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• W2023948035 abstract "In this paper we show that transcription factors Ets-1 and Ets-2 recruit transcription adapter proteins p300 and CBP (cAMP-responsive element-binding protein) during the transcriptional activation of the human stromelysin promoter, which contains palindromic Ets-binding sites. Ets-2 and p300/CBP exist as a complexin vivo. Two regions of p300/CBP between amino acids (a.a.) 328 and 596 and a.a. 1678 and 2370 independently can interact with Ets-1 and Ets-2 in vitro and in vivo. Both these regions of p300/CBP bind to the transactivation domain of Ets-2, whereas the C-terminal region binds only to the DNA binding domain of Ets-2. The N- and the C-terminal regions of CBP (a.a. 1–1097 and 1678–2442, respectively) which lack histone acetylation activity independently are capable of coactivating Ets-2. Other Ets family transcription factors failed to cooperate with p300/CBP in stimulating the stromelysin promoter. The LXXLL sequence, reported to be important in receptor-coactivator interactions, does not appear to play a role in the interaction of Ets-2 with p300/CBP. Previous studies have shown that the stimulation of transcriptional activation activity of Ets-2 requires phosphorylation of threonine 72 by the Ras/mitogen-activated protein kinase signaling pathway. We show that mutation of this site does not affect its capacity to bind to and to cooperate with p300/CBP. In this paper we show that transcription factors Ets-1 and Ets-2 recruit transcription adapter proteins p300 and CBP (cAMP-responsive element-binding protein) during the transcriptional activation of the human stromelysin promoter, which contains palindromic Ets-binding sites. Ets-2 and p300/CBP exist as a complexin vivo. Two regions of p300/CBP between amino acids (a.a.) 328 and 596 and a.a. 1678 and 2370 independently can interact with Ets-1 and Ets-2 in vitro and in vivo. Both these regions of p300/CBP bind to the transactivation domain of Ets-2, whereas the C-terminal region binds only to the DNA binding domain of Ets-2. The N- and the C-terminal regions of CBP (a.a. 1–1097 and 1678–2442, respectively) which lack histone acetylation activity independently are capable of coactivating Ets-2. Other Ets family transcription factors failed to cooperate with p300/CBP in stimulating the stromelysin promoter. The LXXLL sequence, reported to be important in receptor-coactivator interactions, does not appear to play a role in the interaction of Ets-2 with p300/CBP. Previous studies have shown that the stimulation of transcriptional activation activity of Ets-2 requires phosphorylation of threonine 72 by the Ras/mitogen-activated protein kinase signaling pathway. We show that mutation of this site does not affect its capacity to bind to and to cooperate with p300/CBP. The Ets family of transcription factors includes a large number of proteins that perform diverse functions in the cell including the serum stimulation of the c-fos promoter (Elk-1/SAP-1 (1Treisman R. Curr. Opin. Cell Biol. 1996; 8: 205-215Crossref PubMed Scopus (1165) Google Scholar)), activation of herpes simplex virus immediate early promoters (GA-binding protein α and β (2LaMarco K. Thompson C.C. Byers B.P. Walton E.M. McKnight S.L. Science. 1991; 253: 789-792Crossref PubMed Scopus (259) Google Scholar)), regulation of immunoglobulin light chain enhancers (Pu.1/Spi-1 (3Eisenbeis C.F. Singh H. Storb U. Genes Dev. 1995; 9: 1377-1387Crossref PubMed Scopus (417) Google Scholar)), erythroid differentiation (4Metz T. Graf T. Genes Dev. 1991; 5: 369-380Crossref PubMed Scopus (75) Google Scholar), and Drosophila development (5O'Neill E.M. Rebay I. Tjian R. Rubin G.M. Cell. 1994; 78: 137-147Abstract Full Text PDF PubMed Scopus (591) Google Scholar). Constitutively active mutant Ets proteins (v-Ets-1 and -2) are involved in cellular transformation (6Seth A. Watson D.K. Blair D.G. Papas T.S. