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• W2149936418 abstract "Thyroid hormone (triiodothyronine, T3) is known to activate transcription by binding heterodimers of thyroid hormone receptors (TRs) and retinoid X receptors (RXRs). RXR-TRs bind to T3 response elements (TREs) composed of direct repeats of the sequence AGGTCA spaced by four nucleotides (DR-4). In other TREs, however, the half-sites can be arranged as inverted palindromes and palindromes (Pal). Here we show that TR homodimers and monomers activate transcription from representative TREs with alternate half-site placements. TRβ activates transcription more efficiently than TRα at an inverted palindrome (F2), and this correlates with preferential TRβ homodimer formation at F2 in vitro. Furthermore, reconstruction of TR transcription complexes in yeast indicates that TRβ homodimers are active at F2, whereas RXR-TRs are active at DR-4 and Pal. Finally, analysis of TRβ mutations that block homodimer and/or heterodimer formation reveal TRE-selective requirements for these surfaces in mammalian cells, which suggest that TRβ homodimers are active at F2, RXR-TRs at DR-4, and TR monomers at Pal. TRβ requires higher levels of hormone for activation at F2 than other TREs, and this differential effect is abolished by a dimer surface mutation suggesting that it is related to composition of the TR·TRE complex. We propose that interactions of particular TR oligomers with different elements play unappreciated roles in TRE-selective actions of liganded TRs in vivo. Thyroid hormone (triiodothyronine, T3) is known to activate transcription by binding heterodimers of thyroid hormone receptors (TRs) and retinoid X receptors (RXRs). RXR-TRs bind to T3 response elements (TREs) composed of direct repeats of the sequence AGGTCA spaced by four nucleotides (DR-4). In other TREs, however, the half-sites can be arranged as inverted palindromes and palindromes (Pal). Here we show that TR homodimers and monomers activate transcription from representative TREs with alternate half-site placements. TRβ activates transcription more efficiently than TRα at an inverted palindrome (F2), and this correlates with preferential TRβ homodimer formation at F2 in vitro. Furthermore, reconstruction of TR transcription complexes in yeast indicates that TRβ homodimers are active at F2, whereas RXR-TRs are active at DR-4 and Pal. Finally, analysis of TRβ mutations that block homodimer and/or heterodimer formation reveal TRE-selective requirements for these surfaces in mammalian cells, which suggest that TRβ homodimers are active at F2, RXR-TRs at DR-4, and TR monomers at Pal. TRβ requires higher levels of hormone for activation at F2 than other TREs, and this differential effect is abolished by a dimer surface mutation suggesting that it is related to composition of the TR·TRE complex. We propose that interactions of particular TR oligomers with different elements play unappreciated roles in TRE-selective actions of liganded TRs in vivo. Thyroid hormone receptors (TRα 5The abbreviations used are: TR, thyroid hormone receptor; T3, triiodothyronine; RXR, retinoid X receptor; TRE, T3 response element; IP, inverted palindrome; LBD, ligand-binding domain; DBD, DNA-binding domain; CTE, C-terminal extension; Pal, palindrome; NR, nuclear receptor. and TRβ) modulate gene expression by binding to thyroid hormone response elements (TREs) in target gene promoters (1Desvergne B. Mol. Cell. Endocrinol. 1994; 100: 125-131Crossref PubMed Scopus (55) Google Scholar, 2Laudet V. Gronemeyer H. The Nuclear Receptor Facts Book. 1st Ed. Academic Press, London2002: 87-112Google Scholar, 3Yen P.M. Physiol. Rev. 2001; 81: 1097-1142Crossref PubMed Scopus (1542) Google Scholar, 4Zhang J. Lazar M.A. Annu. Rev. Physiol. 