Function of Nuclear Co-repressor Protein on Thyroid Hormone Response Elements Is Regulated by the Receptor A/B Domain
1996; Elsevier BV; Volume: 271; Issue: 45 Linguagem: Inglês
10.1074/jbc.271.45.28516
ISSN1083-351X
AutoresAnthony N. Hollenberg, Tsuyoshi Monden, John P. Madura, Karen Lee, Fredric E. Wondisford,
Tópico(s)Estrogen and related hormone effects
ResumoRecently, a family of nuclear co-repressor proteins (TRACs) have been identified that interact with thyroid hormone (TR) and retinoic acid receptors to mediate ligand-independent repression of gene transcription. In this report, we have cloned and characterized a human TRAC, which when expressed as a truncated protein lacking its repressing domains, can abolish endogenous cellular TRAC activity. Use of this inhibitor has uncovered a differential function of TRACs on negative versus positive thyroid hormone response elements and has demonstrated the importance of the TR A/B domain in modulating TRAC function. Thus, isoform-specific functions of the TR may be mediated by their functional interaction with co-repressor proteins. Recently, a family of nuclear co-repressor proteins (TRACs) have been identified that interact with thyroid hormone (TR) and retinoic acid receptors to mediate ligand-independent repression of gene transcription. In this report, we have cloned and characterized a human TRAC, which when expressed as a truncated protein lacking its repressing domains, can abolish endogenous cellular TRAC activity. Use of this inhibitor has uncovered a differential function of TRACs on negative versus positive thyroid hormone response elements and has demonstrated the importance of the TR A/B domain in modulating TRAC function. Thus, isoform-specific functions of the TR may be mediated by their functional interaction with co-repressor proteins. INTRODUCTIONThe thyroid hormone receptor (TR), 1The abbreviations used are: TRthyroid hormone receptorTREthyroid response elementRXRretinoid X receptorT3triiodothyronineAAamino acid(s)Tracthyroid hormone and retinoic acid-receptor-associated co-repressors. a member of the steroid/thyroid hormone receptor superfamily, mediates gene regulation through its ability to bind to specific thyroid hormone response elements (TREs) (1Desvergne B. Petty K.J. Nikodem V.M. J. Biol. Chem. 1991; 266: 1008-1013Abstract Full Text PDF PubMed Google Scholar, 2Brent G.A. Harney J.W. Chen Y. Warne R.L. Moore D.D. Larsen P.R. Mol. Endocrinol. 1989; 3: 1996-2004Crossref PubMed Scopus (182) Google Scholar, 3Norman M.F. Lavin T.N. Baxter J.D. West B.L. J. Biol. Chem. 1989; 264: 12063-12073Abstract Full Text PDF PubMed Google Scholar, 4Umesono K. Murakami K.K. Thompson C.C. Evans R.M. Cell. 1991; 65: 1255-1266Abstract Full Text PDF PubMed Scopus (1488) Google Scholar) either as a monomer, homodimer, or as a heterodimer with RXR isoforms (5Yu V.C. Delsert C. Andersen B. Holloway J.M. Devary O.V. Naar A.M. Kim S.Y. Boutin J.M. Glass C.K. Rosenfeld M.G. Cell. 1991; 67: 1251-1266Abstract Full Text PDF PubMed Scopus (1051) Google Scholar, 6Heyman R.A. Mangelsdorf D.J. Dyck J.A. Stein R.B. Eichele G. Evans R.M. Thaller C. Cell. 1992; 68: 397-406Abstract Full Text PDF PubMed Scopus (1558) Google Scholar, 7Mangelsdorf D.J. Ong E.S. Dyck J.A. Evans R.M. Nature. 1990; 345: 224-229Crossref PubMed Scopus (1252) Google Scholar). Through its interactions with specific TREs and other protein co-activators, the TR can either positively or negatively regulate gene expression in the presence of its ligand, triiodothyronine (T3) (8Hollenberg A.N. Monden T. Flynn T.R. Boers M.-E. Cohen O. Wondisford F.E. Mol. Endocrinol. 