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 7833-7837Crossref PubMed Scopus (75) Google Scholar, 7Seth A. Papas T.S. Oncogene. 1990; 5: 1761-1767PubMed Google Scholar). A characteristic feature of this class of proteins is a highly conserved 85-amino acid (a.a.) 1The abbreviations used are: a.a., amino acids; CBP, CREB-binding protein; CREB, cAMP-responsive element-binding protein; CMV, cytomegalovirus; HA, hemagglutinin; MAP, mitogen-activated protein; MEK, MAP kinase kinase; TBP, TATA box-binding protein; AP1, activator protein 1; CAT, chloramphenicol acetyltransferase; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; WT, wild type; bp, base pair; NLS, nuclear localization signal; MMP, matrix metalloproteases; TA, transactivation; DB, DNA binding DNA binding domain termed the Ets domain which contains a helix-turn-helix motif. The Ets domain binds to a GGAA purine-rich core sequence found in the promoters and enhancers of viral and cellular genes. Outside the DNA binding domain, the Ets family proteins share limited homology (8Wasylyk B. Soonjung L.H. Giovane A. Eur. J. Biochem. 1993; 211: 7-18Crossref PubMed Scopus (811) Google Scholar, 9Janknecht R. Nordheim A. Biochim. Biophys. Acta. 1993; 1155: 346-356Crossref PubMed Scopus (206) Google Scholar, 10Graves B.J. Peters J.M. Adv. Cancer Res. 1998; 75: 1-55Crossref PubMed Google Scholar). Ets-1 and Ets-2 are ubiquitous proteins that share significant homology (11Watson D.K. McWilliams M.J. Lapis P. Lautenberger J.A. Schweinfest C.W. Papas T.S. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7862-7866Crossref PubMed Scopus (206) Google Scholar). The transactivation and the DNA binding (Ets) domains in both Ets-1 and -2 map to the N- and the C-terminal regions, respectively (12Bhat N.K. Fisher R.J. Fujiwara S. Ascione R. Papas T.S. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 3161-3165Crossref PubMed Scopus (131) Google Scholar, 13Schneikert J. Lutz Y. Wasylyk B. Oncogene. 1992; 7: 249-256PubMed Google Scholar, 14Chumakov A.M. Chen D.L. Chumakova E.A. Koeffler H.P. J. Virol. 1993; 67: 2421-2425Crossref PubMed Google Scholar). The homologous regions include the Ets domain which is 95% conserved between Ets-1 and Ets-2 (11Watson D.K. McWilliams M.J. Lapis P. Lautenberger J.A. Schweinfest C.W. Papas T.S. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 7862-7866Crossref PubMed Scopus (206) Google Scholar) and the Pointed domain located in the transactivation domain (15Klambt C. Development. 1993; 117: 163-176PubMed Google Scholar). The Pointed domain consists of approximately a 100-a.a. region that is conserved within a subgroup of Ets factors including Drosophila Ets factor Pointed P2 and Ets-1 and Ets-2 (15Klambt C. Development. 1993; 117: 163-176PubMed Google Scholar). The Pointed domain contains a MAP kinase phosphorylation site, and Pointed P2 is a target of Ras/MAP kinase signaling pathways in Drosophila (5O'Neill E.M. Rebay I. Tjian R. Rubin G.M. Cell. 1994; 78: 137-147Abstract Full Text PDF PubMed Scopus (591) Google Scholar). Ets-1 and Ets-2 are targets of the Ras signaling pathway (16Galang C.K. Der C.J. Hauser C.A. Oncogene. 