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The CREB-binding Protein (CBP) Cooperates with the Serum Response Factor for Transactivation of the c-fos Serum Response Element

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Idioma: Inglés

Publicado: 01/12/1997

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Abstract:

The serum response element is one of the major promoter elements of the immediate early response to extracellular signals. The serum response element includes two main binding sites for proteins: the Ets box, which binds p62TCF, and the CArG box, which binds p67SRF. These two proteins are direct targets for signal transduction pathways; p62TCF is a nuclear end point of the Ras/mitogen-activated protein kinase pathway, and p67SRF is targeted by the Rho/Rac small G-proteins. The mechanism by which the signal is further transduced from the transcription factors to the basal transcriptional machinery is poorly understood. Recent data have suggested that the cAMP-responsive element-binding protein (CREB)-binding protein, a transcriptional adaptor involved in the transactivation through a wide variety of enhancer elements, participates in p62TCF activity. We here show that the CREB-binding protein also cooperates in the process of transactivation by p67SRF. Cotransfections of expression vectors for the CREB-binding protein increased the expression, in response to serum, of reporters under the control of the c-fos serum response element. Interestingly, the C-terminal moiety of the CREB-binding protein was not necessary to observe this effect. The cooperation did not require the Ets box in the serum response element, and the CArG box was sufficient, indicating that the CREB-binding protein is able to cooperate with p67SRF in the absence of an Ets protein. Co-immunoprecipitation experiments using cell extracts showed that p67SRF could be retained with antibodies directed against the CREB-binding protein, suggesting that the two proteins form a multimolecular complex in live cells. The physical interaction between p67SRF and the CREB-binding protein was further confirmed by two-hybrid assays in mammalian cells. Our results indicate that the CREB-binding protein cooperates with p67SRF and, thus, suggest that the serum response element is regulated by a multimolecular complex, which includes the CREB-binding protein, p67SRF, and p62TCF, with multiple interactions between the components of the complex. The serum response element is one of the major promoter elements of the immediate early response to extracellular signals. The serum response element includes two main binding sites for proteins: the Ets box, which binds p62TCF, and the CArG box, which binds p67SRF. These two proteins are direct targets for signal transduction pathways; p62TCF is a nuclear end point of the Ras/mitogen-activated protein kinase pathway, and p67SRF is targeted by the Rho/Rac small G-proteins. The mechanism by which the signal is further transduced from the transcription factors to the basal transcriptional machinery is poorly understood. Recent data have suggested that the cAMP-responsive element-binding protein (CREB)-binding protein, a transcriptional adaptor involved in the transactivation through a wide variety of enhancer elements, participates in p62TCF activity. We here show that the CREB-binding protein also cooperates in the process of transactivation by p67SRF. Cotransfections of expression vectors for the CREB-binding protein increased the expression, in response to serum, of reporters under the control of the c-fos serum response element. Interestingly, the C-terminal moiety of the CREB-binding protein was not necessary to observe this effect. The cooperation did not require the Ets box in the serum response element, and the CArG box was sufficient, indicating that the CREB-binding protein is able to cooperate with p67SRF in the absence of an Ets protein. Co-immunoprecipitation experiments using cell extracts showed that p67SRF could be retained with antibodies directed against the CREB-binding protein, suggesting that the two proteins form a multimolecular complex in live cells. The physical interaction between p67SRF and the CREB-binding protein was further confirmed by two-hybrid assays in mammalian cells. Our results indicate that the CREB-binding protein cooperates with p67SRF and, thus, suggest that the serum response element is regulated by a multimolecular complex, which includes the CREB-binding protein, p67SRF, and p62TCF, with multiple interactions between the components of the complex. The serum response element (SRE) 1The abbreviations used are: SRE, serum response element; CBP, CREB-binding protein; CREB, cAMP-responsive element-binding protein; CMV, cytomegalovirus; FCS, fetal calf serum; HA, hemagglutinin; MAP, mitogen-activated protein; SRF, serum response factor; TAD, transactivation domain; TCF, ternary complex factor. 1The abbreviations used are: SRE, serum response element; CBP, CREB-binding protein; CREB, cAMP-responsive element-binding protein; CMV, cytomegalovirus; FCS, fetal calf serum; HA, hemagglutinin; MAP, mitogen-activated protein; SRF, serum response factor; TAD, transactivation domain; TCF, ternary complex factor. enhancer (1Treisman R. Semin. Cancer Biol. 1990; 1: 47-58PubMed Google Scholar) is present in the upstream regulatory sequence of a number of immediate early genes such as c-fos (2Treisman R. Cell. 1985; 42: 889-902Abstract Full Text PDF PubMed Scopus (518) Google Scholar, 3Treisman R. Cell. 1986; 46: 567-574Abstract Full Text PDF PubMed Scopus (528) Google Scholar). The SRE is constitutively occupied by a complex of two proteins, p67SRF (4Norman C. Runswick M. Pollock R. Treisman R. Cell. 1988; 55: 989-1003Abstract Full Text PDF PubMed Scopus (695) Google Scholar) and p62TCF (5Shaw P.E. Schrı̂ter H. Nordheim A. Cell. 1989; 56: 563-572Abstract Full Text PDF PubMed Scopus (346) Google Scholar). p67SRF belongs to the MADS box family of proteins (6Shore P. Sharrocks A.D. Nucleic Acids Res. 1995; 23: 4698-4706Crossref PubMed Scopus (77) Google Scholar) and recognizes a CArG box in the SRE (7Phan-Dinh-Tuy F. Tuil D. Schweighoffer F. Pinset C. Kahn A. Minty A. Eur. J. Biochem. 