1995; 9: 540-550Crossref PubMed Google Scholar, 9Saatcioglu F. Deng T. Karin M. Cell. 1993; 75: 1095-1105Abstract Full Text PDF PubMed Scopus (104) Google Scholar, 10Wondisford F.E. Farr E.A. Radovick S. Steinfelder H.J. Moates J.M. McClaskey J.H. Weintraub B.D. J. Biol. Chem. 1989; 264: 14601-14604Abstract Full Text PDF PubMed Google Scholar). The TR also has significant ligand-independent activity in both cell culture and in vitro transcription systems (11Casanova J. Helmer E. Selmi-Ruby S. Qi J.-S. Au-Fliegner M. Desai-Yajnik V. Koudinova N. Yarm F. Raaka B.M. Samuels H.H. Mol. Cell. Biol. 1994; 14: 5756-5765Crossref PubMed Scopus (122) Google Scholar, 12Tong G.-X. Jeyakumar M. Tanen M. Bagchi M.K. Mol. Cell. Biol. 1996; 16: 1909-1920Crossref PubMed Google Scholar). On positive TREs, the TR is a potent repressor of transcription in the absence of ligand (13Baniahmad A. Kohne A.C. Renkawitz R. EMBO J. 1992; 11: 1015-1023Crossref PubMed Scopus (238) Google Scholar, 14Sap J. Munoz A. Schmitt J. Stunnenberg H. Vennstrom B. Nature. 1989; 340: 242-244Crossref PubMed Scopus (295) Google Scholar, 15Damm K. Evans R.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10668-10672Crossref PubMed Scopus (55) Google Scholar), while on negative TREs the TR activates transcription (9Saatcioglu F. Deng T. Karin M. Cell. 1993; 75: 1095-1105Abstract Full Text PDF PubMed Scopus (104) Google Scholar, 16Wondisford F.E. Steinfelder H.J. Nations M. Radovick S. J. Biol. Chem. 1993; 268: 2749-2754Abstract Full Text PDF PubMed Google Scholar, 17Tomie-Canie M. Day D. Samuels H.H. Freedberg I.M. Blumenberg M. J. Biol. Chem. 1996; 271: 1416-1423Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 18Bigler J. Eisenman R.N. EMBO J. 1995; 14: 5710-5723Crossref PubMed Scopus (43) Google Scholar). T3 reverses these ligand-independent activities and causes up-regulation on positive TREs and down-regulation on negative TREs. Recently, two separate proteins, termed nuclear co-repressors (TRAC), have been identified which interact with the TR in the absence of ligand to mediate ligand-independent repression on positive TREs (19Seol W. Choi H.S. Moore D.D. Mol. Endocrinol. 1995; 9: 72-85Crossref PubMed Google Scholar, 20Horlein A.J. Naar A.M. Heinzel T. Torchia J. Gloss B. Kurokawa R. Ryan A. Kamei Y. Soderstrom M. Glass C.K. Rosenfeld M.G. Nature. 1995; 377: 397-404Crossref PubMed Scopus (1695) Google Scholar, 21Chen J.D. Evans R.M. Nature. 1995; 377: 454-457Crossref PubMed Scopus (1699) Google Scholar). These proteins possess a carboxyl-terminal domain which mediates their interaction with the TR and amino-terminal domains, which presumably interact with the initiation complex to mediate ligand-independent repression. Their role in ligand-independent activation on negative TREs is unknown.Further complexity in thyroid hormone signaling is mediated by the presence of 3 separate TR isoforms (α1, β1, and β2) which activate or repress transcription in the presence of ligand (22Lazar M.A. Endocr. Rev. 1993; 14: 184-193Crossref PubMed Scopus (811) Google Scholar). The TRβ isoforms are alternatively spliced variants which differ only in their amino terminus, while TRα1 is the product of a separate gene but shares strong homology in its DNA and ligand binding domains but differs also in its amino terminus. Recent data from a number of different groups suggest that isoform-specific differences in both ligand-independent and -dependent activities exist on both positive and negative TREs and that these differences involve the separate amino termini which allow the TRs to either bind TREs differently, interact with members of the initiation complex with different affinities, or possess separate activation domains as is the case with the TRβ2 amino terminus (23Hadzic E. Desai-Yajnik V. Helmer E. Guo S. Wu S. Koudinova N. Casanova J. Raaka B.M. Samuels H.H. Mol. Cell. Biol. 1995; 15: 4507-4517Crossref PubMed Google Scholar, 