Title: The Steroid Receptor Coactivator-1 Contains Multiple Receptor Interacting and Activation Domains That Cooperatively Enhance the Activation Function 1 (AF1) and AF2 Domains of Steroid Receptors
Abstract: Steroid receptors are ligand-inducible transcription factors, and their association with steroid receptor coactivators (SRCs) upon binding to DNA is necessary for them to achieve full transcriptional potential. To understand the mechanism of SRC-1 action, its ability to interact and enhance the transcriptional activity of steroid receptors was analyzed. First, we show that SRC-1 is a modular coactivator that possesses intrinsic transcriptional activity when tethered to DNA and that it harbors two distinct activation domains, AD1 and AD2, needed for the maximum coactivation function of steroid receptors. We also demonstrate that SRC-1 interacts with both the amino-terminal A/B or AF1-containing domain and the carboxyl-terminal D/E or AF2-containing domain of the steroid receptors. These interactions are carried out by multiple regions of SRC-1, and they are relevant for transactivation. In addition to the inherent histone acetyltransferase activity of SRC-1, the presence of multiple receptor-coactivator interaction sites in SRC-1 and its ability to interact with components of the basic transcriptional machinery appears to be, at least in part, the mechanism by which the individual activation functions of the steroid receptors act cooperatively to achieve full transcriptional activity. Steroid receptors are ligand-inducible transcription factors, and their association with steroid receptor coactivators (SRCs) upon binding to DNA is necessary for them to achieve full transcriptional potential. To understand the mechanism of SRC-1 action, its ability to interact and enhance the transcriptional activity of steroid receptors was analyzed. First, we show that SRC-1 is a modular coactivator that possesses intrinsic transcriptional activity when tethered to DNA and that it harbors two distinct activation domains, AD1 and AD2, needed for the maximum coactivation function of steroid receptors. We also demonstrate that SRC-1 interacts with both the amino-terminal A/B or AF1-containing domain and the carboxyl-terminal D/E or AF2-containing domain of the steroid receptors. These interactions are carried out by multiple regions of SRC-1, and they are relevant for transactivation. In addition to the inherent histone acetyltransferase activity of SRC-1, the presence of multiple receptor-coactivator interaction sites in SRC-1 and its ability to interact with components of the basic transcriptional machinery appears to be, at least in part, the mechanism by which the individual activation functions of the steroid receptors act cooperatively to achieve full transcriptional activity. Steroid receptors belong to a superfamily of transcription factors that regulate hormone-responsive genes and thereby cellular growth and differentiation. In the absence of hormone, the receptor is maintained in an inactive or repressive state by association with heat shock proteins and/or corepressors. Activation of the aporeceptor by ligand binding involves structural and functional changes in the receptor molecule that promote release from the inactive or repressive state to bind specific DNA hormone response elements. In addition, the ligand-bound receptor promotes the recruitment of coactivators to the receptor-DNA complex and thus entitles the receptor to achieve its full transactivation capacity (for review, see Refs. 1Tsai M.J. O'Malley B.W. Annu. Rev. Biochem. 1994; 63: 451-486Crossref PubMed Scopus (2678) Google Scholar, 2Horwitz K.B. Jackson T.A. Bain D.L. Richer J.K. Takimoto G.S. Tung L. Mol. 