1988; 173: 507-515Crossref PubMed Scopus (38) Google Scholar). p62TCF does not bind autonomously to the element, but requires the assistance of p67SRF to efficiently contact the DNA (8Schröter H. Mueller C.G. Meese K. Nordheim A. EMBO J. 1990; 9: 1123-1130Crossref PubMed Scopus (91) Google Scholar, 9Shaw P.E. EMBO J. 1992; 11: 3011-3019Crossref PubMed Scopus (71) Google Scholar). The sequence recognized by p62TCF, located upstream of the CArG box, is in the form CAGGA, a sequence that binds proteins from the Ets family. Several Ets proteins display a TCF activity on the c-fos SRE: ELK-1 (10Hipskind R.A. Rao V.N. Mueller C.G.F. Reddy E.S.P. Nordheim A. Nature. 1991; 354: 531-534Crossref PubMed Scopus (350) Google Scholar), SAP-1 (11Dalton S. Treisman R. Cell. 1992; 68: 597-612Abstract Full Text PDF PubMed Scopus (527) Google Scholar), and SAP-2/NET/ERP (12Lopez M. Oettgen P. Akbarali Y. Dendorfer U. Libermann T.A. Mol. Cell. Biol. 1994; 14: 3292-3309Crossref PubMed Scopus (108) Google Scholar, 13Price M.A. Rogers A.E. Treisman R. EMBO J. 1995; 14: 2589-2601Crossref PubMed Scopus (245) Google Scholar). The SRE is also recognized by oncogenic fusion proteins such as EWS-FLI (14Magnaghi-Jaulin L. Masutani H. Robin P. Lipinski M. Harel-Bellan A. Nucleic Acids Res. 1996; 24: 1052-1058Crossref PubMed Scopus (41) Google Scholar). TCFs can be distinguished by their pattern of expression (15Magnaghi-Jaulin L. Masutani H. Lipinski M. Harel-Bellan A. Magnaghi-Jaulin L. Masutani H. Robin P. Lipinski M. Harel-Bellan A. FEBS Lett. 1996; 391: 247-251Crossref PubMed Scopus (17) Google Scholar, 16Giovane A. Pintzas A. Maira S.M. Sobieszczuk P. Wasylyk B. Genes Dev. 1994; 8: 1502-1513Crossref PubMed Scopus (202) Google Scholar), by their affinity for the c-fos SRE Ets box (6Shore P. Sharrocks A.D. Nucleic Acids Res. 1995; 23: 4698-4706Crossref PubMed Scopus (77) Google Scholar, 17Masutani H. Magnaghi-Jaulin L. Ait-Si Ali S. Groisman R. Robin P. Harel-Bellan A. Oncogene. 1997; 15: 1661-1669Crossref PubMed Scopus (15) Google Scholar), or on a functional basis (13Price M.A. Rogers A.E. Treisman R. EMBO J. 1995; 14: 2589-2601Crossref PubMed Scopus (245) Google Scholar).Both p67SRF and p62TCF contain a transactivation domain (TAD) (18Liu S.H. Ma J.T. Yueh A.Y. Lees-Miller S.P. Anderson C.W. Ng S.Y. J. Biol. Chem. 1993; 268: 21147-21154Abstract Full Text PDF PubMed Google Scholar, 19Johansen F.E. Prywes R. Mol. Cell. Biol. 1993; 13: 4640-4647Crossref PubMed Scopus (112) Google Scholar). Transactivation by TCF TADs is induced by mitogens (20Janknecht R. Ernst W.H. Nordheim A. Oncogene. 1995; 10: 1209-1216PubMed Google Scholar, 21Janknecht R. Ernst W.H. Pingoud V. Nordheim A. EMBO J. 1993; 12: 5097-5104Crossref PubMed Scopus (507) Google Scholar). TCF-TADs are direct targets for the Ras/MAP kinase transduction pathway and are substrates for ERK-1 and ERK-2 (22Hill C.S. Marais R. John S. Wynne J. Dalton S. Treisman R. Cell. 1993; 73: 395-406Abstract Full Text PDF PubMed Scopus (329) Google Scholar, 23Gille H. Sharrocks A.D. Shaw P.E. Nature. 1992; 358: 414-417Crossref PubMed Scopus (812) Google Scholar, 24Gill H. Kortenjann M. Thomae O. Moomaw C. Slaughter C. Cobb M.H. Shaw P.E. EMBO J. 1995; 14: 951-962Crossref PubMed Scopus (584) Google Scholar), suggesting that phosphorylation by MAP kinases activates these domains. p67SRF is a direct target for a poorly defined signal transduction pathway (25Hill C.S. Wynne J. Treisman R. Cell. 1995; 81: 1159-1170Abstract Full Text PDF PubMed Scopus (1199) Google Scholar).The mechanism by which the activating signal, transmitted through the SRE, is further transduced to the transcriptional machinery and the minimal promoter is unknown. Recent data suggest that activation through TCFs could be mediated by a coactivator or adaptor protein, the CREB-binding protein or CBP (26Janknecht R. Nordheim A. Oncogene. 1996; 12: 1961-1969PubMed Google Scholar, 27Janknecht R. Nordheim A. Biochem. Biophys. Res. Commun. 1996; 228: 831-837Crossref PubMed Scopus (172) Google Scholar). The CBP adaptor protein was first characterized as a co-activator for CREB, a cAMP-responsive transcription factor (28Chrivia J.C. Kwok R.P.S. Lamb N. Hagiwara M. Montminy M.R. Goodman R.H. Nature. 1993; 265: 855-859Crossref Scopus (1758) Google Scholar, 29Kwok R.P. Lundblad J.R. Chrivia J.C. Richards J.P. Bachinger H.P. Brennan R.G. Roberts S.G. Green M.R. Goodman R.H. Nature. 1994; 370: 223-226Crossref PubMed Scopus (1279) Google Scholar), but was rapidly shown to be involved in a large variety of responses. CBP is highly homologous to p300, a transcriptional co-activator (30Eckner R. Ewen M.E. Newsome D. Gerdes M. DeCaprio J.A. Lawrence J.B. Livingston D.M. Genes Dev. 1994; 8: 869-884Crossref PubMed Scopus (920) Google Scholar) that is a target for viral transforming proteins such as E1A (31Arany Z. Sellers W.R. Livingston D.M. Eckner R. Cell. 1994; 77: 799-800Abstract Full Text PDF PubMed Scopus (370) Google Scholar); CBP itself is complexed by E1A (31Arany Z. Sellers W.R. Livingston D.M. Eckner R. Cell. 1994; 77: 799-800Abstract Full Text PDF PubMed Scopus (370) Google Scholar, 32Lundblad J.R. Kwok R.P. Laurance M.E. Harter M.L. Goodman R.H. Nature. 1995; 374: 85-88Crossref PubMed Scopus (531) Google Scholar). CBP and p300 (p300/CBP) are involved in the activation of a large variety of transcriptional enhancer elements through various transcription factors (33Janknecht R. Hunter T. Curr. Biol. 1996; 6: 951-954Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar), including c-Jun (34Arias J. Alberts A.S. Brindle P. Claret F.X. Smeal T. Karin M. Feramisco J. Montminy M. Nature. 1994; 370: 226-229Crossref PubMed Scopus (679) Google Scholar, 35Bannister A.J. Oehler T. Wilhelm D. Angel P. Kouzarides T. Oncogene. 1995; 11: 2509-2514PubMed Google Scholar), c-Fos (36Bannister A.J. Kouzarides T. EMBO J. 1995; 14: 4758-4762Crossref PubMed Scopus (319) Google Scholar), c-Myb (37Oelgeschlager M. Janknecht R. Krieg J. Schreek S. Luscher B. EMBO J. 1996; 15: 2771-2780Crossref PubMed Scopus (193) Google Scholar, 38Dai P. Akimaru H. Tanaka Y. Hou D. Yasukawa T. Kanei-Ishii C. Takahashi T. Ishii S. Genes Dev. 1996; 10: 528-540Crossref PubMed Scopus (303) Google Scholar), E2F (39Trouche D. Kouzarides T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1439-1442Crossref PubMed Scopus (93) Google Scholar, 40Trouche D. Cook A. Kouzarides T. Nucleic Acids Res. 1996; 24: 4139-4145Crossref PubMed Scopus (104) Google Scholar), the STAT proteins (41Bhattacharya S. Eckner R. Grossman S. Oldread E. Arany Z. D'Andrea A. Livingston D.M. Nature. 1996; 383: 344-347Crossref PubMed Scopus (419) Google Scholar, 42Zhang J.J. Vinkemeier U. Gu W. Chakravarti D. Horvath C.M. Darnell Jr., J.