2000; 62: 439-466Crossref PubMed Scopus (576) Google Scholar, 5Ribeiro R.C. Apriletti J.W. Wagner R.L. Feng W. Kushner P.J. Nilsson S. Scanlan T.S. West B.L. Fletterick R.J. Baxter J.D. J. Steroid Biochem. Mol. Biol. 1998; 65: 133-141Crossref PubMed Scopus (59) Google Scholar). TR activity is primarily regulated by thyroid hormone (as 3,5,3′-triiodo-l-thyronine (T3)), which alters the conformation of the receptor C-terminal ligand-binding domain (LBD) to promote dissociation of corepressors and association of coactivators (6Glass C.K. Rosenfeld M.G. Genes Dev. 2000; 14: 121-141Crossref PubMed Google Scholar). Nevertheless, TR activity is also influenced by the sequence, arrangement, and promoter context of the TRE (2Laudet V. Gronemeyer H. The Nuclear Receptor Facts Book. 1st Ed. Academic Press, London2002: 87-112Google Scholar, 3Yen P.M. Physiol. Rev. 2001; 81: 1097-1142Crossref PubMed Scopus (1542) Google Scholar). Liganded TRs activate transcription from some TREs and repress transcription at others (reviewed in Ref. 3Yen P.M. Physiol. Rev. 2001; 81: 1097-1142Crossref PubMed Scopus (1542) Google Scholar and see Refs. 7Shibusawa N. Hollenberg A.N. Wondisford F.E. J. Biol. Chem. 2003; 278: 732-738Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar and 8Santos G.M. Afonso V. Barra G.B. Togashi M. Webb P. Neves F.A. Lomri N. Lomri A. Mol. Pharmacol. 2006; 70: 793-800Crossref PubMed Scopus (31) Google Scholar). In addition, TRE sequence and arrangement influence the magnitude of response to unliganded and liganded TRs, T3 concentration dependence, dominant negative activities of mutants that arise in resistance to thyroid hormone syndrome, and the direction and extent of response to TRβ isoform-selective ligands (9Williams G.R. Zavacki A.M. Harney J.W. Brent G.A. Endocrinology. 1994; 134: 1888-1896Crossref PubMed Scopus (29) Google Scholar, 10Yoh S.M. Privalsky M.L. J. Biol. Chem. 2001; 276: 16857-16867Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 11Borngraeber S. Budny M.J. Chiellini G. Cunha-Lima S.T. Togashi M. Webb P. Baxter J.D. Scanlan T.S. Fletterick R.J. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 15358-15363Crossref PubMed Scopus (104) Google Scholar, 12Gloss B. Giannocco G. Swanson E.A. Moriscot A.S. Chiellini G. Scanlan T. Baxter J.D. Dillmann W.H. Endocrinology. 2005; 146: 4926-4933Crossref PubMed Scopus (17) Google Scholar, 13Meier C.A. Parkison C. Chen A. Ashizawa K. Meier-Heusler S.C. Muchmore P. Cheng S.Y. Weintraub B.D. J. Clin. Investig. 1993; 92: 1986-1993Crossref PubMed Scopus (71) Google Scholar). Improved understanding of the molecular basis of these effects could help us to harness these selective activities. TRs bind to DNA either as heterodimers with the closely related retinoid X receptor (RXR) or as homodimers and monomers, and each species exhibits preferences for different TREs (reviewed in Refs. 1Desvergne B. Mol. Cell. Endocrinol. 1994; 100: 125-131Crossref PubMed Scopus (55) Google Scholar and 2Laudet V. Gronemeyer H. The Nuclear Receptor Facts Book. 1st Ed. Academic Press, London2002: 87-112Google Scholar). TREs are composed of degenerate repeats of the sequence AGGTCA, with the half-sites arrayed as direct repeats spaced by four nucleotides (DR-4), inverted repeats spaced by four to six nucleotides (IPs) or palindromes (Pal). Heterodimers of RXRs and TRs bind each of these elements, with a strong preference for DR-4. In contrast, TRβ homodimers bind strongly to IP elements, weakly to DR-4, and not all to Pal (9Williams G.R. Zavacki A.M. Harney J.W. Brent G.A. Endocrinology. 