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The squelching observed between receptor family members and between their various AFs 1The abbreviations used are: AF, activation function; SRC, steroid receptor coactivator; aa, amino acid(s); mAb, monoclonal antibody; PR, progesterone receptor; ER, estrogen receptor; PAGE, polyacrylamide gel electrophoresis; TBP, TATA-binding protein; GR, glucocorticoid receptor.1The abbreviations used are: AF, activation function; SRC, steroid receptor coactivator; aa, amino acid(s); mAb, monoclonal antibody; PR, progesterone receptor; ER, estrogen receptor; PAGE, polyacrylamide gel electrophoresis; TBP, TATA-binding protein; GR, glucocorticoid receptor. suggests that limiting intracellular coactivators are also needed for mediating receptor function (17Shemshedini L. Ji J.W. Brou C. Chambon P. Gronemeyer H. J. Biol. Chem. 1992; 267: 1834-1839Abstract Full Text PDF PubMed Google Scholar, 18Meyer M.E. Gronemeyer H. Turcotte B. Bocquel M.T. Tasset D. Chambon P. Cell. 1989; 57: 433-442Abstract Full Text PDF PubMed Scopus (441) Google Scholar). Furthermore, the synergism observed between the two transactivation functions (AF1 and AF2) of a single receptor suggest that the proper assembly of the individual activation functions of the steroid receptor is required to render the steroid receptor-DNA complex transcriptionally productive. Today, several receptor coactivators have been identified and characterized. Sequence comparisons indicate that they belong to a family of proteins termed steroid receptor coactivators (SRCs) and include SRC-1 (19Onate S.A. Tsai S.Y. Tsai M.J. O'Malley B.W. Science. 1995; 270: 1354-1357Crossref PubMed Scopus (2042) Google Scholar) (or NCoA-1 (20Kamei Y. Xu L. Heinzel T. Torchia J. Kurokawa R. Gloss B. Lin S.C. Heyman R.A. Rose D.W. Glass C.K. Rosenfeld M.G. Cell. 1996; 85: 403-414Abstract Full Text Full Text PDF PubMed Scopus (1916) Google Scholar, 21Torchia J. Rose D.W. Inostroza J. Kamei Y. Westin S. Glass C.K. 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The precise mechanism by which steroid receptor coactivators modulate transactivation function remains to be determined.To better understand the role of coactivators in receptor action, we studied the effect of SRC-1 and SRC-2/TIF2 on the transcriptional activity of several steroid receptors. We show that SRC-1 is a modular coactivator harboring intrinsic independent activation functions required for coactivation in intact cells. We also show that SRC-1 is able to interact with both the amino terminus A/B-C (AF1) and the carboxyl terminus C-D/E (AF2) regions of several steroid receptors. In addition, the cooperativity observed between AF1 and AF2, regions of the steroid receptors, appears to be assisted by SRC-1 and SRC-2/TIF2. We proposed that coactivators provide, in part a mechanism by which the independent AFs of the steroid receptors communicate within a transcription complex on target DNA elements.DISCUSSIONConsiderable understanding of the mechanism by which nuclear receptors activate gene expression has been gained with the cloning and characterization of several steroid receptor coactivators. Modulation of receptor activity by coactivators is a complex multistep process and appears to involve enzymatic remodeling at the chromatin level for access of receptor to DNA (12Beato M. Sanchez-Pacheco A. Endocr. Rev. 1996; 17: 587-609Crossref PubMed Scopus (348) Google Scholar, 24Chen H.W. 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 (1253) Google Scholar, 38Bannister A.J. Kouzarides T. Nature. 1996; 384: 641-643Crossref PubMed Scopus (1523) Google Scholar, 39Ogryzko V.V. Schiltz R.L. Russanova V. Howard B.H. Nakatani Y. Cell. 1996; 87: 953-959Abstract Full Text Full Text PDF PubMed Scopus (2368) Google Scholar, 40Spencer T.E. Jenster G. Burcin M.M. Allis C.D. Zhou J.X. 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 (1054) Google Scholar, 41Jenster G. Spencer T.E. Burcin M.M. Tsai S.Y. Tsai M.J. O'Malley B.W. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7879-7884Crossref PubMed Scopus (231) Google Scholar), as well as interactions with components of the RNA polymerase II transcriptional machinery at the promoter of hormone-responsive genes (6Ing N.H. Beekman J.M. Tsai S.Y. Tsai M.-J. O'Malley B.W. J. Biol. Chem. 1992; 267: 17617-17623Abstract Full Text PDF PubMed Google Scholar, 7Baniahmad A. Ha I. Reinberg D. Tsai S. Tsai M.J. O'Malley B.W. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8832-8836Crossref PubMed Scopus (301) Google Scholar, 8Jacq X. Brou C. Lutz Y. Davidson I. Chambon P. Tora L. Cell. 1994; 79: 107-117Abstract Full Text PDF PubMed Scopus (344) Google Scholar, 9Blanco J.C. Wang I.M. Tsai S.Y. Tsai M.J. O'Malley B.W. Jurutka P.W. Haussler M.R. Ozato K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1535-1539Crossref PubMed Scopus (183) Google Scholar, 10Schulman I.G. Chakravarti D. Juguilon H. Romo A. Evans R.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8288-8292Crossref PubMed Scopus (90) Google Scholar, 11Schwerk C. Klotzbucher M. Sachs M. Ulber V. Klein-Hitpass L. J. Biol. Chem. 1995; 270: 21331-21338Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar, 12Beato M. Sanchez-Pacheco A. Endocr. Rev. 1996; 17: 587-609Crossref PubMed Scopus (348) Google Scholar, 13May M. Mengus G. Lavigne A.C. Chambon P. Davidson I. EMBO J. 1996; 15: 3093-3104Crossref PubMed Scopus (75) Google Scholar, 14McEwan I. Gustafsson J.-A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8485-8490Crossref PubMed Scopus (132) Google Scholar, 15Mengus G. May M. Carre L. Chambon P. Davidson I. Genes Dev. 1997; 11: 1381-1395Crossref PubMed Scopus (132) Google Scholar, 16Rochette-Egly C. Adam S. Rossignol M. Egly J.M. Chambon P. Cell. 1997; 90: 97-107Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar). Consistent with the predicted properties of a coactivator, we show here that SRC-1 activates transcription when tethered to DNA by fusion with a heterologous DNA binding domain. Deletion of either AD1 or AD2 decreases the coactivation function of SRC-1 in intact cells (Fig. 1). Thus, the data highlight the relevance of SRC-1 and its domains in coactivation and steroid receptor function. AD1 includes the basic helix-loop-helix motif, which is highly conserved among the SRC-1 family members (20Kamei Y. Xu L. Heinzel T. Torchia J. Kurokawa R. Gloss B. Lin S.C. Heyman R.A. Rose D.W. Glass C.K. Rosenfeld M.G. Cell. 1996; 85: 403-414Abstract Full Text Full Text PDF PubMed Scopus (1916) Google Scholar, 21Torchia J. Rose D.W. Inostroza J. Kamei Y. Westin S. Glass C.K. Rosenfeld M.G. Nature. 1997; 387: 677-684Crossref PubMed Scopus (1102) Google Scholar, 22Voegel J.J. Heine M.J. Zechel C. Chambon P. Gronemeyer H. EMBO J. 1996; 15: 3667-3675Crossref PubMed Scopus (947) Google Scholar, 23Hong H. Kohli K. Trivedi A. Johnson D.L. Stallcup M.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4948-4952Crossref PubMed Scopus (611) Google Scholar, 24Chen H.W. 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 (1253) Google Scholar, 25Li H. Gomes P.J. Chen J.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8479-8484Crossref PubMed Scopus (499) 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 (1422) Google Scholar). This may explain, at least in part, the intrinsic transcription activity observed in some SRCs family members, including TIF2/GRIP1 (22Voegel J.J. Heine M.J. Zechel C. Chambon P. Gronemeyer H. EMBO J. 1996; 15: 3667-3675Crossref PubMed Scopus (947) Google Scholar, 23Hong H. Kohli K. Trivedi A. Johnson D.L. Stallcup M.R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4948-4952Crossref PubMed Scopus (611) Google Scholar) and RAC3 (25Li H. Gomes P.J. Chen J.