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15092-15096Crossref PubMed Scopus (418) Google Scholar), MyoD (43Eckner R. Yao T.P. Oldread E. Livingston D.M. Genes Dev. 1996; 10: 2478-2490Crossref PubMed Scopus (318) Google Scholar, 44Yuan W. Condorelli G. Caruso M. Felsani A. Giordano A. J. Biol. Chem. 1996; 271: 9009-9013Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar), and the nuclear receptor superfamily (45Kamei Y. Xu L. Heinzel T. Torchia J. Kurokawa R. Gloss B. Lin S.-K. 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, 46Chakravarti 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 (843) Google Scholar, 47Oliner J.D. Andresen J.M. Hansen S.K. Zhou S. Tjian R. Genes Dev. 1996; 10: 2903-2911Crossref PubMed Scopus (140) Google Scholar).Co-activators function, at least in part, as bridges between sequence-specific transcriptional activators and general transcription factors of the basal transcription machinery. CBP directly contacts sequence-specific transactivators via one of two interaction domains located, respectively, in the N-terminal or C-terminal part of the molecule (48Janknecht R. Hunter T. Nature. 1996; 383: 22-23Crossref PubMed Scopus (347) Google Scholar). Once recruited, CBP can modulate the transcription rate through various mechanisms. First, CBP includes two TADs located in the N-terminal and C-terminal parts of the molecule (33Janknecht R. Hunter T. Curr. Biol. 1996; 6: 951-954Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar) that contact two general transcription factors: TATA-binding protein for the N-terminal TAD (26Janknecht R. Nordheim A. Oncogene. 1996; 12: 1961-1969PubMed Google Scholar, 44Yuan W. Condorelli G. Caruso M. Felsani A. Giordano A. J. Biol. Chem. 1996; 271: 9009-9013Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar, 49Swope D.L. Mueller C.L. Chrivia J.C. J. Biol. Chem. 1996; 271: 28138-28145Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar), and TFIIB for the C-terminal TAD (29Kwok R.P. Lundblad J.R. Chrivia J.C. Richards J.P. Bachinger H.P. Brennan R.G. Roberts S.G. Green M.R. Goodman R.H. Nature. 1994; 370: 223-226Crossref PubMed Scopus (1279) Google Scholar). In addition, CBP recruits a protein that displays a histone acetyltransferase activity (50Yang X.J. Ogryzko V.V. Nishikawa J. Howard B.H. Nakatani Y. Nature. 1996; 382: 319-324Crossref PubMed Scopus (1310) Google Scholar). Histone acetyltransferases destabilize the nucleosomal structure by acetylation of the N-terminal histone tails, which protrude from the nucleosome (51Lee D.Y. Hayes J.J. Pruss D. Wolffe A.P. Cell. 1993; 72: 73-84Abstract Full Text PDF PubMed Scopus (955) Google Scholar). CBP not only recruits a histone acetyltransferase, but also displays a histone acetyltransferase enzymatic activity (52Ogryzko 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, 53Bannister A.J. Kouzarides T. Nature. 1996; 384: 641-643Crossref PubMed Scopus (1523) Google Scholar). Thus, CBP may use several mechanisms to activate transcription, either by recruiting proteins of the transcripional machinery or by inducing a nucleosomal remodeling process.CBP has been implicated in the transactivation of the c-fosSRE through the p62TCF protein (26Janknecht R. Nordheim A. Oncogene. 1996; 12: 1961-1969PubMed Google Scholar, 27Janknecht R. Nordheim A. Biochem. Biophys. Res. Commun. 1996; 228: 831-837Crossref PubMed Scopus (172) Google Scholar). We here show that CBP enhances transcriptional activation of the SRE even in the absence of the Ets-binding site, and thus in the absence of p62TCFrecruitment. This result indicates that CBP can also cooperate with p67SRF. Furthermore, we demonstrate the formation of a physical complex between p67SRF and CBP in live cells. In addition, we show that, whereas the transactivation through p62TCF seems to involve the C-terminal TAD, the N-terminal moiety of CBP is sufficient for transactivation through p67SRF. Taken together, our results indicate that CBP participates in c-fos SRE activation both through the p62TCF and the p67SRF proteins, and that this transactivation is mediated through two distinct TADs in the CBP molecule.DISCUSSIONThe SRE, which is a central element of the cell's immediate early response, binds several transcription factors and is targeted by several transduction pathways. In particular, p62TCF is a direct target for the Ras/MAP kinase pathway. p67SRF is required to assist p62TCF binding and is also independently a target for signal transduction pathways involving small G-proteins from the Rho/Rac family. Little is known about the mechanism used by these proteins to further transmit the activation signal to the minimal promoters of the genes controlled by the SRE element. Recently, Janknecht and collaborators (26Janknecht R. Nordheim A. Oncogene. 1996; 12: 1961-1969PubMed Google Scholar) have shown that CBP, a versatile adaptor protein, cooperates with p62TCF for transactivation through the SRE. We here show that, indeed, CBP participates in the transactivation through the SRE, since expression of CBP stimulated a response to serum through this element in a dose-dependent manner. Interestingly, in our study, the effect of CBP was observed in the absence of a functional Ets-binding site, and thus in the absence of TCF binding to the element. In many cell systems, the Ets-binding site is not necessary to observe a significant response to serum (22Hill C.S. Marais R. John S. Wynne J. Dalton S. Treisman R. Cell. 1993; 73: 395-406Abstract Full Text PDF PubMed Scopus (329) Google Scholar,25Hill C.S. Wynne J. Treisman R. Cell. 1995; 81: 1159-1170Abstract Full Text PDF PubMed Scopus (1199) Google Scholar, 59Dalton D. Treisman R. Cell. 1992; 68: 597-612Abstract Full Text PDF PubMed Scopus (534) Google Scholar, 60Johansen F.E. Prywes R. Mol. Cell. Biol. 1994; 14: 5920-5928Crossref PubMed Scopus (123) Google Scholar). Moreover, although F9 cells, which were used in this study, express normal amounts of p62TCF, the ternary complex between p67SRF and p62TCF seems to be inactive in these cells (61König H. Nucleic Acids Res. 1991; 19: 3607-3611Crossref PubMed Scopus (59) Google Scholar). Our result indicates that the participation of CBP in SRE transactivation does not absolutely require the Ets protein p62TCF. In contrast, a functional CArG box was indispensable to observe the stimulation of the SRE response, suggesting that the target protein of CBP in this function was p67SRF. Thus, CBP cooperates both with p62TCF(26Janknecht R. Nordheim A. Oncogene. 