1994; 134: 1888-1896Crossref PubMed Scopus (29) Google Scholar, 14Kurokawa R. Yu V.C. Naar A. Kyakumoto S. Han Z. Silverman S. Rosenfeld M.G. Glass C.K. Genes Dev. 1993; 7: 1423-1435Crossref PubMed Scopus (289) Google Scholar, 15Andersson M.L. Nordstrom K. Demczuk S. Harbers M. Vennstrom B. Nucleic Acids Res. 1992; 20: 4803-4810Crossref PubMed Scopus (76) Google Scholar, 16Miyamoto T. Suzuki S. DeGroot L.J. Mol. Endocrinol. 1993; 7: 224-231PubMed Google Scholar, 17Ribeiro R.C. Feng W. Wagner R.L. Costa C.H. Pereira A.C. Apriletti J.W. Fletterick R.J. Baxter J.D. J. Biol. Chem. 2001; 276: 14987-14995Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar), and TRα homodimers bind TREs, at best, only weakly (18Lazar M.A. Berrodin T.J. Harding H.P. Mol. Cell. Biol. 1991; 11: 5005-5015Crossref PubMed Scopus (129) Google Scholar, 19Darling D.S. Carter R.L. Yen P.M. Welborn J.M. Chin W.W. Umeda P.K. J. Biol. Chem. 1993; 268: 10221-10227Abstract Full Text PDF PubMed Google Scholar). Finally, both TRs can bind to DNA elements as monomers. The fact that different oligomeric forms of TR bind to DNA and exhibit clear preferences for different TREs raises the possibility that the nature of the response element could influence TR action and T3 response by recruitment of distinct TR transcription complexes with unique activities. It is clear that RXR-TRs are important mediators of T3 action. TR binding to RXRs in vivo has been verified by observations that TRs consistently copurify as complexes with RXRs from cell and tissue extracts (1Desvergne B. Mol. Cell. Endocrinol. 1994; 100: 125-131Crossref PubMed Scopus (55) Google Scholar, 3Yen P.M. Physiol. Rev. 2001; 81: 1097-1142Crossref PubMed Scopus (1542) Google Scholar). Moreover, analysis of RXR knock-out mice reveals defective T3 responses (3Yen P.M. Physiol. Rev. 2001; 81: 1097-1142Crossref PubMed Scopus (1542) Google Scholar, 20O'Shea P.J. Williams G.R. J. Endocrinol. 2002; 175: 553-570Crossref PubMed Scopus (174) Google Scholar), and RXRs and TRs synergize at direct repeats in cotransfection experiments in some cell types (21Leng X. Blanco J. Tsai S.Y. Ozato K. O'Malley B.W. Tsai M.J. J. Biol. Chem. 1994; 269: 31436-31442Abstract Full Text PDF PubMed Google Scholar, 22Liang F. Webb P. Marimuthu A. Zhang S. Gardner D.G. J. Biol. Chem. 2003; 278: 15073-15083Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Furthermore, native TREs are often comprised of DR-4 elements, which bind preferentially to RXR-TRs (1Desvergne B. Mol. Cell. Endocrinol. 1994; 100: 125-131Crossref PubMed Scopus (55) Google Scholar, 3Yen P.M. Physiol. Rev. 2001; 81: 1097-1142Crossref PubMed Scopus (1542) Google Scholar), and in vitro DNA binding properties of RXR-TRs are consistent with roles in T3 response; RXR-TRs form stable complexes with DNA that persist in the presence of hormone (23Yen P.M. Darling D.S. Carter R.L. Forgione M. Umeda P.K. Chin W.W. J. Biol. Chem. 1992; 267: 3565-3568Abstract Full Text PDF PubMed Google Scholar, 24Ribeiro R.C. Kushner P.J. Apriletti J.W. West B.L. Baxter J.D. Mol. Endocrinol. 1992; 6: 1142-1152PubMed Google Scholar, 25Yen P.M. Sugawara A. Chin W.W. J. Biol. Chem. 1992; 267: 23248-23252Abstract Full Text PDF PubMed Google Scholar). Finally, chromatin immunoprecipitation analysis confirms that RXR-TRs occupy DR-4 elements in the Xenopus laevis TRβ and thyroid hormone/bZIP promoters at late stages of embryogenesis (26Sachs L.M. Shi Y.B. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 13138-13143Crossref PubMed Scopus (105) Google Scholar). Potential roles of other TR oligomers are not as well understood. An RXR gene knock-out fails to affect TR action in ear development, implying that alternate TR species could regulate this process (27Barros A.C. Erway L.C. Krezel W. Curran T. Kastner P. Chambon P. Forrest D. Neuroreport. 