1994; 9: 2913-2921PubMed Google Scholar), and Ras-mediated activation of Ets-1 and -2 transactivation activity requires phosphorylation of Ets-2 threonine 72 and the corresponding Ets-1 threonine 38, which are also conserved in Pointed P2 and Yan (a Drosophila repressor (17Yang B. Hauser C.A. Henkel G. Colman M.S. Beveren C.V. Stacy L.J. Hume D.A. Maki R.A. Ostrowski M.C. Mol. Cell. Biol. 1996; 16: 538-547Crossref PubMed Scopus (318) Google Scholar, 18Galang C.K. Garcia-Ramirez J. Solski P.A. Westwick J.K. Der C.J. Neznanov N.N. Oshima R.G. Hauser C.A. J. Biol. Chem. 1996; 271: 7992-7998Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar, 19McCarthy S.A. Chen D. Yang B.-S. Garcı́a-Ramı́rez J.J. Cherwinski H. Chen X.-R. Klagsbrun M.L. Hauser C.A. Ostrowski M.C. McMahon M. Mol. Cell. Biol. 1997; 17: 2401-2412Crossref PubMed Scopus (151) Google Scholar)). The conserved MAP kinase site (threonine 72) in Ets-2 is phosphorylated by MAP kinase (17Yang B. Hauser C.A. Henkel G. Colman M.S. Beveren C.V. Stacy L.J. Hume D.A. Maki R.A. Ostrowski M.C. Mol. Cell. Biol. 1996; 16: 538-547Crossref PubMed Scopus (318) Google Scholar). Ets-binding sites, as well as AP-1-binding sites, are often found in the promoters of Ras-induced genes (20Bortner D.M. Langer S.J. Ostrowski M.C. Crit. Rev. Oncol. 1993; 4: 137-160Google Scholar), and Ets-2 and AP-1 cooperate in gene expression (21Wasylyk B. Wasylyk C. Flores P. Begue A. Leprince D. Stehelin D. Nature. 1990; 346: 191-193Crossref PubMed Scopus (416) Google Scholar, 22Nerlov C. Rorth P. Blasi F. Johnsen M. Oncogene. 1991; 6: 1583-1592PubMed Google Scholar, 23Kramer B. Wiegmann K. Kronke M. J. Biol. Chem. 1995; 270: 6577-6583Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). p300 and the CREB-binding protein (CBP) are two highly homologous, conserved nuclear phosphoproteins that function as transcriptional coactivators by bridging a very large number of DNA-bound transcription factors with basal transcription complex (24Janknecht R. Hunter R. Curr. Biol. 1996; 6: 951-954Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar, 25Goldman P.S. Tran V.K. Goodman R.H. Recent Prog. Horm. Res. 1997; 52: 103-119PubMed Google Scholar, 26Shikama N. Lyon J. La Thangue N.B. Trends Cell Biol. 1997; 7: 230-237Abstract Full Text PDF PubMed Scopus (432) Google Scholar). p300/CBP also binds to a number of proteins that are not transcription factors including viral oncogene products (27Eckner 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 (927) Google Scholar, 28Lill N.L. Tevethia M.J. Eckner R. Livingston D.M. Modjtahedi N. J. Virol. 1997; 7: 129-137Crossref Google Scholar), SRC1 (29Smith C.L. Onate S.A. Tsai M.-J. O'Malley B.W. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8884-8888Crossref PubMed Scopus (370) Google Scholar), Cdk2 (30Perkins N.D. Felzien L.K. Betts J.C. Leung K. Beach D. Nabel G.J. Science. 1997; 275: 523-527Crossref PubMed Scopus (666) Google Scholar), and a protein containing enzymatic activity as histone acetyltransferase, p300/CBP-associated factor (31Yang X.-J. Ogryzko V.V. Nishikawa J. Howard B.H. Nakatani Y. Nature. 1996; 382: 319-324Crossref PubMed Scopus (1320) Google Scholar). Recent studies show that p300/CBP also has enzyme activity as a histone acetyltransferase, linking chromatin remodeling with transcription (32Grant P.A. Sterner D.E. Duggan L.J. Workman J.L. Berger S.L. Trends Cell Biol. 1998; 8: 193-197Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). It has been suggested that the amount of p300/CBP in cells may be rate-limiting and that different transcription factors may compete for rate-limiting amounts of these coactivators and thus provide mechanisms for cross-talk in the regulation of gene expression (33Cheng X. Reginato M.J. Andrews N.C. Lazar M.A. Mol. Cell. Biol. 1997; 17: 1407-1416Crossref PubMed Scopus (97) Google Scholar, 34Kamei Y. Xu L. Heinzel T. Torchia J. Kurokawa R. Gloss D.W. Glass C.K. Rosenfeld M.G. Cell. 1996; 85: 403-414Abstract Full Text Full Text PDF PubMed