24Hollenberg A.N. Monden T. Wondisford F.E. J. Biol. Chem. 1995; 270: 14274-14280Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 25Sjoberg M. Vennstrom B. Mol. Cell. Biol. 1995; 15: 4718-4726Crossref PubMed Google Scholar). These isoforms are also differentially expressed such that TRβ2 is primarily expressed in the hypothalamus and pituitary, suggesting that its primary role may be in mediating negative regulation by T3.To explore the role of nuclear co-repressors in both negative regulation and isoform specificity, we isolated, using the yeast two-hybrid system (26Fields S. Song O. Nature. 1989; 340: 245-247Crossref PubMed Scopus (4822) Google Scholar, 27Chien C.T. Bartel P.L. Sternglanz R. Fields S. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9578-9582Crossref PubMed Scopus (1222) Google Scholar), 3.1 kilobases of the carboxyl-terminal portion of the human analog of NCoR originally identified by Seol et al. (19Seol W. Choi H.S. Moore D.D. Mol. Endocrinol. 1995; 9: 72-85Crossref PubMed Google Scholar) and Horlein et al. (20Horlein A.J. Naar A.M. Heinzel T. Torchia J. Gloss B. Kurokawa R. Ryan A. Kamei Y. Soderstrom M. Glass C.K. Rosenfeld M.G. Nature. 1995; 377: 397-404Crossref PubMed Scopus (1695) Google Scholar). In this study, we report that this portion of NCoR functions as a powerful dominant inhibitor of endogenous cellular co-repressor activity on TREs and substantially increases ligand-dependent negative regulation by the TRβ1 and α1 isoforms. However, this function was isoform-specific in that the ligand-independent and -dependent activity of the β2 isoform was unaffected by the presence of this inhibitor. Gel-mobility shift assays demonstrate that the β2 amino terminus does not impair the ability of TRβ2 to interact physically with NCoR, but a functional interaction is prevented, perhaps by coactivators which interact with the β2 amino terminus (25Sjoberg M. Vennstrom B. Mol. Cell. Biol. 1995; 15: 4718-4726Crossref PubMed Google Scholar).RESULTS AND DISCUSSIONUsing the yeast two-hybrid system, as described above, we isolated a 3.1-kilobase cDNA clone from a human placental library which showed strong nucleic acid and amino acid homology to the carboxyl-terminal region of murine NCoR (20Horlein A.J. Naar A.M. Heinzel T. Torchia J. Gloss B. Kurokawa R. Ryan A. Kamei Y. Soderstrom M. Glass C.K. Rosenfeld M.G. Nature. 1995; 377: 397-404Crossref PubMed Scopus (1695) Google Scholar) (Fig. 1). Functional analysis of this clone in yeast demonstrated that 1μM T3 could prevent it from interacting with the carboxyl terminus of TRβ. As this original clone contained the interacting domain of NCoR and surrounding amino acids (AA 1539-2453), but lacked the two repressor domains present between AA 1-1120 of murine NCoR, we hypothesized that it would function as a dominant inhibitor of endogenous TRAC activity and thus termed it, NCoRI. The function of this inhibitor in eukaryotic cell lines was then tested on both positive and negative TREs.As demonstrated in Fig. 2A, TRβ1 represses basal transcription on a positive TRE reporter containing two copies of a DR+4 upstream of TK109 in the absence of ligand. Co-transfection of NCoRI, as expected, competes for endogenous co-repressor activity and causes ligand-independent repression to be lost and in the case of the DR+4 reporter leads to partial activation. Ligand-dependent activation was also blocked on the DR+4 reporter (expressed as fold T3 activation) in the presence of NCoRI, primarily because of the loss of ligand-independent repression, suggesting that relief of repression is an important mechanism of thyroid hormone action (data not shown). To rule out an independent activating function of NCoRI, we cloned AA 1539-2453 in-frame with the Gal4 DNA-binding domain and showed