1998; 9: 2933-2937Crossref PubMed Scopus (22) Google Scholar). Moreover, the fact that unliganded TRs repress transcription more efficiently at IP elements than DR-4 elements has been attributed to preferential recruitment of homodimers, which bind corepressors more efficiently than RXR-TRs (9Williams G.R. Zavacki A.M. Harney J.W. Brent G.A. Endocrinology. 1994; 134: 1888-1896Crossref PubMed Scopus (29) Google Scholar, 10Yoh S.M. Privalsky M.L. J. Biol. Chem. 2001; 276: 16857-16867Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 28Hollenberg 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 Scholar, 29Liu Y. Takeshita A. Misiti S. Chin W.W. Yen P.M. Endocrinology. 1998; 139: 4197-4204Crossref PubMed Scopus (60) Google Scholar, 30Cohen R.N. Putney A. Wondisford F.E. Hollenberg A.N. Mol. Endocrinol. 2000; 14: 900-914Crossref PubMed Scopus (90) Google Scholar). Accordingly, it has been shown that TR can bind to an unusual TRE in the human immunodeficiency virus type 1 in the absence of RXR in the frog oocyte chromatin assembly system (31Hsia S.C. Shi Y.B. Mol. Cell. Biol. 2002; 22: 4043-4052Crossref PubMed Scopus (55) Google Scholar). Roles of alternate forms of TR in T3 activation are even less clear. Although the fact that TR homodimers dissociate rapidly from cognate TREs and T3 suppresses homodimer formation on DNA has been taken as an argument against a role for this species in T3 activation (1Desvergne B. Mol. Cell. Endocrinol. 1994; 100: 125-131Crossref PubMed Scopus (55) Google Scholar), other studies reveal that TR-TR·DNA complexes are stabilized by coactivators (32Diallo E.M. Thompson D.L. Koenig R.J. Protein Expression Purif. 2005; 40: 292-298Crossref PubMed Scopus (10) Google Scholar) and that T3 response at IP-6 elements is often independent of coexpressed RXR in transfections (9Williams G.R. Zavacki A.M. Harney J.W. Brent G.A. Endocrinology. 1994; 134: 1888-1896Crossref PubMed Scopus (29) Google Scholar, 33Piedrafita F.J. Bendik I. Ortiz M.A. Pfahl M. Mol. Endocrinol. 1995; 9: 563-578PubMed Google Scholar). Most strikingly, a recent study revealed that RXR expression was only required for optimal T3 response at subsets of positively regulated genes in a mouse preneuronal cell line (34Diallo E.M. Wilhelm Jr., K.G. Thompson D.L. Koenig R.J. Mol. Cell. Endocrinol. 2007; 264: 149-156Crossref PubMed Scopus (13) Google Scholar). Thus, there are several indications that alternate TR oligomers contribute to T3 induction. Study of the actions of different TR oligomers in mammalian cells is often complicated by endogenous expression of RXR and NR coregulators. To overcome this difficulty, we (35Walfish P.G. Yoganathan T. Yang Y.F. Hong H. Butt T.R. Stallcup M.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3697-3702Crossref PubMed Scopus (47) Google Scholar, 36Anafi M. Yang Y.F. Barlev N.A. Govindan M.V. Berger S.L. Butt T.R. Walfish P.G. Mol. Endocrinol. 2000; 14: 718-732Crossref PubMed Scopus (50) Google Scholar, 37Meng X. Yang Y.F. Cao X. Govindan M.V. Shuen M. Hollenberg A.N. Mymryk J.S. Walfish P.G. Mol. Endocrinol. 2003; 17: 1095-1105Crossref PubMed Scopus (17) Google Scholar, 38Meng X. Webb P. Yang Y.F. Shuen M. Yousef A.F. Baxter J.D. Mymryk J.S. Walfish P.G. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 6267-6272Crossref PubMed Scopus (17) Google Scholar) and others (39Wu Y. Xu B. Koenig R.J. J. Biol. Chem. 2001; 276: 3929-3936Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) have examined TR action in the yeast Saccharomyces cerevisiae. This species is devoid of known NRs and NR coregulators and can be used to reconstruct defined TR transcription complexes in a eukaryotic cell background. These studies indicate that TRs indeed activate transcription at selected TREs in the absence of RXR (35Walfish P.G. Yoganathan T. Yang Y.F. Hong H. Butt T.R. Stallcup M.