Scopus (1928) Google Scholar). Targeted gene disruption studies have confirmed that p300 function is essential for normal embryonic cellular proliferation, morphogenesis, and development with double knockouts resulting in 100% embryonic lethality (35Yao T.P. Oh S.P. Fuchs M. Zhou N.D. Chang L.E. Newsome D. Bronson R.T.E.L. Livingston D.M. Eckner R. Cell. 1998; 93: 361-372Abstract Full Text Full Text PDF PubMed Scopus (827) Google Scholar). Normal levels of CBP in these mice did not substitute for the p300 functions suggesting that the double knock-out phenotype may be either due to gene dosage effect or the loss of specific functions provided by p300. Likewise, haploinsufficiency of CBP gives rise to severe developmental abnormalities characteristic of the Rubinstein-Taybi syndrome, including mental retardation, craniofacial abnormalities, skeletal abnormalities, and increased cancer incidence (36Giles R.H. Peters D.J. Breuning M.H. Trends Genet. 1998; 14: 178-183Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar). These studies suggest that both proteins are required for embryonic development. Stromelysin is an important member of a family of matrix metalloproteases (MMPs) which degrade extracellular matrix during a variety of normal and pathological processes. In this paper, we show that two important members of the Ets family, Ets-1 and Ets-2, recruit p300/CBP in the activation of the stromelysin promoter, and this recruiting involves multiple protein-protein interactions. Consistent with these multiple interactions, the N- and the C-terminal halves of p300/CBP independently can coactivate Ets-2 to stimulate the stromelysin promoter. Other Ets family transcription factors do not cooperate with p300/CBP in the stimulation of the stromelysin promoter. We also show that mutation of the Ets-2 MAP kinase phosphorylation site, important for Ras-mediated regulation of Ets-2, does not affect its ability to bind to p300/CBP or its ability to cooperate with p300/CBP in the transcriptional activation. pSK200 is a promoter-reporter plasmid that contains 542 bp upstream from the cap site of the human stromelysin gene fused to the CAT reporter gene. pSK201 and pSK202 are two mutant versions of pSK200 in which the AP-1 site, located between −64 and −71, is mutated (pSK201) or the two palindromic Ets sites deleted (pSK202) (37Buttice G. Kurkinen M. J. Biol. Chem. 1993; 268: 7196-7204Abstract Full Text PDF PubMed Google Scholar). E18pal is a promoter-reporter plasmid in which two palindromic Ets sites were cloned upstream of a c-fosminimal promoter followed by the CAT gene. c-fos minimal promoter contains 56 bp upstream of the cap site of the c-fos gene (16Galang C.K. Der C.J. Hauser C.A. Oncogene. 1994; 9: 2913-2921PubMed Google Scholar). Plasmids pFNEts-1 and pFNEts-2 are two expression plasmids that express mouse ets-1 andets-2, respectively (17Yang B. Hauser C.A. Henkel G. Colman M.S. Beveren C.V. Stacy L.J. Hume D.A. Maki R.A. Ostrowski M.C. Mol. Cell. Biol. 1996; 16: 538-547Crossref PubMed Scopus (318) Google Scholar). 2C. A. Hauser, unpublished results. The Ets-1 and Ets-2 protein coding sequences in these plasmids are tagged with FLAG epitope followed by the SV40 large T nuclear localization signal (NLS) at their N-terminal end. These plasmids are in pcDNA3 background. Plasmid pFNEts-2A72 is a derivative of pFNEts-2 in which the MAP kinase substrate Thr at 72 residue is changed to Ala (T72A mutation (17Yang B. Hauser C.A. Henkel G. Colman M.S. Beveren C.V. Stacy L.J. Hume D.A. Maki R.A. Ostrowski M.C. Mol. Cell. Biol. 1996; 16: 538-547Crossref PubMed Scopus (318) Google Scholar)). Plasmid p300CHA expresses hemagglutinin epitope-tagged p300 from