that it not alter expression of a UAS-TK Luc reporter construct (data not shown). NCoRI itself also had little effect on basal activity in CV-1 cells or on a cytomegalovirus promoter when co-transfected in isolation.Fig. 2A dominant inhibitor of the nuclear co-repressor family effects ligand-independent activity of the TR in an isoform-specific fashion on positive and negative thyroid hormone response elements. CV-1 cells were co-transfected with 1.67 μg of reporter and 83 ng of pKCR2-TR and/or 83 ng of pKCR2 and/or 83 ng of pKCR2-NCoRI alone to determine ligand-independent activity and -dependent activity. TRΔββ lacks the A/B domain (24Hollenberg A.N. Monden T. Wondisford F.E. J. Biol. Chem. 1995; 270: 14274-14280Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar) while TRE220R and TRAHT contain amino acid changes in the hinge region (20Horlein A.J. Naar A.M. Heinzel T. Torchia J. Gloss B. Kurokawa R. Ryan A. Kamei Y. Soderstrom M. Glass C.K. Rosenfeld M.G. Nature. 1995; 377: 397-404Crossref PubMed Scopus (1695) Google Scholar) of TRβ1. A, ligand-independent activity was determined on the DR+4-luciferase reporter using a number of TR isoforms and mutants. The data are quantified as fold repression on the DR+4 reporter (mean ± S.E.) In this case, 1 represents basal activity either in the presence of pKCR2 alone (stippled bars) or pKCR2-NCoRI (black bars). B, ligand-independent activity was determined, as above, on the TRH luciferase promoter. The data are quantified as fold activation on the TRH reporter (mean ± S.E.). In this case, 1 represents basal activity either in the presence of pKCR2 alone (stippled bars) or pKCR2-NCoRI (black bars). C, the TRH-luciferase reporter was transfected in a similar paradigm as above with the three TR isoforms. 24 h after transfection, 10 nM T3 was added. The data are presented as fold basal activity (mean ± S.E.) where 1 (indicated by the arrow) is the basal reporter activity either in the presence of pKCR2 alone (stippled bars) or pKCR2-NCoRI (black bars). The numbering within the graph represents fold repression in the presence of ligand.View Large Image Figure ViewerDownload Hi-res image Download (PPT)As we have demonstrated previously, TRα1 is a more potent ligand-independent repressor than TRβ1 on positive TREs. Yet, as shown in Fig. 2A, co-transfection of NCoRI again competes completely for endogenous co-repressor activity on the DR+4 element in the presence of TRα1. While TRβ2 is relatively weaker than the other two isoforms in ligand-independent activity on positive TREs, its activity is not appreciably changed by NCoRI. In contrast, TRΔββ, which lacks the amino terminus of the TRβ isoforms and is comparable to TRβ2 in ligand-independent activity, becomes a significant activator in the presence of co-transfected NCoRI (3-fold activation). These data indicate that TRΔββ interacts functionally with NCoRI and that the introduction of the TRβ2 amino terminus reverses this functional interaction. In addition, the TRβ1 and TRα1 amino termini permit a functional interaction with NCoRI. Mutagenesis of the hinge region (TRβAHT) also impairs functional interactions with NCoRI based upon its inability to bind NCoR, while TRβE220R fully interacts with NCoRI and is known to bind the wild-type NCoR (20Horlein A.J. Naar A.M. Heinzel T. Torchia J. Gloss B. Kurokawa R. Ryan A. Kamei Y. Soderstrom M. Glass C.K. Rosenfeld M.G. Nature. 1995; 377: 397-404Crossref PubMed Scopus (1695) Google Scholar). Similar results with each wild-type and mutant TR isoform were obtained on either an inverted (F2Lys) (36Hsu J.H. Zavacki A.M. Harney J.W. Brent G.A. Endocrinology. 