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3697-3702Crossref PubMed Scopus (47) Google Scholar, 39Wu Y. Xu B. Koenig R.J. J. Biol. Chem. 2001; 276: 3929-3936Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) and that the extent of TR homodimer formation to different DR-4 elements in vitro correlates with T3 response (39Wu Y. Xu B. Koenig R.J. J. Biol. Chem. 2001; 276: 3929-3936Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). RXR-TR heterodimer and TR homodimer surfaces have also been defined. There are contacts between respective LBDs and DBDs of RXRs and TRs (1Desvergne B. Mol. Cell. Endocrinol. 1994; 100: 125-131Crossref PubMed Scopus (55) Google Scholar), whereas TR homodimer formation at DR-4 and IP-6 relies exclusively on LBD-LBD contacts (14Kurokawa R. Yu V.C. Naar A. Kyakumoto S. Han Z. Silverman S. Rosenfeld M.G. Glass C.K. Genes Dev. 1993; 7: 1423-1435Crossref PubMed Scopus (289) Google Scholar, 40Zechel C. Shen X.Q. Chen J.Y. Chen Z.P. Chambon P. Gronemeyer H. EMBO J. 1994; 13: 1425-1433Crossref PubMed Scopus (232) Google Scholar, 41Zechel C. Shen X.Q. Chambon P. Gronemeyer H. EMBO J. 1994; 13: 1414-1424Crossref PubMed Scopus (174) Google Scholar, 42Perlmann T. Umesono K. Rangarajan P.N. Forman B.M. Evans R.M. Mol. Endocrinol. 1996; 10: 958-966PubMed Google Scholar). We used x-ray structure-directed mutagenesis to define these surfaces and showed that RXR-TRβ heterodimer and TRβ homodimer formation requires a small hydrophobic patch at the junction of helices (H) 10 and H11 in the LBD (17Ribeiro R.C. Feng W. Wagner R.L. Costa C.H. Pereira A.C. Apriletti J.W. Fletterick R.J. Baxter J.D. J. Biol. Chem. 2001; 276: 14987-14995Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 43Togashi M. Nguyen P. Fletterick R. Baxter J.D. Webb P. J. Biol. Chem. 2005; 280: 25665-25673Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). This surface (LBD dimer surface) resembles those seen in x-ray structures of other NR LBD heterodimers and homodimers (reviewed in Ref. 2Laudet V. Gronemeyer H. The Nuclear Receptor Facts Book. 1st Ed. Academic Press, London2002: 87-112Google Scholar). The TRβ interface (DBD heterodimer surface) that contacts the RXR DBD was detected in an x-ray structure of an RXR·TRβ DBD complex on a DR-4 element (44Rastinejad F. Perlmann T. Evans R.M. Sigler P.B. Nature. 1995; 375: 203-211Crossref PubMed Scopus (473) Google Scholar), and mutation of this surface inhibits RXR-TR heterodimer formation at DR-4 in vitro (17Ribeiro R.C. Feng W. Wagner R.L. Costa C.H. Pereira A.C. Apriletti J.W. Fletterick R.J. Baxter J.D. J. Biol. Chem. 2001; 276: 14987-14995Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). The same structure reveals another difference between TRs and RXRs; the TRβ DBD contains an α-helical C-terminal extension (CTE) that is absent from RXR and mediates additional DNA contacts (44Rastinejad F. Perlmann T. Evans R.M. Sigler P.B. Nature. 1995; 375: 203-211Crossref PubMed Scopus (473) Google Scholar). The requirements for these surfaces in T3 response in vivo are not known. In this study, we compare actions of wild type and mutant TRs at different TREs in yeast and in mammalian cells. Although the data confirm that RXR-TRs activate transcription at DR-4, they suggest that TRβ homodimers and monomers mediate T3 response at IP-6 and Pal, respectively. We propose that TRE half-site orientation dictates the composition of the active form of TR in vivo and that this effect should be considered in analysis of TR activity and design of selective TR modulators. Plasmids—The following plasmids have been described previously: T3-inducible β-galactosidase reporters for yeast transcription assays, TRE-F2x1, TRE-DR-4x1, and TRE-Palx1 and yeast (YEp56) expression vectors for wild type TRβ, RXR, and GRIP1 (35Walfish P.G. Yoganathan T. Yang Y.F. Hong H. Butt T.R. Stallcup M.