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• W2003349141 abstract "Although PGC-1 (peroxisome proliferator-activated receptor-γcoactivator-1) has been previously shown to enhance thyroid hormone receptor (TR)/retinoid X receptor-mediated ucp-1 gene expression in a ligand-induced manner in rat fibroblast cells, the precise mechanism of PGC-1 modulation of TR function has yet to be determined. In this study, we show that PGC-1 can potentiate TR-mediated transactivation of reporter genes driven by natural thyroid hormone response elements both in a ligand-dependent and ligand-independent manner and that the extent of coactivation is a function of the thyroid hormone response element examined. Our data also show that PGC-1 stimulation of TR activity in terms of Gal4 DNA-binding domain fusion is strictly ligand-dependent. In addition, an E457A AF-2 mutation had no effect on the ligand-induced PGC-1 enhancement of TR activity, indicating that the conserved charged residue in AF-2 is not essential for this PGC-1 function. Furthermore, GST pull-down and mammalian two-hybrid assays demonstrated that the PGC-1 LXXLL motif is required for ligand-induced PGC-1/TR interaction. This agonist-dependent PGC-1/TR interaction also requires both helix 1 and the AF-2 region of the TR ligand-binding domain. Taken together, these results support the notion that PGC-1 is a bona fide TR coactivator and that PGC-1 modulates TR activity via a mechanism different from that utilized with peroxisome proliferator activator receptor-γ. Although PGC-1 (peroxisome proliferator-activated receptor-γcoactivator-1) has been previously shown to enhance thyroid hormone receptor (TR)/retinoid X receptor-mediated ucp-1 gene expression in a ligand-induced manner in rat fibroblast cells, the precise mechanism of PGC-1 modulation of TR function has yet to be determined. In this study, we show that PGC-1 can potentiate TR-mediated transactivation of reporter genes driven by natural thyroid hormone response elements both in a ligand-dependent and ligand-independent manner and that the extent of coactivation is a function of the thyroid hormone response element examined. Our data also show that PGC-1 stimulation of TR activity in terms of Gal4 DNA-binding domain fusion is strictly ligand-dependent. In addition, an E457A AF-2 mutation had no effect on the ligand-induced PGC-1 enhancement of TR activity, indicating that the conserved charged residue in AF-2 is not essential for this PGC-1 function. Furthermore, GST pull-down and mammalian two-hybrid assays demonstrated that the PGC-1 LXXLL motif is required for ligand-induced PGC-1/TR interaction. This agonist-dependent PGC-1/TR interaction also requires both helix 1 and the AF-2 region of the TR ligand-binding domain. Taken together, these results support the notion that PGC-1 is a bona fide TR coactivator and that PGC-1 modulates TR activity via a mechanism different from that utilized with peroxisome proliferator activator receptor-γ. thyroid hormone thyroid hormone receptor DNA-binding domain ligand-binding domain thyroid hormone response element peroxisome proliferator-activated receptor glucocorticoid receptor glutathione S-transferase Thyroid hormone (T3)1 plays profound roles in development, homeostasis, and metabolism. These biological activities of T3 are mediated by thyroid hormone receptors (TRs), which, along with the receptors for steroid hormones, retinoids, and vitamin D, belong to the nuclear hormone receptor superfamily of ligand-activated transcription factors (1Mangelsdorf D.J. Thummel C. Beato M. Herrlich P. Schutz G. Umesono K. Blumberg B. Kastner P. Mark M. Chambon P. Evans R.M. Cell. 1995; 83: 835-839Abstract Full Text PDF PubMed Scopus (6088) Google Scholar, 2Burris T.P. Burris