Y. A. Refetoff S. J. Endocrinol. 1997; PubMed Google T3 binding to hinge mutant TRs in binding of are the a of the three tested hinge mutant TRs to wt TR. in a are the a of the three tested hinge mutant TRs to wt TR. mutant TR binding to the nuclear corepressor, NCoR, a gel-shift The three hinge mutants (A234T, R243Q, and a mutant A. Tsai S.Y. O'Malley B.W. Tsai M.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: PubMed Scopus Google and hinge mutant to not with corepressor binding A.J. Naar A.M. Heinzel T. Torchia J. Gloss B. Kurokawa R. Ryan A. Kamel Y. Soderstrom M. Glass C.K. Rosenfeld M.G. Nature. 1995; 377: 397-404Crossref PubMed Scopus (1714) Google tested in A of NCoR, the TR domains but the termed was in the A.N. Monden T. Madura J.P. Lee K. Wondisford F.E. J. Biol. Chem. 1996; 271: 28516-28520Abstract Full Text Full Text PDF PubMed Scopus (86) Google TRs a in the absence of a Addition of in caused the to to a and of the of to the binding the of binding of to of the mutants was to wt TR Addition of or to the binding in the of a and a in the not In as in T3 dissociation of from mutant TRs was from wt TR the wt TR, T3 in dissociation of the The hinge mutant with T3 binding, a dissociation The mutant however, concentration of T3 to did wt TR. The and mutants and T3 concentration to wt TR to the which bind T3, not dissociated from at the T3 concentration. the chicken lysozyme and not On the a T3 concentration was to from wt TR of from and was to the and mutant TRs a did wt TR As expected, not dissociated from the mutant at very high T3 is a as in of TRs with NCoR binding a of T3 concentrations as Hinge mutant TRs T3 to the of dissociation as TRs are and To hinge mutants normally SRC-1, the of SRC-1 A. Yen P.M. Misiti S. Cardona G.R. Liu Y. Chin W.W. Endocrinology. 1996; 137: 3594-3597Crossref PubMed Scopus (147) Google Scholar, Y. Qi C. C. J.K. 1996; Google was to the protein and in and in a gel-shift of wt and mutant TR binding to a In the absence of T3, SRC-1 did not a with wt TR A wt was at and was at These results T3 is to the TR and prevent its binding to DNA. The presence of a that the in the binding of TR DNA in the presence of The three hinge mutants also recruited the of SRC-1 with increasing T3 concentrations. The mutant to wt TR in its ability to SRC-1 the T3 concentrations The and mutants T3 to wt TR, to to In these in the presence of was to bind to both wt TR and wt as by the in of both and the presence of a of not however, the in SRC-1 caused by the hinge mutants was to the data Thus, hinge mutants a in dissociation from to the of avidity of TRs SRC-1 is not by the presence of The TRs with and SRC-1 the of T3 concentrations The hinge mutants in recruitment of SRC-1 with the T3 concentration as that by wt TR. are and To determine NCoR binding with SRC-1 the gel-shift assays TR domains from both In the wt TR and hinge mutants, and are the and at a that the the in and the protein to the In the absence of T3 to bind to the wt TR, was and the and of the was not by of the at high T3 concentration bind to TR, a was which was by the of the A from the to the was the tested no was a with binding both and to wt TR. as the the from the to the was at a T3 concentration of both and of binding wt TR when tested At concentration and in the absence of the wt TR bound in a with its binding to Addition of the the of the to a the with of the these data suggest that of from the TR in solution to bind to the wt TR. The result of the impaired dissociation from hinge mutants is also in and at T3 was dissociated from the hinge mutants to the of the The of that at T3 the hinge mutants bound to At T3, bound to the hinge mutants dissociation of the by the result is with the data showing that the mutant recruited at T3 concentrations the data was with the mutant Thus, in NCoR dissociation by T3, as with the hinge mutants, SRC-1 is the hinge mutants in dissociation from in the absence of in solution binding to the and mutants T3 concentrations to did the mutant despite the that these very T3 binding To the gel-shift assays in or the wt and mutant In and the to except and of was In TRs R243Q, and with and in and of In the was except that the was by a and was As by M. R. V. E. C. E. K. G. L. R.J. Parker M.G. Chatterjee V.K.K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: PubMed Scopus Google the wt was by T3 and the was not despite of these the result as indicating that of TR by T3 the which is not dissociated by T3, T3 bound to TR is to the and a at that of the of T3 the we not determine the wt was The the mutant was by T3 by and the R243Q, and mutants not the mutant not to bind T3; however, results