1996; 12: 1961-1969PubMed Google Scholar, 27Janknecht R. Nordheim A. Biochem. Biophys. Res. Commun. 1996; 228: 831-837Crossref PubMed Scopus (172) Google Scholar) and with p67SRF (this study).CBP is a large molecule that includes several sites of interaction with various sequence-specific transcription factors and has two TADs. To determine which of these functional domains was involved in SRE transactivation, we have used deletion mutants of the protein. Interestingly, the N-terminal moiety of CBP was sufficient to stimulate the SRE response to serum. A C-terminal transactivation domain of CBP seems to be involved in the cooperation with p62TCF (26Janknecht R. Nordheim A. Oncogene. 1996; 12: 1961-1969PubMed Google Scholar). Thus, CBP uses two TADs for transactivation through the SRE, the N-terminal and the C-terminal TAD. In addition, these data demonstrate that CBP can transactivate the SRE in the absence of the domain bearing the histone acetyltransferase activity, which is located in the C-terminal part of the molecule, indicating that this intramolecular activity is not absolutely required in this system. A similar result has been obtained in the CREB model system (49Swope D.L. Mueller C.L. Chrivia J.C. J. Biol. Chem. 1996; 271: 28138-28145Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar).In addition, we also show that CBP and p67SRF form a complex in live cells, since the two proteins can be co-immunoprecipitated from cell extracts. The interaction between p67SRF and CBP does not require that the transcription factor be bound to its target DNA sequence, contrary to what has been observed with MyoD (43Eckner R. Yao T.P. Oldread E. Livingston D.M. Genes Dev. 1996; 10: 2478-2490Crossref PubMed Scopus (318) Google Scholar). The physical interaction between p67SRF and CBP is detected by co-immunoprecipitation assays, which require a high affinity between the proteins, suggesting that this interaction is strong. The interaction between the N-terminal part of CBP and p67SRF was confirmed in a two-hybrid assay in mammalian cells. However, analysis of various subregions of the N-terminal CBP did not allow us to determine more precisely the site of interaction in CBP. This suggests that CBP and p67SRF do not interact through the previously characterized domains of interaction (amino acids 1–101 for the glucocorticoid receptor; amino acid 461–661 for various transcription factors). A possibility is that two physically separate sequences are required for this interaction. p67SRF is not the only member of the MADS box family which is able to interact with CBP. Indeed, CBP is also able to contact, through an undetermined region, MEF-2, a member of this family of proteins that is involved in muscle cell differentiation (43Eckner R. Yao T.P. Oldread E. Livingston D.M. Genes Dev. 1996; 10: 2478-2490Crossref PubMed Scopus (318) Google Scholar, 62Sartorelli V. Huang J. Hamamori Y. Kedes L. Mol. Cell. Biol. 1997; 17: 1010-1026Crossref PubMed Scopus (319) Google Scholar).For SRE transactivation, p67SRF cooperates with p62TCF. Both p67SRF (this study) and p62TCF (26Janknecht R. Nordheim A. Oncogene. 1996; 12: 1961-1969PubMed Google Scholar) physically interact with CBP. Thus, transactivation through the SRE might involve a multimolecular complex including CBP, p67SRF, and p62TCF, stabilized by multiple interactions between the partners in the complex. Interestingly, p67SRF is also involved in other processes such as muscle-cell terminal differentiation (63Vandromme M. Gauthier-Rouviäre C. Carnac G. Lamb N. Fernandez A. J. Cell Biol. 1992; 118: 1489-1500Crossref PubMed Scopus (85) Google Scholar). Indeed, p67SRF cooperates with the myogenic differentiation factors MyoD and myogenin for some muscle promoters' transactivation (64Catala F. Wanner R. Barton P. Cohen A. Wright W. Buckingham M. Mol. Cell. Biol. 1995; 15: 4585-4596Crossref PubMed Scopus (82) Google Scholar) and is able to interact physically with these myogenic bHLHs (65Groisman R. Masutani H. Leibovitch M.-P. Robin P. Soudant I. Trouche D. Harel-Bellan A. J. Biol. Chem. 1996; 271: 5258-5264Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). CBP interacts both with MyoD (43Eckner R. Yao T.P. Oldread E. Livingston D.M. Genes Dev. 1996; 10: 2478-2490Crossref PubMed Scopus (318) Google Scholar, 62Sartorelli V. Huang J. Hamamori Y. Kedes L. Mol. Cell. Biol. 1997; 17: 1010-1026Crossref PubMed Scopus (319) Google Scholar) and p67SRF (this study). Thus, the cooperation between MyoD and p67SRF on muscle cell differentiation also might involve the cooperative recruitment of CBP resulting in the formation, on muscle promoters, of a multimolecular complex including CBP, p67SRF and MyoD, and which would be stabilized by multiple interactions between the various partners of the complex. The serum response element (SRE) 1The abbreviations used are: SRE, serum response element; CBP, CREB-binding protein; CREB, cAMP-responsive element-binding protein; CMV, cytomegalovirus; FCS, fetal calf serum; HA, hemagglutinin; MAP, mitogen-activated protein; SRF, serum response factor; TAD, transactivation domain; TCF, ternary complex factor. 1The abbreviations used are: SRE, serum response element; CBP, CREB-binding protein; CREB, cAMP-responsive element-binding protein; CMV, cytomegalovirus; FCS, fetal calf serum; HA, hemagglutinin; MAP, mitogen-activated protein; SRF, serum response factor; TAD, transactivation domain; TCF, ternary complex factor. enhancer (1Treisman R. Semin. Cancer Biol. 1990; 1: 47-58PubMed Google Scholar) is present in the upstream regulatory sequence of a number of immediate early genes such as c-fos (2Treisman R. Cell. 1985; 42: 889-902Abstract Full Text PDF PubMed Scopus (518) Google Scholar, 3Treisman R. Cell. 1986; 46: 567-574Abstract Full Text PDF PubMed Scopus (528) Google Scholar). The SRE is constitutively occupied by a complex of two proteins, p67SRF (4Norman C. Runswick M. Pollock R. Treisman R. Cell. 1988; 55: 989-1003Abstract Full Text PDF PubMed Scopus (695) Google Scholar) and p62TCF (5Shaw P.E. Schrı̂ter H. Nordheim A. Cell. 1989; 56: 563-572Abstract Full Text PDF PubMed Scopus (346) Google Scholar). p67SRF belongs to the MADS box family of proteins (6Shore P. Sharrocks A.D. Nucleic Acids Res. 1995; 23: 4698-4706Crossref PubMed Scopus (77) Google Scholar) and recognizes a CArG box in the SRE (7Phan-Dinh-Tuy F. Tuil D. Schweighoffer F. Pinset C. Kahn A. Minty A. Eur. J. Biochem. 