T.P. McCabe E.R.B. Nuclear Receptors and Genetic Disease: The Nuclear Receptor Superfamily. Academic Press, New York2000: 1-57Google Scholar). In mammals, two distinct genes, TRα and TRβ, produce several TR isoforms (3Lazar M.A. Endocr. Rev. 1993; 14: 184-193Crossref PubMed Scopus (811) Google Scholar). Like other nuclear receptors, TRs exhibit a modular structure with distinct functional domains. These include a highly variable N-terminal A/B domain harboring a constitutive activation function (AF-1) (1Mangelsdorf D.J. Thummel C. Beato M. Herrlich P. Schutz G. Umesono K. Blumberg B. Kastner P. Mark M. Chambon P. Evans R.M. Cell. 1995; 83: 835-839Abstract Full Text PDF PubMed Scopus (6088) Google Scholar, 4Wilkinson J.R. Towle H.C. J. Biol. Chem. 1997; 272: 23824-23832Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar), a highly conserved DNA-binding domain (DBD) containing two zinc fingers (5Umesono K. Evans R.M. Cell. 1989; 57: 1139-1146Abstract Full Text PDF PubMed Scopus (725) Google Scholar), and a C-terminal ligand-binding domain (LBD) containing a ligand-dependent activation function (AF-2) and a major receptor dimerization interface (6Spanjaard R.A. Darling D.S. Chin W.W. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8587-8591Crossref PubMed Scopus (43) Google Scholar, 7Qi J.S. Desai-Yajnik V. Greene M.E. Raaka B.M. Samuels H.H. Mol. Cell. Biol. 1995; 15: 1817-1825Crossref PubMed Google Scholar, 8Barettino D. Vivanco Ruiz M.M. Stunnenberg H.G. EMBO J. 1994; 13: 3039-3049Crossref PubMed Scopus (291) Google Scholar, 9Baniahmad A. Leng X. Burris T.P. Tsai S.Y. Tsai M.J. O'Malley B.W. Mol. Cell. Biol. 1995; 15: 76-86Crossref PubMed Google Scholar). In addition to those three major functional domains, a hinge region separating the TR DBD from the LBD also appears to be important for receptor function (10Miyamoto T. Kakizawa T. Ichikawa K. Nishio S. Takeda T. Suzuki S. Kaneko A. Kumagai M. Mori J. Yamashita K. Sakuma T. Hashizume K. Mol. Cell. Endocrinol. 2001; 181: 229-238Crossref PubMed Scopus (27) Google Scholar). Control of gene expression is a dynamic and complex process. Gene transcription involves assembly of multiple transcription factors at the distal enhancer region and the basal transcriptional machinery, including RNA polymerase II, at the core promoter of the target gene. Like other transcription factors, TRs exert their effects on gene regulation via binding to specific DNA sequences referred to as thyroid hormone response elements (TREs) in the regulatory regions of T3 target genes (11Williams G.R. Brent G.A. Weintraub B.D. Molecular Endocrinology: Basic Concepts and Clinical Correlations. Raven Press, Ltd., New York1995: 217-239Google Scholar). Unlike the classical steroid receptors, TRs can positively or negatively regulate T3-responsive gene expression, depending on the nature of the TREs and auxiliary proteins (12Wu Y. Koenig R.J. Trends Endocrinol. Metab. 2000; 11: 207-211Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). In the past few years, there has been enormous progress in elucidating the molecular mechanisms of nuclear receptor-mediated gene expression. In the current model, TR-modulated gene expression involves the sequential assembly of an array of coregulatory proteins, including coactivators and corepressors (13Rosenfeld M.G. Glass C.K. J. Biol. Chem. 2001; 276: 36865-36868Abstract Full Text Full Text PDF PubMed Scopus (432) Google Scholar, 14Glass C.K. Rosenfeld M.G. Genes Dev. 2000; 14: 121-141Crossref PubMed Google Scholar). In general, the binding of unliganded TRs to “positive” TREs results in repression of basal transcription, and this silencing is mediated by interaction of TR with a corepressor complex that contains histone deacetylase activity (15Horlein 