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8479-8484Crossref PubMed Scopus (499) Google Scholar). In view of the role of basic helix-loop-helix domains in dimerization and DNA binding for several activators (49Baxevanis A.D. Vinson C.R. Curr. Opin. Genet. Dev. 1993; 3: 278-285Crossref PubMed Scopus (176) Google Scholar), the intrinsic transcription activity of AD1 may be the result of the direct interaction(s) with general transcription factors and/or the recruitment of intermediary factors capable of dimerization and consequent stabilization of the preinitiation complex. In contrast, sequence comparison of AD2 failed to reveal homology to any protein canonical domain, including SRC-1 family members. Therefore, mapping of the activation domain and elucidation of the downstream interacting protein(s) for the SRCs are critical for understanding the mechanism by which SRC-1 modulates steroid receptor function.At least two AF domains have been identified for the steroid/nuclear hormone receptors: a poorly characterized AF1 located at the amino-terminal A/B region and the ligand-inducible AF2 located at the D/E region of the steroid receptors. The AF2 domain contains an amphiphatic α-helix (helix 12) that is highly conserved between the steroid receptor superfamily members (50Danielian P.S. White R. Lees J.A. Parker M.G. EMBO J. 1992; 11: 1025-1033Crossref PubMed Scopus (716) Google Scholar). Here, we have shown that SRC-1 is capable of interacting with both the A/B and D/E regions via multiple receptor interaction sites. The original receptor interacting region for SRC-1 was found at the carboxyl terminus (19Onate S.A. Tsai S.Y. Tsai M.J. O'Malley B.W. Science. 1995; 270: 1354-1357Crossref PubMed Scopus (2042) Google Scholar). Our present data indicate that this region in SRC-1 (hereafter termed SRB2) is largely responsible for interaction with the ligand binding D/E domain of the receptor; it does not interact with the A/B region of steroid (Fig. 2). Hence, the carboxyl-terminal region in SRC-1, aa 1139–1441, is proposed to provide specificity for ligand-bound AF2.Consistent with previous findings, the middle region of SRC-1, aa 361–1139, also interacts with the D/E region of the receptor in a ligand-dependent manner (Fig. 2). Three highly conserved hydrophobic LXXLL motifs within aa 570–833 in SRC-1 family members and other nuclear receptor interacting proteins have been shown to be relevant for the interactions with ER, retinoic acid receptor, and TR ligand binding domains (45Henttu P.M. Kalkhoven E. Parker M.G. Mol Cell. Biol. 1997; 17: 1832-1839Crossref PubMed Scopus (192) Google Scholar, 51Heery D.M. Kalkhoven E. Hoare S. Parker M.G. Nature. 1997; 387: 733-736Crossref PubMed Scopus (1755) Google Scholar). A unique observation of this study is that the SRC-1 region from aa 361 to aa 1139, hereafter termed SRB1, also interacts with the ligand-independent AF1 in the A/B region of PR (Fig. 2). Interactions of SRB1 with the D/E region were observed to have about 15 the efficiency observed with the A/B region of PR. In present experiments, we observe that several subregions of SRC-1 are involved in the interactions with the A/B region of PR in intact cells (Fig. 4). Thus, aa 633–772, containing a cluster of three LXXLL motifs, and aa 361–633 bind to the N terminus and full-length PR. However, the region comprising aa 782–1139 fails to interact with PR in vitro, but is active in a mammalian two-hybrid interaction assay (Fig. 2), suggesting that this region in SRC-1 requires additional cellular components for interaction with the A/B region of PR in intact cells. The interaction of SRB1 with the AF1- and AF2-containing regions of the receptor is not limited to PR. Other receptors, including ER, GR, and AR, exhibited similar patterns of interaction in intact cells. Therefore, there are multiple interaction sites in the SRC-1 and in the receptor molecule that are important for receptor-coactivator function.Additional variants for SRC-1 containing distinct amino-terminal and carboxyl-terminal sequences, reflecting alternative initiation and splicing events, have been proposed (20Kamei Y. Xu L. Heinzel T. Torchia J. Kurokawa R. Gloss B. Lin S.C. Heyman R.A. Rose D.W. Glass C.K. Rosenfeld M.G. Cell. 