1988; 173: 507-515Crossref PubMed Scopus (38) Google Scholar). p62TCF does not bind autonomously to the element, but requires the assistance of p67SRF to efficiently contact the DNA (8Schröter H. Mueller C.G. Meese K. Nordheim A. EMBO J. 1990; 9: 1123-1130Crossref PubMed Scopus (91) Google Scholar, 9Shaw P.E. EMBO J. 1992; 11: 3011-3019Crossref PubMed Scopus (71) Google Scholar). The sequence recognized by p62TCF, located upstream of the CArG box, is in the form CAGGA, a sequence that binds proteins from the Ets family. Several Ets proteins display a TCF activity on the c-fos SRE: ELK-1 (10Hipskind R.A. Rao V.N. Mueller C.G.F. Reddy E.S.P. Nordheim A. Nature. 1991; 354: 531-534Crossref PubMed Scopus (350) Google Scholar), SAP-1 (11Dalton S. Treisman R. Cell. 1992; 68: 597-612Abstract Full Text PDF PubMed Scopus (527) Google Scholar), and SAP-2/NET/ERP (12Lopez M. Oettgen P. Akbarali Y. Dendorfer U. Libermann T.A. Mol. Cell. Biol. 1994; 14: 3292-3309Crossref PubMed Scopus (108) Google Scholar, 13Price M.A. Rogers A.E. Treisman R. EMBO J. 1995; 14: 2589-2601Crossref PubMed Scopus (245) Google Scholar). The SRE is also recognized by oncogenic fusion proteins such as EWS-FLI (14Magnaghi-Jaulin L. Masutani H. Robin P. Lipinski M. Harel-Bellan A. Nucleic Acids Res. 1996; 24: 1052-1058Crossref PubMed Scopus (41) Google Scholar). TCFs can be distinguished by their pattern of expression (15Magnaghi-Jaulin L. Masutani H. Lipinski M. Harel-Bellan A. Magnaghi-Jaulin L. Masutani H. Robin P. Lipinski M. Harel-Bellan A. FEBS Lett. 1996; 391: 247-251Crossref PubMed Scopus (17) Google Scholar, 16Giovane A. Pintzas A. Maira S.M. Sobieszczuk P. Wasylyk B. Genes Dev. 1994; 8: 1502-1513Crossref PubMed Scopus (202) Google Scholar), by their affinity for the c-fos SRE Ets box (6Shore P. Sharrocks A.D. Nucleic Acids Res. 1995; 23: 4698-4706Crossref PubMed Scopus (77) Google Scholar, 17Masutani H. Magnaghi-Jaulin L. Ait-Si Ali S. Groisman R. Robin P. Harel-Bellan A. Oncogene. 1997; 15: 1661-1669Crossref PubMed Scopus (15) Google Scholar), or on a functional basis (13Price M.A. Rogers A.E. Treisman R. EMBO J. 1995; 14: 2589-2601Crossref PubMed Scopus (245) Google Scholar). Both p67SRF and p62TCF contain a transactivation domain (TAD) (18Liu S.H. Ma J.T. Yueh A.Y. Lees-Miller S.P. Anderson C.W. Ng S.Y. J. Biol. Chem. 1993; 268: 21147-21154Abstract Full Text PDF PubMed Google Scholar, 19Johansen F.E. Prywes R. Mol. Cell. Biol. 1993; 13: 4640-4647Crossref PubMed Scopus (112) Google Scholar). Transactivation by TCF TADs is induced by mitogens (20Janknecht R. Ernst W.H. Nordheim A. Oncogene. 1995; 10: 1209-1216PubMed Google Scholar, 21Janknecht R. Ernst W.H. Pingoud V. Nordheim A. EMBO J. 1993; 12: 5097-5104Crossref PubMed Scopus (507) Google Scholar). TCF-TADs are direct targets for the Ras/MAP kinase transduction pathway and are substrates for ERK-1 and ERK-2 (22Hill C.S. Marais R. John S. Wynne J. Dalton S. Treisman R. Cell. 1993; 73: 395-406Abstract Full Text PDF PubMed Scopus (329) Google Scholar, 23Gille H. Sharrocks A.D. Shaw P.E. Nature. 1992; 358: 414-417Crossref PubMed Scopus (812) Google Scholar, 24Gill H. Kortenjann M. Thomae O. Moomaw C. Slaughter C. Cobb M.H. Shaw P.E. EMBO J. 1995; 14: 951-962Crossref PubMed Scopus (584) Google Scholar), suggesting that phosphorylation by MAP kinases activates these domains. p67SRF is a direct target for a poorly defined signal transduction pathway (25Hill C.S. Wynne J. Treisman R. Cell. 1995; 81: 1159-1170Abstract Full Text PDF PubMed Scopus (1199) Google Scholar). The mechanism by which the activating signal, transmitted through the SRE, is further transduced to the transcriptional machinery and the minimal promoter is unknown. Recent data suggest that activation through TCFs could be mediated by a coactivator or adaptor protein, the CREB-binding protein or CBP (26Janknecht R. Nordheim A. Oncogene. 1996; 12: 1961-1969PubMed Google Scholar, 27Janknecht R. Nordheim A. Biochem. Biophys. Res. Commun. 1996; 228: 831-837Crossref PubMed Scopus (172) Google Scholar). The CBP adaptor protein was first characterized as a co-activator for CREB, a cAMP-responsive transcription factor (28Chrivia J.C. Kwok R.P.S. Lamb N. Hagiwara M. Montminy M.R. Goodman R.H. Nature. 1993; 265: 855-859Crossref Scopus (1758) Google Scholar, 29Kwok R.P. Lundblad J.R. Chrivia J.C. Richards J.P. Bachinger H.P. Brennan R.G. Roberts S.G. Green M.R. Goodman R.H. Nature. 1994; 370: 223-226Crossref PubMed Scopus (1279) Google Scholar), but was rapidly shown to be involved in a large variety of responses. CBP is highly homologous to p300, a transcriptional co-activator (30Eckner R. Ewen M.E. Newsome D. Gerdes M. DeCaprio J.A. Lawrence J.B. Livingston D.M. Genes Dev. 1994; 8: 869-884Crossref PubMed Scopus (920) Google Scholar) that is a target for viral transforming proteins such as E1A (31Arany Z. Sellers W.R. Livingston D.M. Eckner R. Cell. 1994; 77: 799-800Abstract Full Text PDF PubMed Scopus (370) Google Scholar); CBP itself is complexed by E1A (31Arany Z. Sellers W.R. Livingston D.M. Eckner R. Cell. 1994; 77: 799-800Abstract Full Text PDF PubMed Scopus (370) Google Scholar, 32Lundblad J.R. Kwok R.P. Laurance M.E. Harter M.L. Goodman R.H. Nature. 1995; 374: 85-88Crossref PubMed Scopus (531) Google Scholar). CBP and p300 (p300/CBP) are involved in the activation of a large variety of transcriptional enhancer elements through various transcription factors (33Janknecht R. Hunter T. Curr. Biol. 1996; 6: 951-954Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar), including c-Jun (34Arias J. Alberts A.S. Brindle P. Claret F.X. Smeal T. Karin M. Feramisco J. Montminy M. Nature. 1994; 370: 226-229Crossref PubMed Scopus (679) Google Scholar, 35Bannister A.J. Oehler T. Wilhelm D. Angel P. Kouzarides T. Oncogene. 1995; 11: 2509-2514PubMed Google Scholar), c-Fos (36Bannister A.J. Kouzarides T. EMBO J. 1995; 14: 4758-4762Crossref PubMed Scopus (319) Google Scholar), c-Myb (37Oelgeschlager M. Janknecht R. Krieg J. Schreek S. Luscher B. EMBO J. 1996; 15: 2771-2780Crossref PubMed Scopus (193) Google Scholar, 38Dai P. Akimaru H. Tanaka Y. Hou D. Yasukawa T. Kanei-Ishii C. Takahashi T. Ishii S. Genes Dev. 1996; 10: 528-540Crossref PubMed Scopus (303) Google Scholar), E2F (39Trouche D. Kouzarides T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1439-1442Crossref PubMed Scopus (93) Google Scholar, 40Trouche D. Cook A. Kouzarides T. Nucleic Acids Res. 1996; 24: 4139-4145Crossref PubMed Scopus (104) Google Scholar), the STAT proteins (41Bhattacharya S. Eckner R. Grossman S. Oldread E. Arany Z. D'Andrea A. Livingston D.M. Nature. 