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 (1712) Google Scholar, 16Nagy L. Kao H.Y. Chakravarti D. Lin R.J. Hassig C.A. Ayer D.E. Schreiber S.L. Evans R.M. Cell. 1997; 89: 373-380Abstract Full Text Full Text PDF PubMed Scopus (1107) Google Scholar). Binding of ligand triggers significant conformational changes (17Bourguet W. Germain P. Gronemeyer H. Trends Pharmacol. Sci. 2000; 21: 381-388Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar), including the repositioning of the amphipathic helix 12 containing the core of AF-2 in the TR LBD (14Glass C.K. Rosenfeld M.G. Genes Dev. 2000; 14: 121-141Crossref PubMed Google Scholar, 18Wagner R.L. Apriletti J.W. McGrath M.E. West B.L. Baxter J.D. Fletterick R.J. Nature. 1995; 378: 690-697Crossref PubMed Scopus (811) Google Scholar), that result in release of the corepressor complex and recruitment of a coactivator complex (19Onate S.A. Tsai S.Y. Tsai M.J. O'Malley B.W. Science. 1995; 270: 1354-1357Crossref PubMed Scopus (2058) Google Scholar). A coactivator complex is usually associated with histone acetyltransferase activity, through which the chromatin structure may be modified, leading to transcriptional activation (20Spencer T.E. Jenster G. Burcin M.M. Allis C.D. Zhou J. Mizzen C.A. McKenna N.J. Onate S.A. Tsai S.Y. Tsai M.J. O'Malley B.W. Nature. 1997; 389: 194-198Crossref PubMed Scopus (1066) Google Scholar, 21Marmorstein R. Roth S.Y. Curr. Opin. Genet. Dev. 2001; 11: 155-161Crossref PubMed Scopus (314) Google Scholar). It appears that ligand-dependent recruitment of coactivators is critical for TR and other nuclear receptor-mediated transcriptional activation. To date, a large number of coactivators for TR have been defined and characterized. They include at least several proteins: 1) three members of the structurally and functionally related p160 family, SRC-1/NCoA-1 (19Onate S.A. Tsai S.Y. Tsai M.J. O'Malley B.W. Science. 1995; 270: 1354-1357Crossref PubMed Scopus (2058) Google Scholar), TIF2/GRIP-1/NCoA-2 (22Hong H. Kohli K. Trivedi A. Johnson D.L. Stallcup M.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4948-4952Crossref PubMed Scopus (614) Google Scholar, 23Voegel J.J. Heine M.J. Zechel C. Chambon P. Gronemeyer H. EMBO J. 1996; 15: 3667-3675Crossref PubMed Scopus (952) Google Scholar), and p/CIP/ACTR/AIB1/RAC3/TRAM-1 (24Torchia J. Rose D.W. Inostroza J. Kamei Y. Westin S. Glass C.K. Rosenfeld M.G. Nature. 1997; 387: 677-684Crossref PubMed Scopus (1107) Google Scholar, 25Chen H. Lin R.J. Schiltz R.L. Chakravarti D. Nash A. Nagy L. Privalsky M.L. Nakatani Y. Evans R.M. Cell. 1997; 90: 569-580Abstract Full Text Full Text PDF PubMed Scopus (1268) Google Scholar, 26Anzick S.L. Kononen J. Walker R.L. Azorsa D.O. Tanner M.M. Guan X.Y. Sauter G. Kallioniemi O.P. Trent J.M. Meltzer P.S. Science. 1997; 277: 965-968Crossref PubMed Scopus (1432) Google Scholar, 27Li H. Gomes P.J. Chen J.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8479-8484Crossref PubMed Scopus (504) Google Scholar, 28Takeshita A. Cardona G.R. Koibuchi N. Suen C.S. Chin W.W. J. Biol. Chem. 1997; 272: 27629-27634Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar); 2) p300/CBP (cAMP response element-binding protein-binding protein) (29Chakravarti D. LaMorte V.J. Nelson M.C. Nakajima T. Schulman I.G. Juguilon H. Montminy M. Evans R.M. Nature. 1996; 383: 99-103Crossref PubMed Scopus (850) Google Scholar) and p/CAF (p300/CBP-associated factor) (30Blanco J.C. Minucci S. Lu J. Yang X.J. Walker K.K. Chen H. Evans R.M. Nakatani Y. Ozato K. Genes Dev. 1998; 12: 1638-1651Crossref PubMed Scopus (340) Google Scholar); and 3) TRAP/DRIP/ARC (TR-associated proteins) (31Fondell J.D. Ge H. Roeder R.G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8329-8333Crossref PubMed Scopus (462) Google Scholar). Most of the characterized coactivators interact with the LBDs of ligand-bound nuclear receptors, including TRs, through helical LXXLL motifs (where X is any amino acid) present within p160 family members and other coactivators (32Heery D.M. Kalkhoven E. Hoare S. Parker M.G. Nature. 