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3697-3702Crossref PubMed Scopus (47) Google Scholar, 36Anafi M. Yang Y.F. Barlev N.A. Govindan M.V. Berger S.L. Butt T.R. Walfish P.G. Mol. Endocrinol. 2000; 14: 718-732Crossref PubMed Scopus (50) Google Scholar, 37Meng X. Yang Y.F. Cao X. Govindan M.V. Shuen M. Hollenberg A.N. Mymryk J.S. Walfish P.G. Mol. Endocrinol. 2003; 17: 1095-1105Crossref PubMed Scopus (17) Google Scholar, 38Meng X. Webb P. Yang Y.F. Shuen M. Yousef A.F. Baxter J.D. Mymryk J.S. Walfish P.G. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 6267-6272Crossref PubMed Scopus (17) Google Scholar); T3-inducible reporters containing two copies of each TRE driving luciferase expression and a similar 9-cis-retinoic acid-responsive reporter containing two copies of a DR-1 element and mammalian (pCMX) expression vectors for TRβ,TRβL422R, and RXR (17Ribeiro R.C. Feng W. Wagner R.L. Costa C.H. Pereira A.C. Apriletti J.W. Fletterick R.J. Baxter J.D. J. Biol. Chem. 2001; 276: 14987-14995Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). New TR mutants reported in these studies (pCMX vectors TRβ4XDBD (D104A, Y117A, R120A, and D177A), TRβ3XLBD (L400R, L422R, and M423R), TRβ4XDBD3XLBD, TRβ T-box deletion (deletion of 6 amino acids from 175 to 180 in the T-box region, -ATDLVL) and the yeast Yep56 vector TRβL422R were created in existing vectors using QuikChange site-directed mutagenesis kits (Stratagene). The presence of the mutation(s) was verified by DNA sequencing using Sequenase kits (Stratagene). FLAG and Myc-tagged TR expression vectors were created by PCR amplification of the TRβ cDNA and insertion into FLAG and Myc vectors (Clontech) at appropriate restriction sites. Mammalian Cell Culture, Electroporation, and Luciferase Assays—Human promonocyte U937, HEK, HeLa, and U2-OS cells were maintained at 37 °C, 5% CO2 subcultured in media RPMI 1640 with 10% newborn bovine serum, 2 mm glutamine, 50 units/ml penicillin, and 50 μg/ml streptomycin. Transfections were carried out as described previously (45Leitman D.C. Costa C.H. Graf H. Baxter J.D. Ribeiro R.C. J. Biol. Chem. 1996; 271: 21950-21955Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). After incubation for 24 h at 37 °C with ethanol or T3, cells were collected by centrifugation, and the pellets were solubilized by addition of 150 μl of 0.25 m Tris-HCl, pH 7.6, containing 0.1% Triton X-100. Luciferase activity was analyzed by standard methods (luciferase assay system, Promega). Gel Shifts—For Fig. 1, TRs were labeled with [35S]methionine, and complex formation was measured at unlabeled TREs in a 20-μl reaction with 1 μg of poly(dI-dC) (Amersham Biosciences) (17Ribeiro R.C. Feng W. Wagner R.L. Costa C.H. Pereira A.C. Apriletti J.W. Fletterick R.J. Baxter J.D. J. Biol. Chem. 2001; 276: 14987-14995Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). The binding buffer contained 25 mm HEPES, 50 mm KCl, 1 mm dithiothreitol, 10 μm ZnSO4, 0.1% Nonidet P-40, 5% glycerol. After 30 min at room temperature, the mixture was loaded onto a 5% nondenaturing polyacrylamide gel that was previously run for 30 min at 200 V. TR and RXR·TR·DNA complexes were visualized as follows: the gel was run at 4 °C for 120–180 min at 200 V in a running buffer containing 45 mm Tris borate, pH 8.0, and 1 mm EDTA, fixed, dried, and exposed to autoradiography. For other figures, binding of TRs and RXR-TRs to DNA was assayed as described previously (43Togashi M. Nguyen P. Fletterick R. Baxter J.D. Webb P. J. Biol. Chem. 2005; 280: 25665-25673Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar), by mixing 20 fmol of TRs ± RXRs with 300,000 cpm of [γ-32P]ATP-radiolabeled TRE oligonucleotide. Yeast Strains and Transcriptional Analysis—The S. cerevisiae strain YPH499 (MATα, ura3, lys2, ade2, trp1, his3, leu2) was used for most transformations. For reporter assays, transformants were grown in minimal medium (0.67% yeast nitrogen base, 2% glucose) supplemented with adenine and lysine (both at 40 mg/liter), as described previously (35Walfish P.G. Yoganathan T. Yang Y.F. Hong H. Butt T.R. Stallcup M.