1996; 383: 344-347Crossref PubMed Scopus (419) Google Scholar, 42Zhang J.J. Vinkemeier U. Gu W. Chakravarti D. Horvath C.M. Darnell Jr., J.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 15092-15096Crossref PubMed Scopus (418) Google Scholar), MyoD (43Eckner R. Yao T.P. Oldread E. Livingston D.M. Genes Dev. 1996; 10: 2478-2490Crossref PubMed Scopus (318) Google Scholar, 44Yuan W. Condorelli G. Caruso M. Felsani A. Giordano A. J. Biol. Chem. 1996; 271: 9009-9013Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar), and the nuclear receptor superfamily (45Kamei Y. Xu L. Heinzel T. Torchia J. Kurokawa R. Gloss B. Lin S.-K. 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, 46Chakravarti 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 (843) Google Scholar, 47Oliner J.D. Andresen J.M. Hansen S.K. Zhou S. Tjian R. Genes Dev. 1996; 10: 2903-2911Crossref PubMed Scopus (140) Google Scholar). Co-activators function, at least in part, as bridges between sequence-specific transcriptional activators and general transcription factors of the basal transcription machinery. CBP directly contacts sequence-specific transactivators via one of two interaction domains located, respectively, in the N-terminal or C-terminal part of the molecule (48Janknecht R. Hunter T. Nature. 1996; 383: 22-23Crossref PubMed Scopus (347) Google Scholar). Once recruited, CBP can modulate the transcription rate through various mechanisms. First, CBP includes two TADs located in the N-terminal and C-terminal parts of the molecule (33Janknecht R. Hunter T. Curr. Biol. 1996; 6: 951-954Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar) that contact two general transcription factors: TATA-binding protein for the N-terminal TAD (26Janknecht R. Nordheim A. Oncogene. 1996; 12: 1961-1969PubMed Google Scholar, 44Yuan W. Condorelli G. Caruso M. Felsani A. Giordano A. J. Biol. Chem. 1996; 271: 9009-9013Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar, 49Swope D.L. Mueller C.L. Chrivia J.C. J. Biol. Chem. 1996; 271: 28138-28145Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar), and TFIIB for the C-terminal TAD (29Kwok R.P. Lundblad J.R. Chrivia J.C. Richards J.P. Bachinger H.P. Brennan R.G. Roberts S.G. Green M.R. Goodman R.H. Nature. 1994; 370: 223-226Crossref PubMed Scopus (1279) Google Scholar). In addition, CBP recruits a protein that displays a histone acetyltransferase activity (50Yang X.J. Ogryzko V.V. Nishikawa J. Howard B.H. Nakatani Y. Nature. 1996; 382: 319-324Crossref PubMed Scopus (1310) Google Scholar). Histone acetyltransferases destabilize the nucleosomal structure by acetylation of the N-terminal histone tails, which protrude from the nucleosome (51Lee D.Y. Hayes J.J. Pruss D. Wolffe A.P. Cell. 1993; 72: 73-84Abstract Full Text PDF PubMed Scopus (955) Google Scholar). CBP not only recruits a histone acetyltransferase, but also displays a histone acetyltransferase enzymatic activity (52Ogryzko 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, 53Bannister A.J. Kouzarides T. Nature. 1996; 384: 641-643Crossref PubMed Scopus (1523) Google Scholar). Thus, CBP may use several mechanisms to activate transcription, either by recruiting proteins of the transcripional machinery or by inducing a nucleosomal remodeling process. CBP has been implicated in the transactivation of the c-fosSRE through the p62TCF protein (26Janknecht R. Nordheim A. Oncogene. 1996; 12: 1961-1969PubMed Google Scholar, 27Janknecht R. Nordheim A. Biochem. Biophys. Res. Commun. 1996; 228: 831-837Crossref PubMed Scopus (172) Google Scholar). We here show that CBP enhances transcriptional activation of the SRE even in the absence of the Ets-binding site, and thus in the absence of p62TCFrecruitment. This result indicates that CBP can also cooperate with p67SRF. Furthermore, we demonstrate the formation of a physical complex between p67SRF and CBP in live cells. In addition, we show that, whereas the transactivation through p62TCF seems to involve the C-terminal TAD, the N-terminal moiety of CBP is sufficient for transactivation through p67SRF. Taken together, our results indicate that CBP participates in c-fos SRE activation both through the p62TCF and the p67SRF proteins, and that this transactivation is mediated through two distinct TADs in the CBP molecule. DISCUSSIONThe SRE, which is a central element of the cell's immediate early response, binds several transcription factors and is targeted by several transduction pathways. In particular, p62TCF is a direct target for the Ras/MAP kinase pathway. p67SRF is required to assist p62TCF binding and is also independently a target for signal transduction pathways involving small G-proteins from the Rho/Rac family. Little is known about the mechanism used by these proteins to further transmit the activation signal to the minimal promoters of the genes controlled by the SRE element. Recently, Janknecht and collaborators (26Janknecht R. Nordheim A. Oncogene. 1996; 12: 1961-1969PubMed Google Scholar) have shown that CBP, a versatile adaptor protein, cooperates with p62TCF for transactivation through the SRE. We here show that, indeed, CBP participates in the transactivation through the SRE, since expression of CBP stimulated a response to serum through this element in a dose-dependent manner. Interestingly, in our study, the effect of CBP was observed in the absence of a functional Ets-binding site, and thus in the absence of TCF binding to the element. In many cell systems, the Ets-binding site is not necessary to observe a significant response to serum (22Hill C.S. Marais R. John S. Wynne J. Dalton S. Treisman R. Cell. 1993; 73: 395-406Abstract Full Text PDF PubMed Scopus (329) Google Scholar,25Hill C.S. Wynne J. Treisman R. Cell. 1995; 81: 1159-1170Abstract Full Text PDF PubMed Scopus (1199) Google Scholar, 59Dalton D. Treisman R. Cell. 1992; 68: 597-612Abstract Full Text PDF PubMed Scopus (534) Google Scholar, 60Johansen F.E. Prywes R. Mol. Cell. Biol. 1994; 14: 5920-5928Crossref PubMed Scopus (123) Google Scholar). Moreover, although F9 cells, which were used in this study, express normal amounts of p62TCF, the ternary complex between p67SRF and p62TCF seems to be inactive in these cells (61König H. Nucleic Acids Res. 1991; 19: 3607-3611Crossref PubMed Scopus (59) Google Scholar). Our result indicates that the participation of CBP in SRE transactivation does not absolutely require the Ets protein p62TCF. In contrast, a functional CArG box was indispensable to observe the stimulation of the SRE response, suggesting that the target protein of CBP in this function was p67SRF. Thus, CBP cooperates both with p62TCF(26Janknecht R. Nordheim A. Oncogene. 