1997; 387: 733-736Crossref PubMed Scopus (1772) Google Scholar). p160 family proteins and other coactivators contain one or more copies of the LXXLL motif in their central nuclear receptor interaction domains (33Leo C. Chen J.D. Gene (Amst.). 2000; 245: 1-11Crossref PubMed Scopus (440) Google Scholar). Thus, TRs and other nuclear receptors may differentially interact with various combinations of these multiple LXXLL motifs to assemble distinct coactivator complexes (34McInerney E.M. Rose D.W. Flynn S.E. Westin S. Mullen T.M. Krones A. Inostroza J. Torchia J. Nolte R.T. Assa-Munt N. Milburn M.V. Glass C.K. Rosenfeld M.G. Genes Dev. 1998; 12: 3357-3368Crossref PubMed Scopus (528) Google Scholar, 35Westin S. Kurokawa R. Nolte R.T. Wisely G.B. McInerney E.M. Rose D.W. Milburn M.V. Rosenfeld M.G. Glass C.K. Nature. 1998; 395: 199-202Crossref PubMed Scopus (303) Google Scholar), possibly leading to selective expression of target genes. However, emerging evidence indicates that tissue-specific or inducible coactivators may play a critical role in achieving selective and tissue-specific gene expression (36Muller J.M. Isele U. Metzger E. Rempel A. Moser M. Pscherer A. Breyer T. Holubarsch C. Buettner R. Schule R. EMBO J. 2000; 19: 359-369Crossref PubMed Scopus (288) Google Scholar, 37Puigserver P. Wu Z. Park C.W. Graves R. Wright M. Spiegelman B.M. Cell. 1998; 92: 829-839Abstract Full Text Full Text PDF PubMed Scopus (3086) Google Scholar). Unlike most characterized coactivators that are often expressed ubiquitously, PGC-1 (PPARγcoactivator-1) displays a pattern of tissue-specific expression and is inducible by various stimuli, including exposure to cold temperature and exercise (37Puigserver P. Wu Z. Park C.W. Graves R. Wright M. Spiegelman B.M. Cell. 1998; 92: 829-839Abstract Full Text Full Text PDF PubMed Scopus (3086) Google Scholar, 38Goto M. Terada S. Kato M. Katoh M. Yokozeki T. Tabata I. Shimokawa T. Biochem. Biophys. Res. Commun. 2000; 274: 350-354Crossref PubMed Scopus (210) Google Scholar). Although PGC-1 was initially identified as a coactivator for the nuclear receptor PPARγ, accumulating data have shown that PGC-1 plays a broader role in mediating transactivation by other nuclear receptors and transcription factors (39Knutti D. Kralli A. Trends Endocrinol. Metab. 2001; 12: 360-365Abstract Full Text Full Text PDF PubMed Scopus (249) Google Scholar). Most importantly, PGC-1 has been implicated in the regulation of many important physiological processes, including adaptive thermogenesis (37Puigserver P. Wu Z. Park C.W. Graves R. Wright M. Spiegelman B.M. Cell. 1998; 92: 829-839Abstract Full Text Full Text PDF PubMed Scopus (3086) Google Scholar, 40Rosen E.D. Spiegelman B.M. Annu. Rev. Cell Dev. Biol. 2000; 16: 145-171Crossref PubMed Scopus (1053) Google Scholar) and hepatic gluconeogenesis (41Yoon J.C. Puigserver P. Chen G. Donovan J. Wu Z. Rhee J. Adelmant G. Stafford J. Kahn C.R. Granner D.K. Newgard C.B. Spiegelman B.M. Nature. 