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3697-3702Crossref PubMed Scopus (47) Google Scholar, 36Anafi M. Yang Y.F. Barlev N.A. Govindan M.V. Berger S.L. Butt T.R. Walfish P.G. Mol. Endocrinol. 2000; 14: 718-732Crossref PubMed Scopus (50) Google Scholar, 37Meng X. Yang Y.F. Cao X. Govindan M.V. Shuen M. Hollenberg A.N. Mymryk J.S. Walfish P.G. Mol. Endocrinol. 2003; 17: 1095-1105Crossref PubMed Scopus (17) Google Scholar, 38Meng X. Webb P. Yang Y.F. Shuen M. Yousef A.F. Baxter J.D. Mymryk J.S. Walfish P.G. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 6267-6272Crossref PubMed Scopus (17) Google Scholar). The yeast transformants were isolated and grown in the appropriate minimal medium with added supplements as required. Cells were grown overnight with T3 at a final concentration of 1 μm, harvested, washed, resuspended in Z buffer, and lysed with glass beads (425–600 μm) before centrifugation. The supernatant was collected, and the protein concentration was determined by the Lowry method using bovine serum albumin as a standard. Twenty micrograms of protein were used for the β-galactosidase assay, and transcriptional activities were expressed as Miller units/mg of protein, as described previously (35Walfish P.G. Yoganathan T. Yang Y.F. Hong H. Butt T.R. Stallcup M.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3697-3702Crossref PubMed Scopus (47) Google Scholar, 36Anafi M. Yang Y.F. Barlev N.A. Govindan M.V. Berger S.L. Butt T.R. Walfish P.G. Mol. Endocrinol. 2000; 14: 718-732Crossref PubMed Scopus (50) Google Scholar, 37Meng X. Yang Y.F. Cao X. Govindan M.V. Shuen M. Hollenberg A.N. Mymryk J.S. Walfish P.G. Mol. Endocrinol. 2003; 17: 1095-1105Crossref PubMed Scopus (17) Google Scholar, 38Meng X. Webb P. Yang Y.F. Shuen M. Yousef A.F. Baxter J.D. Mymryk J.S. Walfish P.G. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 6267-6272Crossref PubMed Scopus (17) Google Scholar). Data shown were pooled from three independent experiments and calculated as the means ± S.E. Cell Extracts, Immunoprecipitation, and Western Blotting— HeLa cells were cotransfected with 5 μg of pCMV-Myc-TRβ1 and 5 μg of pCMV-Tag2B-FLAG-TRβ1 by electroporation at 240 V and 950 microfarads. 24 h after transfection, cells were washed with phosphate-buffered saline and harvested. Samples of whole cell extracts were lysed in RIPA buffer (50 mm Tris, pH 8.1, 150 mm NaCl, 0.2% SDS, 1% sodium deoxycholate, 1% Nonidet P-40, 5 mm EDTA) containing protease inhibitor mixture (Calbiochem) and 1 mm phenylmethylsulfonyl fluoride at 4 °C for 1 h, sonicated, and then centrifuged at 20,000 × g for 15 min. 100 μl (400 μg of total protein) of supernatant was diluted with 500 μl of phosphate-buffered saline, and 4 μg of rabbit anti-FLAG antibody (Rockland, Inc.) was added. After incubation overnight at 4 °C, 50 μl of protein G-Sepharose beads (Amersham Biosciences) were added to each sample, and the lysates were incubated for 1 h at 4°C. The beads then were washed six times with TBS-T (Tris-buffered saline; 25 mm Tris, pH 8.3, 192 mm glycine, containing 0.05% Tween 20), suspended in 2× SDS sample buffer containing 5% β-mercaptoethanol, heated for 10 min at 95 °C, and resolved on 8% denaturing polyacrylamide