1995; 136: 421-430Crossref PubMed Scopus (40) Google Scholar) or palindromic TRE (data not shown) demonstrating that the TRβ2 amino terminus blocked functional interactions with NCoRI.To further address the functional role of the co-repressor family on thyroid hormone action, we investigated the effect of co-transfected NCoRI on the negative TREs present in the human TRH promoter. In contrast to its ligand-independent repressive activity on positive TREs, the TR causes ligand-independent activation on negative TREs. As shown in Fig. 2B, basal ligand-independent activation by both TRβ1 and TRα1 is increased dramatically by NCoRI (from 2-fold and 1.5-fold, respectively, to 9-fold). In contrast, the 3-fold ligand-independent activation by TRβ2 is not affected by co-transfected NCoRI, consistent with its isoform-specific effect seen on the positive TREs. Similarly, TRΔββ, which is a poor ligand-independent activator, becomes a potent activator (from 1.2-fold to 10-fold) in the presence of NCoRI, demonstrating that the TRβ2 amino terminus again either impairs NCoR-TR interactions or modulates the function of the repressing domains of the co-repressor family.From these data it also appears that ligand-independent activation is important for negative regulation as increased ligand-independent effects due to co-transfected NCoRI lead to a doubling of fold repression by TRs which interact functionally with NCoRI (see upper and middle panels, Fig. 2C); however, fold repression is only marginally affected by NCoRI in context of TRβ2. Furthermore, as NCoRI can increase both ligand-independent activation and ligand-dependent repression of the TRH reporter construct by the TRα1 and TRβ1 isoforms, the converse indicates that increased levels of endogenous nuclear co-repressor activity could block ligand-independent and -dependent regulation of negative TREs by these isoforms leaving TRβ2 as the only TR isoform capable of mediating negative regulation. This would be consistent with TRβ2 being expressed primarily in the thyrotroph and hypothalamus, tissues where negative regulation by T3 is paramount (37Hodin R.A. Lazar M.A. Wintman B.I. Darling D.S. Koenig R.J. Larsen P.R. Moore D.D. Chin W.W. Science. 1989; 244: 76-79Crossref PubMed Scopus (410) Google Scholar).As our functional data suggest that isoform-specific interactions occur with the nuclear co-repressor family, we next asked whether the different TR isoforms could interact with NCoRI when bound to DNA. In vitro translation of the NCoRI cDNA generates a primary transcript of approximately 130 kDa consistent with its predicted size (data not shown). Incubation of in vitro translated NCoRI with in vitro translated TRβ1 in the presence of a DR+4 probe generates a slower migrating band (as compared to TR homodimer, lane 4, Fig. 3) and a diminution in the TR homodimer, consistent with a TRβ1-NCoRI interaction. This interaction can be disrupted in the presence of 100 nM T3 (lane 5, Fig. 3). Control lanes demonstrate that NCoRI does not bind the element in the absence of TR (not shown). Furthermore, TRβ2 (Fig. 4A) as well as TRα1 (not shown) also interact with NCoRI as does TRβE220R (Fig. 4B). As expected, TRβAHT does not bind NCoRI well (Fig. 4C). These data indicate that although functional interactions between TRβ2 and NCoRI do not appear to occur, the two proteins can still physically interact on the response element consistent with the ligand-binding domain of the TR being the primary region responsible for physical interaction (20Horlein A.J. Naar A.M. Heinzel T. Torchia J. Gloss B. Kurokawa R. Ryan A. Kamei Y. Soderstrom M. Glass C.K. Rosenfeld M.G. Nature. 1995; 377: 397-404Crossref PubMed Scopus (1695) Google Scholar, 21Chen J.D. Evans R.M. Nature. 