2001; 413: 131-138Crossref PubMed Scopus (1515) Google Scholar). Given that T3 is a key regulator of energy metabolism and that the major target tissues of T3 in mammals overlap with the selective distribution of PGC-1, a role of PGC-1 as an important physiological modulator for TR action is suggested. Previous observations that PGC-1 stimulates TR/retinoid X receptor-mediateducp-1 (uncouplingprotein-1) gene transcription in a ligand-induced manner in rat fibroblast cells has strongly supported this notion (37Puigserver P. Wu Z. Park C.W. Graves R. Wright M. Spiegelman B.M. Cell. 1998; 92: 829-839Abstract Full Text Full Text PDF PubMed Scopus (3086) Google Scholar). However, the precise mechanism of PGC-1 coactivation of TR has yet to be determined. Studies of several other nuclear receptors, including PPARγ, PPARα, estrogen receptor-α, and GR, have suggested that PGC-1 may adopt distinct mechanisms of action, depending on the identity of the nuclear receptor in question (37Puigserver P. Wu Z. Park C.W. Graves R. Wright M. Spiegelman B.M. Cell. 1998; 92: 829-839Abstract Full Text Full Text PDF PubMed Scopus (3086) Google Scholar,42Tcherepanova I. Puigserver P. Norris J.D. Spiegelman B.M. McDonnell D.P. J. Biol. Chem. 2000; 275: 16302-16308Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 43Knutti D. Kaul A. Kralli A. Mol. Cell. Biol. 2000; 20: 2411-2422Crossref PubMed Scopus (242) Google Scholar, 44Vega R.B. Huss J.M. Kelly D.P. Mol. Cell. Biol. 2000; 20: 1868-1876Crossref PubMed Scopus (942) Google Scholar). For example, PGC-1 interacts with PPARγ in a ligand-independent fashion via the hinge region of the receptor (37Puigserver P. Wu Z. Park C.W. Graves R. Wright M. Spiegelman B.M. Cell. 1998; 92: 829-839Abstract Full Text Full Text PDF PubMed Scopus (3086) Google Scholar), whereas the ligand-induced interaction with PPARα, estrogen receptor-α, and GR depends on the AF-2 region of the receptor (42Tcherepanova I. Puigserver P. Norris J.D. Spiegelman B.M. McDonnell D.P. J. Biol. Chem. 2000; 275: 16302-16308Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 43Knutti D. Kaul A. Kralli A. Mol. Cell. Biol. 2000; 20: 2411-2422Crossref PubMed Scopus (242) Google Scholar, 44Vega R.B. Huss J.M. Kelly D.P. Mol. Cell. Biol. 2000; 20: 1868-1876Crossref PubMed Scopus (942) Google Scholar). In this report, utilizing GST pull-down and mammalian two-hybrid assays, we provide evidence that PGC-1 interacts with TRβ1 in a largely ligand-dependent manner. The LXXLL motif of PGC-1 and the intact AF-2 region of the receptor mediate the agonist-dependent PGC-1/TR interaction. Surprisingly, helix 1 of the TR LBD is also essential for this process. Furthermore, functional studies demonstrate that the effect of PGC-1 on TR transcriptional activation can be ligand-dependent and ligand-independent, depending on the structure of the response element with which TR interacts. The 1XTRE-tk-Luc and 3XTRE-tk-Luc reporters, containing one and three copies, respectively, of a characterized direct repeat TRE derived from the rat α-myosin heavy chain (45Brent G.A. Williams G.R. Harney J.W. Forman B.M. Samuels H.H. Moore D.D. Larsen P.R. Mol. Endocrinol. 1992; 6: 502-514Crossref PubMed Scopus (38) Google Scholar), were generated by inserting double-stranded oligonucleotide response elements upstream of a minimal thymidine kinase promoter linked to a luciferase gene (pTAL, CLONTECH). The DR4-tk-Luc reporter contains a copy of a direct repeat TRE from human 5′-deiodinase type I, TRE2 (46Toyoda N. Zavacki A.M. Maia A.L. Harney J.W. Larsen P.R. Mol. Cell. Biol. 1995; 15: 5100-5112Crossref PubMed Scopus (124) Google Scholar). The IP6-tk-Luc reporter contains a single copy of an inverted palindrome TRE from mouse myelin basic protein (47Jeannin E. Robyr D. Desvergne B. J. Biol. Chem. 1998; 273: 24239-24248Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). PCR-amplified full-length human TRβ1 and TRα1 and rat TRβ2 cDNAs were cloned into the pcDNA3.1(−) expression vector (Invitrogen). The TRβ1 E457A mutant was generated by PCR-based mutagenesis. pcDNA3-PGC-1 expressing full-length human PGC-1 was a generous gift from Dr. A. Kralli and has been previously described (43Knutti D. Kaul A. Kralli A. Mol. Cell. Biol. 2000; 20: 2411-2422Crossref PubMed Scopus (242) Google Scholar). Gal4TRβ1 and the various deletions and mutations were created by cloning PCR-amplified DNA fragments corresponding to the different TR regions into the EcoRI andSalI sites of the pM vector (CLONTECH). These varieties of DNA fragments were also cloned into pcDNA3. Gal4 DBD-PPARγ LBD was described previously (48Hourton D. Delerive P. Stankova J. Staels B. Chapman M.J. Ninio E. Biochem. J. 2001; 354: 225-232Crossref PubMed Scopus (29) Google Scholar). VP16-PGC-1 and the LXXLL motif mutant VP16-PGC-1AXXAL were generated by cloning PCR-amplified DNA encoding PGC-1 amino acids 100–411 into theEcoRI and XhoI sites of the VP16 vector. The GRIP-1 expression vector was obtained from Dr. R. J. Koenig. To ensure the fidelity of the resulting constructs, all predicted mutations and PCR-based constructs were verified by DNA sequencing. Hela and CV-1 cells were routinely maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Hyclone Laboratories). Prior to transfection, the cells were seeded in 24-well plates at a density of 5 × 104 cells/well in Dulbecco's modified Eagle's medium supplemented with 10% serum. After 16 h of growth at 37 °C and 5% CO2, cells were transfected with LipofectAMINE 2000 reagent (Invitrogen) according to the manufacturer's protocol. The next morning, the transfected cells were washed with phosphate-buffered saline, and fresh media containing 10% charcoal-stripped serum and 10−6mT3, if indicated, were added. After 24 h, the cells were washed with phosphate-buffered saline and harvested with a cell culture lysis buffer (Promega). Luciferase activity was measured in a Dynex luminometer. Generally, all transfections included 50 ng of expression vector for receptors, 100 ng of expression vector for coactivators, 250 ng of pG5-Luc reporter, and 100 ng of TRE-Luc reporter. All experiments were done at least twice in triplicate. GST pull-down assays were performed as previously described (49Ko L. Cardona G.R. Chin W.W. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6212-6217Crossref PubMed Scopus (128) Google Scholar) with minor modifications. Bacterially expressed GST fusion proteins bound to glutathione-Sepharose 4B beads were incubated with in vitro translated35S-labeled receptors in binding buffer containing 20 mm Tris (pH 7.5), 75 mm KCl, 50 mmNaCl, 1 mm EDTA (pH 8.0), 0.05% Nonidet P-40, 10% glycerol, 1 mm dithiothreitol, and one tablet of protease inhibitor mixture (Roche Molecular Biochemicals). After incubation for 1–2 h at room temperature in the presence or absence of 10−6m T3, the beads were extensively washed with binding buffer, and the bound proteins were analyzed by SDS-PAGE and visualized by autoradiography. To determine the ability of PGC-1 to coactivate TR-dependent transactivation from different TREs in various cell lines, we performed transient transfection experiments in HeLa and CV-1 cells using various luciferase reporter constructs. The 1XTRE-tk-Luc and 3XTRE-tk-Luc reporters contain one and three copies, respectively, of a direct repeat TRE derived from the rat α-myosin heavy chain gene upstream of a minimal thymidine kinase promoter linked to a luciferase reporter gene, whereas the DR4-tk-Luc construct contains a single copy of a direct repeat TRE from the human 5′-deiodinase type I gene. The IP6-tk-Luc reporter contains a single copy of an inverted palindrome TRE from the mouse myelin basic protein. Fig.1 shows that, in the presence of T3, PGC-1 potently augmented the transcription of 1XTRE-tk-Luc by TRβ1 4.6-fold. This PGC-1-mediated activation was further elevated to 8.7-fold with the 3XTRE-tk-Luc reporter. Similarly, the activities of the TR-dependent DR4-tk-Luc and IP6-tk-Luc reporters were stimulated by PGC-1 ∼5.2- and 6.9-fold, respectively. Intriguingly, expression