1996; 12: 1961-1969PubMed Google Scholar, 27Janknecht R. Nordheim A. Biochem. Biophys. Res. Commun. 1996; 228: 831-837Crossref PubMed Scopus (172) Google Scholar) and with p67SRF (this study).CBP is a large molecule that includes several sites of interaction with various sequence-specific transcription factors and has two TADs. To determine which of these functional domains was involved in SRE transactivation, we have used deletion mutants of the protein. Interestingly, the N-terminal moiety of CBP was sufficient to stimulate the SRE response to serum. A C-terminal transactivation domain of CBP seems to be involved in the cooperation with p62TCF (26Janknecht R. Nordheim A. Oncogene. 1996; 12: 1961-1969PubMed Google Scholar). Thus, CBP uses two TADs for transactivation through the SRE, the N-terminal and the C-terminal TAD. In addition, these data demonstrate that CBP can transactivate the SRE in the absence of the domain bearing the histone acetyltransferase activity, which is located in the C-terminal part of the molecule, indicating that this intramolecular activity is not absolutely required in this system. A similar result has been obtained in the CREB model system (49Swope D.L. Mueller C.L. Chrivia J.C. J. Biol. Chem. 1996; 271: 28138-28145Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar).In addition, we also show that CBP and p67SRF form a complex in live cells, since the two proteins can be co-immunoprecipitated from cell extracts. The interaction between p67SRF and CBP does not require that the transcription factor be bound to its target DNA sequence, contrary to what has been observed with MyoD (43Eckner R. Yao T.P. Oldread E. Livingston D.M. Genes Dev. 1996; 10: 2478-2490Crossref PubMed Scopus (318) Google Scholar). The physical interaction between p67SRF and CBP is detected by co-immunoprecipitation assays, which require a high affinity between the proteins, suggesting that this interaction is strong. The interaction between the N-terminal part of CBP and p67SRF was confirmed in a two-hybrid assay in mammalian cells. However, analysis of various subregions of the N-terminal CBP did not allow us to determine more precisely the site of interaction in CBP. This suggests that CBP and p67SRF do not interact through the previously characterized domains of interaction (amino acids 1–101 for the glucocorticoid receptor; amino acid 461–661 for various transcription factors). A possibility is that two physically separate sequences are required for this interaction. p67SRF is not the only member of the MADS box family which is able to interact with CBP. Indeed, CBP is also able to contact, through an undetermined region, MEF-2, a member of this family of proteins that is involved in muscle cell differentiation (43Eckner R. Yao T.P. Oldread E. Livingston D.M. Genes Dev. 1996; 10: 2478-2490Crossref PubMed Scopus (318) Google Scholar, 62Sartorelli V. Huang J. Hamamori Y. Kedes L. Mol. Cell. Biol. 1997; 17: 1010-1026Crossref PubMed Scopus (319) Google Scholar).For SRE transactivation, p67SRF cooperates with p62TCF. Both p67SRF (this study) and p62TCF (26Janknecht R. Nordheim A. Oncogene. 1996; 12: 1961-1969PubMed Google Scholar) physically interact with CBP. Thus, transactivation through the SRE might involve a multimolecular complex including CBP, p67SRF, and p62TCF, stabilized by multiple interactions between the partners in the complex. Interestingly, p67SRF is also involved in other processes such as muscle-cell terminal differentiation (63Vandromme M. Gauthier-Rouviäre C. Carnac G. Lamb N. Fernandez A. J. Cell Biol. 1992; 118: 1489-1500Crossref PubMed Scopus (85) Google Scholar). Indeed, p67SRF cooperates with the myogenic differentiation factors MyoD and myogenin for some muscle promoters' transactivation (64Catala F. Wanner R. Barton P. Cohen A. Wright W. Buckingham M. Mol. Cell. Biol. 1995; 15: 4585-4596Crossref PubMed Scopus (82) Google Scholar) and is able to interact physically with these myogenic bHLHs (65Groisman R. Masutani H. Leibovitch M.-P. Robin P. Soudant I. Trouche D. Harel-Bellan A. J. Biol. Chem. 1996; 271: 5258-5264Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). CBP interacts both with MyoD (43Eckner R. Yao T.P. Oldread E. Livingston D.M. Genes Dev. 1996; 10: 2478-2490Crossref PubMed Scopus (318) Google Scholar, 62Sartorelli V. Huang J. Hamamori Y. Kedes L. Mol. Cell. Biol. 1997; 17: 1010-1026Crossref PubMed Scopus (319) Google Scholar) and p67SRF (this study). Thus, the cooperation between MyoD and p67SRF on muscle cell differentiation also might involve the cooperative recruitment of CBP resulting in the formation, on muscle promoters, of a multimolecular complex including CBP, p67SRF and MyoD, and which would be stabilized by multiple interactions between the various partners of the complex. The SRE, which is a central element of the cell's immediate early response, binds several transcription factors and is targeted by several transduction pathways. In particular, p62TCF is a direct target for the Ras/MAP kinase pathway. p67SRF is required to assist p62TCF binding and is also independently a target for signal transduction pathways involving small G-proteins from the Rho/Rac family. Little is known about the mechanism used by these proteins to further transmit the activation signal to the minimal promoters of the genes controlled by the SRE element. Recently, Janknecht and collaborators (26Janknecht R. Nordheim A. Oncogene. 