1995; 377: 454-457Crossref PubMed Scopus (1699) Google Scholar). Functionally, however, the TRβ2 amino terminus either through the recruitment of other proteins or through changes in the conformation of the TR prevents the repressing function of the nuclear co-repressor from occurring.Fig. 3The nuclear co-repressor inhibitor binds to TRβ1 in a ligand-independent fashion in the presence of a TRE. Gel-mobility shift assays were carried out using 3-4 μl of in vitro translated proteins from rabbit reticulocyte lysate and a 32P-radiolabeled DR+4 oligonucleotide probe. The concentration of T3 used was 100 nM. The labeled DR+4 probe was incubated with in vitro translated TRβ1, RXRα, and NCoRI or unprogrammed reticulocyte lysate (UP). D, homodimer; HD, heterodimer.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 4The nuclear co-repressor binds to all TR isoforms. The labeled DR+4 probe was incubated with in vitro translated wild-type and mutant TR isoforms, RXR, and NCoRI as indicated. A, TRβ2; B, TRE220R; C, TRAHT.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Analysis of the interactions between NCoRI and the TR in the gel-mobility shift assay demonstrate that NCoRI appears to principally interact with the TR homodimer or with TR monomer as the TR-RXR complex (lane 6, Fig. 3) is not altered in either intensity or position by NCoRI. Furthermore, the intensity of the TR-NCoRI band is diminished consistent with RXR competing with NCoRI for binding to the TR. To test this possibility, we performed a time course competition between TR-NCoRI and RXR. As Fig. 5 demonstrates, the addition of RXR competes effectively for the TR binding to NCoRI and diminishes the TR-NCoRI complex at every time point. These data imply that the TR-RXR heterodimer does not interact well with NCoRI in the presence of DNA in this assay, and that the interaction between TR homodimer or monomer and NCoR may be important for co-repressor activity.Fig. 5The nuclear co-repressor inhibitor (NCoRI) preferentially binds the TR homodimer. The DR+4 probe was incubated with TRβ1 and NCoRI. At the indicated time points during the 20-min incubation, an equal amount of RXRα was added. The resulting complexes were resolved on a 5% nondenaturing gel acrylamide gel followed by autoradiography. D, homodimer; HD, heterodimer.View Large Image Figure ViewerDownload Hi-res image Download (PPT)In summary, we have demonstrated that endogenous nuclear co-repressor activity can be competed for with a dominant negative form of NCoR. This inhibitor, which lacks the two repressing domains, blocked ligand-independent repression by the TR on positive TREs and enhanced ligand-independent activity on negative TREs. However, co-repressor activity appears to be isoform-specific as the ligand-independent function of TRβ2 is not affected by NCoRI. This is most notably demonstrated on negative TREs where ligand-independent activation and repression by TRβ2 is not appreciably changed by NCoRI even though its physical interaction with NCoRI is maintained. Thus, the A/B domain of the TRβ2 can inactivate nuclear co-repressor function through either structural alteration or the recruitment of novel factors which modulate the function of the co-repressor.This study supports the notion that endogenous anti-repressors may exist which could further modulate thyroid hormone and retinoic acid action in a tissue-specific fashion. Recently, an endogenous truncated isoform of the co-repressor SMRT was identified (38Sande S. Privalsky M.L. Mol. Endocrinol. 1996; 10: 813-825Crossref PubMed Scopus (207) Google Scholar). This protein appears to function as an antagonist to wild type SMRT. Further studies will be directed at identifying other isoforms of the human NCoR family. INTRODUCTIONThe thyroid hormone receptor (TR), 1The abbreviations used are: TRthyroid hormone receptorTREthyroid response elementRXRretinoid X receptorT3triiodothyronineAAamino acid(s)Tracthyroid hormone and retinoic acid-receptor-associated co-repressors. a member of the steroid/thyroid hormone receptor superfamily, mediates gene regulation through its ability to bind to specific thyroid hormone response elements (TREs) (1Desvergne B. Petty K.J. Nikodem V.M. J. Biol. Chem. 1991; 266: 1008-1013Abstract Full Text PDF PubMed Google Scholar, 2Brent G.A. Harney J.W. Chen Y. Warne R.L. Moore D.D. Larsen P.R. Mol. Endocrinol. 