of PGC-1 also resulted in a 3.7-fold ligand-independent increase in TR-mediated DR4-tk-Luc reporter activation relative to a vector control containing no TRE, whereas a mild increase (1.5-fold) was also observed with either the 1XTRE-tk-Luc or IP6-tk-Luc reporter. In contrast, no such ligand-independent PGC-1 enhancement was obtained with 3XTRE-tk-Luc. Similar results were obtained using CV-1 cells (data not shown). Thus, the effect of PGC-1 on TR-mediated transactivation has both ligand-dependent and ligand-independent components that are a function of the TRE utilized. Furthermore, these effects do not appear to be dependent on the cell type utilized. There are at least three functional TR isoforms: TRα1, TRβ1, and TRβ2. Although they are structurally and functionally related, expression of TRα1 and TRβ (TRβ1 and TRβ2) in distinct but overlapping patterns suggests that the TR isoforms may have both distinct and common functional roles (3Lazar M.A. Endocr. Rev. 1993; 14: 184-193Crossref PubMed Scopus (811) Google Scholar, 50Zhang J. Lazar M.A. Annu. Rev. Physiol. 2000; 62: 439-466Crossref PubMed Scopus (576) Google Scholar). PGC-1 also displays a tissue-specific pattern of expression and has been shown to be highly expressed in cold-induced brown fat cells and in skeletal muscle (37Puigserver P. Wu Z. Park C.W. Graves R. Wright M. Spiegelman B.M. Cell. 1998; 92: 829-839Abstract Full Text Full Text PDF PubMed Scopus (3086) Google Scholar). Because both PGC-1 and TRs exhibit tissue-selective expression, we investigated whether PGC-1 function on TR exhibits an isoform specificity. To address this possibility, the effect of PGC-1 on the transcriptional activity of the three TR isoforms fused to a Gal4 DBD was examined by mammalian one-hybrid analysis. As shown in Fig.2, PGC-1 stimulated the transcriptional activation of all three Gal4-TR constructs to a similar level. This indicates that PGC-1 exhibits no preference for TR isoforms, at least within our experimental conditions. Unlike the effect of PGC-1 on TR-mediated reporter gene expression, coactivation of the transcriptional activity from the Gal4-TR constructs by PGC-1 was entirely ligand-dependent. However, in contrast to TR, coactivation of the transcriptional activity of Gal4-PPARγ by PGC-1 was ligand-independent, which is in agreement with a previous report (37Puigserver P. Wu Z. Park C.W. Graves R. Wright M. Spiegelman B.M. Cell. 1998; 92: 829-839Abstract Full Text Full Text PDF PubMed Scopus (3086) Google Scholar). Taken together, these observations confirm that PGC-1 is a potent coactivator for TR action and suggest that PGC-1 coactivation of TR is influenced by the structure of the promoter with which TR interacts. Biochemical and x-ray crystallographic studies have established that the AF-2 domain within helix 12 of TR and other nuclear receptors plays a critical role in mediating ligand-induced transcriptional activation (17Bourguet W. Germain P. Gronemeyer H. Trends Pharmacol. Sci. 2000; 21: 381-388Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar, 18Wagner R.L. Apriletti J.W. McGrath M.E. West B.L. Baxter J.D. Fletterick R.J. Nature. 1995; 378: 690-697Crossref PubMed Scopus (811) Google Scholar). Mutational analysis has revealed that highly conserved residues such as Glu457 and Leu454 in this domain are very important for the recruitment of coactivators such as p160 proteins (51Feng W. Ribeiro R.C. Wagner R.L. Nguyen H. Apriletti J.W. Fletterick R.J. Baxter J.D. Kushner P.J. West B.L. Science. 