with the hinge mutants their solution T3 These results that solution and binding of T3 by hinge mutants of the TR not As a thyroid hormone of a positive thyroid hormone response element of a in assays of On the element activation by the wt TR was at T3 and a at T3 of element by the hinge mutant was to wt TR not The hinge mutants defective in activation with wt TR. by the mutant was at T3, transactivation by the mutant TRs was at At very high T3 however, transactivation by mutants except that of wt TR. hinge mutant wt TR the element not results with the element from the chicken lysozyme of NCoR inhibitor expression pTREs A.N. Monden T. Madura J.P. Lee K. Wondisford F.E. J. Biol. Chem. 1996; 271: 28516-28520Abstract Full Text Full Text PDF PubMed Scopus (86) Google we to determine of the in transactivation with the hinge In of by the wt TR the element and of activation in the presence of corepressors (NCoR and SMRT) and are from wt TR at T3 by corepressors is selectively by recruitment and In in are data with of the hinge mutants, that but did not transactivation except at Importantly, these studies are with gel-shift studies showing that TR hinge mutants are markedly defective in their ability to and SRC-1 Our of hinge mutations of the TR of transactivation and the RTH hinge mutants of the TR a defective of NCoR in response to with studies that both mutants, and to a the bound to NCoR at concentrations of NCoR from wt TR. S.M. Chatterjee V.K.K. Privalsky M.L. Mol. Endocrinol. 1997; 11: 470-480Crossref PubMed Scopus (160) Google that RTH mutants interaction with nuclear As expected, mutations which bound failed to from corepressor except at very high T3 concentrations. Our results with the are in with their mutants located in the and to have a in dissociation from corepressor due to their in The that a in the terminus of TR caused by the by T3 binding from corepressor to with the hinge These mutants solution T3binding a failed to from NCoR in gel-shift and to silence transcription in studies except at very high T3 concentrations. Importantly, however, we the ability of these mutants to bind to T3 when complexed to DNA. To these mutants as bound T3 at in Our results are to the TR not in due to the dissociated when bound by T3, and T3 the these data suggest that binding of T3 in solution and DNA not as by mutations in the hinge region of TR. the of hinge mutants affect T3 binding R.L. J.W. J.D. R.J. Nature. 1995; PubMed Scopus Google Scholar). These mutations are near which is to the which binds of the hinge are to DNA M.J. O'Malley B.W. 1994; PubMed Scopus Google Scholar, F. T. Evans R.M. Nature. 1995; PubMed Scopus Google and to mediate ligand binding C. P. Mol. Endocrinol. PubMed Scopus Google is that a in the mutant hinge as to prevent ligand to the bound TR or prevent of the TR. The result is that corepressor to bind to TR and prevent transactivation despite the presence of T3 in solution. is solution and binding of hinge mutants or also with RTH mutants in the In TR and their in the of the of corepressors and coactivators in the of RTH impaired ligand-dependent associated with impaired S.M. Chatterjee V.K.K. Privalsky M.L. Mol. Endocrinol. 1997; 11: 470-480Crossref PubMed Scopus (160) Google corepressor or with defective recruitment M. R. V. E. C. E. K. G. L. R.J. Parker M.G. Chatterjee V.K.K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: PubMed Scopus Google Scholar, Y. A. T. A. Chin W.W. Yen P.M. Mol. Endocrinol. 1998; PubMed Scopus Google Scholar). Our data suggest that impaired corepressor is the in RTH with hinge mutations and with is of RTH mutations affect T3 binding to TR in solution or DNA, as is the with hinge result in impaired of corepressor at T3 concentrations and NCoR and SRC-1 not bind to TR, at are Addition of T3 dissociation of NCoR recruitment of SRC-1 the binding of the that both TR or the TR when NCoR is bound is that SRC-1 binding is not result is the NCoR inhibitor in of with in a of pTREs caused by the the mutant TR is to transcription except at very high T3 concentration. mutant TR binding to is also to SRC-1 or coactivators are not recruited to the TR due to of binding to the corepressor TR, a of the wt TR, in the absence of ligand, binds corepressor and transcription is Addition of T3 results in dissociation of corepressor, recruitment of coactivators and the hinge mutant TRs bind corepressor normally in the absence of ligand in T3 to the corepressor and recruitment is T3 binding by the hinge mutant TRs is or near-normal in their T3 binding is defective when complexed to DNA. Our data that the ability of hinge mutants to bind T3, novel mechanism of resistance by of RTH