1996; 85: 403-414Abstract Full Text Full Text PDF PubMed Scopus (1916) Google Scholar). To test the hypothesis that truncated forms of SRC-1 may be of physiological relevance, deletion mutants for SRC-1 were tested for their ability to coactivate PR and PR domains. We observed that a carboxyl-terminally truncated form of SRC-1, lacking the ligand-dependent steroid receptor interacting region, was as efficient as the wild type SRC-1 to coactivate PR (Fig. 5). Therefore, one might speculate that different isoforms of SRC-1 could be relevant for ligand-independent activation of target gene expression. Because steroid receptors are phosphoproteins and the major phosphorylation sites are at the amino-terminal A/B domain (52Zhang Y. Beck C.A. Poletti A. Edwards D.P. Weigel N.L. Mol. Endocrinol. 1995; 9: 1029-1040Crossref PubMed Google Scholar, 53Zhang Y. Beck C.A. Poletti A. Clement IV J.P. Prendergast P. Yip T.-T. Hutchens T.W. Edwards D.P. Weigel N.L. Mol. Endocrinol. 1997; 11: 823-832Crossref PubMed Scopus (76) Google Scholar), the present data raise the interesting possibility that phosphorylation of steroid receptors may modulate SRC-1 interactions. It will be of interest to determine whether ligand-dependent and/or independent phosphorylation of the A/B region of steroid receptors is directly involved in receptor-coactivator interactions and transactivation.Previous reports indicate that the interactions and coactivation of steroid receptors by SRC-1 and SRC-2 (TIF2/GRIP1) are specific for AF2-mediated transactivation of the steroid receptors (22Voegel J.J. Heine M.J. Zechel C. Chambon P. Gronemeyer H. EMBO J. 1996; 15: 3667-3675Crossref PubMed Scopus (947) Google Scholar, 45Henttu P.M. Kalkhoven E. Parker M.G. Mol Cell. Biol. 1997; 17: 1832-1839Crossref PubMed Scopus (192) Google Scholar, 51Heery D.M. Kalkhoven E. Hoare S. Parker M.G. Nature. 1997; 387: 733-736Crossref PubMed Scopus (1755) Google Scholar, 54Hong H. Kohli K. Garabedian M.J. Stallcup M.R. Mol. Cell. Biol. 1997; 17: 2735-2744Crossref PubMed Scopus (493) Google Scholar). The failure of coactivators to enhance the transcription activity of the A/B region has been ascribed to the ability of AF1 to activate transcription by a mechanism dependent on coactivator(s), presumably different from those described for AF2-interacting coactivators (54Hong H. Kohli K. Garabedian M.J. Stallcup M.R. Mol. Cell. Biol. 1997; 17: 2735-2744Crossref PubMed Scopus (493) Google Scholar). However, we observed here that there are functional interactions of SRC-1 and SRC-2/TIF2 with the AF1 and AF2 domains for several steroid receptors (Fig. 6). These differences support the hypothesis that additional accessory proteins, which appear to be absent in the yeast systems previously employed for analysis, are relevant for AF1 interactions and coactivation in mammalian cells. We observed that SRC-1 can directly interact with the receptor more efficiently when both AF1 and AF2 are present, as compared with the interactions with the individual AFs (Fig. 3). Similarly, SRC-1 coactivates more efficiently when individual AF1 and AF2 of the steroid receptors were expressed simultaneously in intact cells (Fig. 7). Although the mechanism by which the AFs act in a synergistic manner remains unclear, the assembly of their functional domains likely depends on coactivators. Taken together, the existence of multiple coactivators and receptor-coactivator interaction sites may contribute to a mechanism by which steroid receptors achieve their specificity and the diversity of target gene expression. Steroid receptors belong to a superfamily of transcription factors that regulate hormone-responsive genes and thereby cellular growth and differentiation. In the absence of hormone, the receptor is maintained in an inactive or repressive state by association with heat shock proteins and/or corepressors. 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