gels. After transferring to Immun-Blot polyvinylidene difluoride membranes (Bio-Rad), the membranes were blocked with 5% nonfat milk in TBS-T and incubated with primary antibody, mouse Myc-TRβ1 antibody (Clontech), at 1:1000 (w/v) dilution in 1% nonfat milk in TBS-T. After washing with TBS-T, immunoblots were incubated with horseradish peroxidase-conjugated goat anti-mouse-IgG (Santa Cruz Biotechnology) in 1% TBS-T, and proteins were visualized using enhanced chemiluminescence according to the manufacturer's instructions (Amersham Biosciences). TRβ, but Not TRα, Is Superactive at an IP-6 Element (F2)— First, we first compared the actions of two major hormone-binding TR isoforms (TRα1 and TRβ1) at TRE-dependent reporters in cell culture (Fig. 1A). TRβ gave significantly larger T3 responses at an IP-6 element (F2) than TRα, 30–40-fold versus 6-fold. By contrast, the two TR isoforms gave comparable T3 responses at a DR-4 element (4–5-fold), and T3 responses were weaker with TRβ at Pal (2–3-fold versus 4–5-fold for TRα). Western blotting of transfected cell extracts with an antibody against an N-terminal FLAG tag in both TRs revealed similar expression levels (Fig. 1A, inset). Moreover, varying the amounts of TR expression vector confirmed that differential effects were not related to differences in amounts of TR required for optimal activation at each element (not shown). Analysis of DNA binding preferences of TRs and RXR-TRs confirms that TRβ homodimerizes on DNA in vitro and that TRα does not (Fig. 1B). TRβ homodimers bind strongly to F2 and, to a lesser extent, to DR-4. As expected, T3 inhibited TRβ homodimer formation at both elements, although significant residual binding of liganded TRβ at F2 was detected in the presence of hormone. In the same gels, TRβ and TRα bound weakly to all three TREs as monomers. Moreover, in parallel, both TRs formed heterodimers with RXR at all three TREs, with preferential binding to DR-4 (DR-4 > F2 > Pal). Thus, TRβ activates transcription more efficiently at F2 than TRα, and this correlates with enhanced homodimer formation at F2 in vitro. TRβ Homodimers Activate Transcription at F2 in Yeast—To determine the extent to which different TRβ oligomers activate transcription in eukaryotes, we assembled different TR transcription complexes in yeast stably transfected with TRE-dependent reporters and examined the effects of a mutation in the TR LBD dimer surface (TRβL422R) in each context (17Ribeiro R.C. Feng W. Wagner R.L. Costa C.H. Pereira A.C. Apriletti J.W. Fletterick R.J. Baxter J.D. J. Biol. Chem. 2001; 276: 14987-14995Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). TRβ homodimers and RXR-TRs were both active in yeast but exhibited distinct TRE preferences (Fig. 2, A–C). In accordance with our previous results, TRβ gave weak T3 response at all three elements, regardless of the presence of coexpressed RXR, and these responses were enhanced by GRIP1 (35Walfish P.G. Yoganathan T. Yang Y.F. Hong H. Butt T.R. Stallcup M.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3697-3702Crossref PubMed Scopus (47) Google Scholar). With TRβ and GRIP1, large T3 responses were obtained at F2, and these were inhibited by the TRβL422R mutation (Fig. 2A). By contrast, T3 responses obtained with TRβ and GRIP1 at DR-4 and Pal were smaller and were enhanced by the same mutation (Fig. 2, B and C). Thus, TR-TR homodimers are active at F2, whereas homodimer formation places a constraint on T3 response at other elements. Although RXR did not enh" @default.
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• W2149936418 title "Thyroid Hormone Response Element Organization Dictates the Composition of Active Receptor" @default.
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