1996; 12: 1961-1969PubMed Google Scholar) have shown that CBP, a versatile adaptor protein, cooperates with p62TCF for transactivation through the SRE. We here show that, indeed, CBP participates in the transactivation through the SRE, since expression of CBP stimulated a response to serum through this element in a dose-dependent manner. Interestingly, in our study, the effect of CBP was observed in the absence of a functional Ets-binding site, and thus in the absence of TCF binding to the element. In many cell systems, the Ets-binding site is not necessary to observe a significant response to serum (22Hill C.S. Marais R. John S. Wynne J. Dalton S. Treisman R. Cell. 1993; 73: 395-406Abstract Full Text PDF PubMed Scopus (329) Google Scholar,25Hill C.S. Wynne J. Treisman R. Cell. 1995; 81: 1159-1170Abstract Full Text PDF PubMed Scopus (1199) Google Scholar, 59Dalton D. Treisman R. Cell. 1992; 68: 597-612Abstract Full Text PDF PubMed Scopus (534) Google Scholar, 60Johansen F.E. Prywes R. Mol. Cell. Biol. 1994; 14: 5920-5928Crossref PubMed Scopus (123) Google Scholar). Moreover, although F9 cells, which were used in this study, express normal amounts of p62TCF, the ternary complex between p67SRF and p62TCF seems to be inactive in these cells (61König H. Nucleic Acids Res. 1991; 19: 3607-3611Crossref PubMed Scopus (59) Google Scholar). Our result indicates that the participation of CBP in SRE transactivation does not absolutely require the Ets protein p62TCF. In contrast, a functional CArG box was indispensable to observe the stimulation of the SRE response, suggesting that the target protein of CBP in this function was p67SRF. Thus, CBP cooperates both with p62TCF(26Janknecht R. Nordheim A. Oncogene. 1996; 12: 1961-1969PubMed Google Scholar, 27Janknecht R. Nordheim A. Biochem. Biophys. Res. Commun. 1996; 228: 831-837Crossref PubMed Scopus (172) Google Scholar) and with p67SRF (this study). CBP is a large molecule that includes several sites of interaction with various sequence-specific transcription factors and has two TADs. To determine which of these functional domains was involved in SRE transactivation, we have used deletion mutants of the protein. Interestingly, the N-terminal moiety of CBP was sufficient to stimulate the SRE response to serum. A C-terminal transactivation domain of CBP seems to be involved in the cooperation with p62TCF (26Janknecht R. Nordheim A. Oncogene. 1996; 12: 1961-1969PubMed Google Scholar). Thus, CBP uses two TADs for transactivation through the SRE, the N-terminal and the C-terminal TAD. In addition, these data demonstrate that CBP can transactivate the SRE in the absence of the domain bearing the histone acetyltransferase activity, which is located in the C-terminal part of the molecule, indicating that this intramolecular activity is not absolutely required in this system. A similar result has been obtained in the CREB model system (49Swope D.L. Mueller C.L. Chrivia J.C. J. Biol. Chem. 1996; 271: 28138-28145Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar). In addition, we also show that CBP and p67SRF form a complex in live cells, since the two proteins can be co-immunoprecipitated from cell extracts. The interaction between p67SRF and CBP does not require that the transcription factor be bound to its target DNA sequence, contrary to what has been observed with MyoD (43Eckner R. Yao T.P. Oldread E. Livingston D.M. Genes Dev. 1996; 10: 2478-2490Crossref PubMed Scopus (318) Google Scholar). The physical interaction between p67SRF and CBP is detected by co-immunoprecipitation assays, which require a high affinity between the proteins, suggesting that this interaction is strong. The interaction between the N-terminal part of CBP and p67SRF was confirmed in a two-hybrid assay in mammalian cells. However, analysis of various subregions of the N-terminal CBP did not allow us to determine more precisely the site of interaction in CBP. This suggests that CBP and p67SRF do not interact through the previously characterized domains of interaction (amino acids 1–101 for the glucocorticoid receptor; amino acid 461–661 for various transcription factors). A possibility is that two physically separate sequences are required for this interaction. p67SRF is not the only member of the MADS box family which is able to interact with CBP. Indeed, CBP is also able to contact, through an undetermined region, MEF-2, a member of this family of proteins that is involved in muscle cell differentiation (43Eckner R. Yao T.P. Oldread E. Livingston D.M. Genes Dev. 1996; 10: 2478-2490Crossref PubMed Scopus (318) Google Scholar, 62Sartorelli V. Huang J. Hamamori Y. Kedes L. Mol. Cell. Biol. 1997; 17: 1010-1026Crossref PubMed Scopus (319) Google Scholar). For SRE transactivation, p67SRF cooperates with p62TCF. Both p67SRF (this study) and p62TCF (26Janknecht R. Nordheim A. Oncogene. 1996; 12: 1961-1969PubMed Google Scholar) physically interact with CBP. Thus, transactivation through the SRE might involve a multimolecular complex including CBP, p67SRF, and p62TCF, stabilized by multiple interactions between the partners in the complex. Interestingly, p67SRF is also involved in other processes such as muscle-cell terminal differentiation (63Vandromme M. Gauthier-Rouviäre C. Carnac G. Lamb N. Fernandez A. J. Cell Biol. 1992; 118: 1489-1500Crossref PubMed Scopus (85) Google Scholar). Indeed, p67SRF cooperates with the myogenic differentiation factors MyoD and myogenin for some muscle promoters' transactivation (64Catala F. Wanner R. Barton P. Cohen A. Wright W. Buckingham M. Mol. Cell. Biol. 1995; 15: 4585-4596Crossref PubMed Scopus (82) Google Scholar) and is able to interact physically with these myogenic bHLHs (65Groisman R. Masutani H. Leibovitch M.-P. Robin P. Soudant I. Trouche D. Harel-Bellan A. J. Biol. Chem. 1996; 271: 5258-5264Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). CBP interacts both with MyoD (43Eckner R. Yao T.P. Oldread E. Livingston D.M. Genes Dev. 1996; 10: 2478-2490Crossref PubMed Scopus (318) Google Scholar, 62Sartorelli V. Huang J. Hamamori Y. Kedes L. Mol. Cell. Biol. 1997; 17: 1010-1026Crossref PubMed Scopus (319) Google Scholar) and p67SRF (this study). Thus, the cooperation between MyoD and p67SRF on muscle cell differentiation also might involve the cooperative recruitment of CBP resulting in the formation, on muscle promoters, of a multimolecular complex including CBP, p67SRF and MyoD, and which would be stabilized by multiple interactions between the various partners of the complex. We thank Linda Pritchard for helpful discussion. The CREB-binding protein (CBP) cooperates with the serum response factor for transactivation of the c-fosserum response element.Journal of Biological ChemistryVol. 274Issue 25PreviewDr. Ait-Si-Ali's name was printed incorrectly. The correct version is shown above. Full-Text PDF Open Access

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Virus-based gene therapy research

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FuenteJournal of Biological Chemistry	Cuartil año de publicaciónNo disponible	Volumen272
Issue49	Páginas31016 - 31021	pISSN0021-9258
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