1989; 3: 1996-2004Crossref PubMed Scopus (182) Google Scholar, 3Norman M.F. Lavin T.N. Baxter J.D. West B.L. J. Biol. Chem. 1989; 264: 12063-12073Abstract Full Text PDF PubMed Google Scholar, 4Umesono K. Murakami K.K. Thompson C.C. Evans R.M. Cell. 1991; 65: 1255-1266Abstract Full Text PDF PubMed Scopus (1488) Google Scholar) either as a monomer, homodimer, or as a heterodimer with RXR isoforms (5Yu V.C. Delsert C. Andersen B. Holloway J.M. Devary O.V. Naar A.M. Kim S.Y. Boutin J.M. Glass C.K. Rosenfeld M.G. Cell. 1991; 67: 1251-1266Abstract Full Text PDF PubMed Scopus (1051) Google Scholar, 6Heyman R.A. Mangelsdorf D.J. Dyck J.A. Stein R.B. Eichele G. Evans R.M. Thaller C. Cell. 1992; 68: 397-406Abstract Full Text PDF PubMed Scopus (1558) Google Scholar, 7Mangelsdorf D.J. Ong E.S. Dyck J.A. Evans R.M. Nature. 1990; 345: 224-229Crossref PubMed Scopus (1252) Google Scholar). Through its interactions with specific TREs and other protein co-activators, the TR can either positively or negatively regulate gene expression in the presence of its ligand, triiodothyronine (T3) (8Hollenberg A.N. Monden T. Flynn T.R. Boers M.-E. Cohen O. Wondisford F.E. Mol. Endocrinol. 1995; 9: 540-550Crossref PubMed Google Scholar, 9Saatcioglu F. Deng T. Karin M. Cell. 1993; 75: 1095-1105Abstract Full Text PDF PubMed Scopus (104) Google Scholar, 10Wondisford F.E. Farr E.A. Radovick S. Steinfelder H.J. Moates J.M. McClaskey J.H. Weintraub B.D. J. Biol. Chem. 1989; 264: 14601-14604Abstract Full Text PDF PubMed Google Scholar). The TR also has significant ligand-independent activity in both cell culture and in vitro transcription systems (11Casanova J. Helmer E. Selmi-Ruby S. Qi J.-S. Au-Fliegner M. Desai-Yajnik V. Koudinova N. Yarm F. Raaka B.M. Samuels H.H. Mol. Cell. Biol. 1994; 14: 5756-5765Crossref PubMed Scopus (122) Google Scholar, 12Tong G.-X. Jeyakumar M. Tanen M. Bagchi M.K. Mol. Cell. Biol. 1996; 16: 1909-1920Crossref PubMed Google Scholar). On positive TREs, the TR is a potent repressor of transcription in the absence of ligand (13Baniahmad A. Kohne A.C. Renkawitz R. EMBO J. 1992; 11: 1015-1023Crossref PubMed Scopus (238) Google Scholar, 14Sap J. Munoz A. Schmitt J. Stunnenberg H. Vennstrom B. Nature. 1989; 340: 242-244Crossref PubMed Scopus (295) Google Scholar, 15Damm K. Evans R.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10668-10672Crossref PubMed Scopus (55) Google Scholar), while on negative TREs the TR activates transcription (9Saatcioglu F. Deng T. Karin M. Cell. 1993; 75: 1095-1105Abstract Full Text PDF PubMed Scopus (104) Google Scholar, 16Wondisford F.E. Steinfelder H.J. Nations M. Radovick S. J. Biol. Chem. 1993; 268: 2749-2754Abstract Full Text PDF PubMed Google Scholar, 17Tomie-Canie M. Day D. Samuels H.H. Freedberg I.M. Blumenberg M. J. Biol. Chem. 1996; 271: 1416-1423Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 18Bigler J. Eisenman R.N. EMBO J. 1995; 14: 5710-5723Crossref PubMed Scopus (43) Google Scholar). T3 reverses these ligand-independent activities and causes up-regulation on positive TREs and down-regulation on negative TREs. Recently, two separate proteins, termed nuclear co-repressors (TRAC), have been identified which interact with the TR in the absence of ligand to mediate ligand-independent repression on positive TREs (19Seol W. Choi H.S. Moore D.D. Mol. Endocrinol. 