1998; 280: 1747-1749Crossref PubMed Scopus (516) Google Scholar, 52Tone Y. Collingwood T.N. Adams M. Chatterjee V.K. J. Biol. Chem. 1994; 269: 31157-31161Abstract Full Text PDF PubMed Google Scholar). To assess whether the PGC-1 effect on TR also depends on these highly conserved residues, we employed a TRβ1 E457A mutant fused to the Gal4 DBD and performed transient transfection analysis in HeLa cells. As expected, in the absence of T3, expression of wild-type Gal4-TRβ1 repressed basal transcription. Addition of T3 resulted in a 5-fold induction of Gal4-TRβ1 activity (Fig. 3). The ligand-induced transcriptional activation of Gal4-TRβ1 was diminished ∼3-fold by the E457A mutation when no coactivators were ectopically expressed in the cells (Fig. 3). The decreased Gal4-TRβ1 activity is likely due to impaired interaction with endogenous p160 or other coactivators. As observed earlier, a 20-fold increase in the ligand-dependent transcriptional activity of Gal4-TRβ1 was achieved when PGC-1 was coexpressed (Fig. 3). Surprisingly, the E457A mutant did not alter the ability of PGC-1 to stimulate the ligand-dependent transcriptional activity of Gal4-TRβ1 (Fig. 3). However, enhancement of Gal4-TRβ1 activity by overexpressing SRC-1 was severely impaired (>70%) by this E457A mutant (data not shown). Therefore, these data indicate that the requirement of critical amino acid residues in the TR AF-2 region for PGC-1 function is distinct from that seen with p160 coactivators. Initial domain analysis of PPARγ revealed that the interaction of PGC-1 with this receptor is ligand-independent and mediated by a region spanning part of the DBD and the hinge region (37Puigserver P. Wu Z. Park C.W. Graves R. Wright M. Spiegelman B.M. Cell. 1998; 92: 829-839Abstract Full Text Full Text PDF PubMed Scopus (3086) Google Scholar). The AF-2 domain was suggested not to be involved in PGC-1 recruitment. However, studies with PPARα and GR revealed that the effect of PGC-1 on these receptors is ligand- and AF-2-dependent (42Tcherepanova I. Puigserver P. Norris J.D. Spiegelman B.M. McDonnell D.P. J. Biol. Chem. 2000; 275: 16302-16308Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 43Knutti D. Kaul A. Kralli A. Mol. Cell. Biol. 2000; 20: 2411-2422Crossref PubMed Scopus (242) Google Scholar, 44Vega R.B. Huss J.M. Kelly D.P. Mol. Cell. Biol. 2000; 20: 1868-1876Crossref PubMed Scopus (942) Google Scholar). To determine the functional domain(s) responsible for PGC-1 coactivation of TR, a series of TR LBD deletion mutants fused to the Gal4 DBD were used to perform mammalian one-hybrid assays. As shown earlier, PGC-1 had no effect on the Gal4 DBD itself either in the presence or absence of T3. PGC-1 greatly enhanced the T3-dependent transcriptional activity of full-length TRβ1 fused to the Gal4 DBD (Fig. 4 B). Surprisingly, the N-terminally truncated mutant containing only the intact TRβ LBD (Gal4-TR216) not only increased its transcriptional activity without coexpression of PGC-1, but also exhibited a significant stimulation of the transcription by PGC-1 compared with the wild-type receptor (Fig. 4 B). This unexpected finding indicates that the deleted region may have an inhibitory effect on the receptor activity modulated by PGC-1 or other coactivators (53Liu Y. Takeshita A. Nagaya T. Baniahmad A. Chin W.W. Yen P.M. Mol. Endocrinol. 1998; 12: 34-44Crossref PubMed Scopus (11) Google Scholar). However, as shown in Fig. 4 B, the T3-activated PGC-1 enhancement was completely abolished by the deletion mutant Gal4-TR233, which lacks helix 1, but contai" @default.
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• W2003349141 date "2002-03-01" @default.
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• W2003349141 title "Requirement of Helix 1 and the AF-2 Domain of the Thyroid Hormone Receptor for Coactivation by PGC-1" @default.
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• W2003349141 doi "https://doi.org/10.1074/jbc.m110761200" @default.
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