1995; 9: 72-85Crossref PubMed Google Scholar, 20Horlein A.J. Naar A.M. Heinzel T. Torchia J. Gloss B. Kurokawa R. Ryan A. Kamei Y. Soderstrom M. Glass C.K. Rosenfeld M.G. Nature. 1995; 377: 397-404Crossref PubMed Scopus (1695) Google Scholar, 21Chen J.D. Evans R.M. Nature. 1995; 377: 454-457Crossref PubMed Scopus (1699) Google Scholar). These proteins possess a carboxyl-terminal domain which mediates their interaction with the TR and amino-terminal domains, which presumably interact with the initiation complex to mediate ligand-independent repression. Their role in ligand-independent activation on negative TREs is unknown.Further complexity in thyroid hormone signaling is mediated by the presence of 3 separate TR isoforms (α1, β1, and β2) which activate or repress transcription in the presence of ligand (22Lazar M.A. Endocr. Rev. 1993; 14: 184-193Crossref PubMed Scopus (811) Google Scholar). The TRβ isoforms are alternatively spliced variants which differ only in their amino terminus, while TRα1 is the product of a separate gene but shares strong homology in its DNA and ligand binding domains but differs also in its amino terminus. Recent data from a number of different groups suggest that isoform-specific differences in both ligand-independent and -dependent activities exist on both positive and negative TREs and that these differences involve the separate amino termini which allow the TRs to either bind TREs differently, interact with members of the initiation complex with different affinities, or possess separate activation domains as is the case with the TRβ2 amino terminus (23Hadzic E. Desai-Yajnik V. Helmer E. Guo S. Wu S. Koudinova N. Casanova J. Raaka B.M. Samuels H.H. Mol. Cell. Biol. 1995; 15: 4507-4517Crossref PubMed Google Scholar, 24Hollenberg A.N. Monden T. Wondisford F.E. J. Biol. Chem. 1995; 270: 14274-14280Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 25Sjoberg M. Vennstrom B. Mol. Cell. Biol. 1995; 15: 4718-4726Crossref PubMed Google Scholar). These isoforms are also differentially expressed such that TRβ2 is primarily expressed in the hypothalamus and pituitary, suggesting that its primary role may be in mediating negative regulation by T3.To explore the role of nuclear co-repressors in both negative regulation and isoform specificity, we isolated, using the yeast two-hybrid system (26Fields S. Song O. Nature. 1989; 340: 245-247Crossref PubMed Scopus (4822) Google Scholar, 27Chien C.T. Bartel P.L. Sternglanz R. Fields S. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9578-9582Crossref PubMed Scopus (1222) Google Scholar), 3.1 kilobases of the carboxyl-terminal portion of the human analog of NCoR originally identified by Seol et al. (19Seol W. Choi H.S. Moore D.D. Mol. Endocrinol. 1995; 9: 72-85Crossref PubMed Google Scholar) and Horlein et al. (20Horlein A.J. Naar A.M. Heinzel T. Torchia J. Gloss B. Kurokawa R. Ryan A. Kamei Y. Soderstrom M. Glass C.K. Rosenfeld M.G. Nature. 1995; 377: 397-404Crossref PubMed Scopus (1695) Google Scholar). In this study, we report that this portion of NCoR functions as a powerful dominant inhibitor of endogenous cellular co-repressor activity on TREs and substantially increases ligand-dependent negative regulation by the TRβ1 and α1 isoforms. However, this function was isoform-specific in that the ligand-independent and -dependent activity of the β2 isoform was unaffected by the presence of this inhibitor. Gel-mobility shift assays demonstrate that the β2 amino terminus does not impair the ability of TRβ2 to interact physically with NCoR, but a functional interaction is prevented, perhaps by coactivators which interact with the β2 amino terminus (25Sjoberg M. Vennstrom B. Mol. Cell. Biol. 1995; 15: 4718-4726Crossref PubMed Google Scholar).
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