the CMV promoter (27Eckner 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 (927) Google Scholar). Plasmids GST-p300N, GST-p300M, and GST-p300C contain a.a. 1–596, 744–1571, and 1572–2370, respectively, fused in frame with glutathione S-transferase coding sequences and kind gifts of Dr. D. Livingston of Harvard Medical School (27Eckner 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 (927) Google Scholar). p300N, p300M, and p300C are plasmids in which p300 cDNA sequences corresponding a.a. 1–746, 743–1572, and 1572–2414 respectively, cloned downstream of the T7 phage promoter in Bluescript SK. Full-length and truncated murine CBP coding sequences are expressed from a CMV promoter-based expression plasmid (pCMV2N3-T (38Ramirez S. Ali S.A.S. Robin P. Trouche D. Harel-Bellan A. J. Biol. Chem. 1997; 272: 31016-31021Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar)) in which the coding sequences are tagged at their N-terminal end with two copies of SV40 nuclear localization signals followed by three copies of HA epitope (38Ramirez S. Ali S.A.S. Robin P. Trouche D. Harel-Bellan A. J. Biol. Chem. 1997; 272: 31016-31021Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). These plasmids are kind gifts of Dr. A. Harel-Bellan (CNRS, Villejuif, France). To identify the Ets-2·CBP complex in live cells, proliferating human 293 cells were pelleted and lysed on ice in a lysis buffer (27Eckner 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 (927) Google Scholar) for 40 min, and the protein was quantitated by the Bradford method (39Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (217544) Google Scholar). The cell lysate, equivalent to 2 mg of protein, was concentrated by the addition of 5 volumes of ice-cold acetone for 30 min on ice and then centrifuged for 30 min at 15,000 rpm. The pellet was dissolved in 500 μl of the lysis buffer containing 0.3% Nonidet P-40, and it was then incubated with 40 μl of the anti Ets-2 rabbit polyclonal antibody (Santa Cruz Biotechnology, SC-351) overnight at 4 °C. 50 μl of the protein A-agarose beads were then added, and the incubation was continued for another 2 h at 4 °C. The beads were then pelleted, washed three times with lysis buffer containing 0.3% Nonidet P-40, subjected to SDS-8% PAGE, and electrophoresed in Tris glycine buffer for 5 h as described (40Somasundaram K. Jayaraman G. Williams T. Moran E. Frisch S. Thimmapaya B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3088-3093Crossref PubMed Scopus (69) Google Scholar). The immunoprecipitates were then assayed in Western blots using an anti-CBP rabbit polyclonal antibody (Santa Cruz Biotechnology, SC-583). For the negative control experiment, 2 mg of the protein from the above lysate was immunoprecipitated using an anti-PKR polyclonal antibody raised in our laboratory. For direct immunoprecipitation, cell lysate corresponding to 500 μg of the protein was incubated with anti-CBP antibody, and the immunoprecipitated polypeptides were subjected to SDS-PAGE followed by Western blot as described above. For the detection of the complexes containing various forms of Ets and the truncated forms of CBP, 100-cm dishes of 293 cells were transfected with 15 μg of each of the plasmids indicated in the figure legends using the calcium phosphate precipitation method, and cell extracts were subjected to immunoprecipitations followed by Western blots as described above with appropriate antibodies. The construction of the GST-Ets2 plasmid expressing GST full-length fusion protein was described previously (41Basuyaux J.P. Ferreira E. Stehelin D. Buttice G. J. Biol. Chem. 1997; 272: 26188-26195Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Other GST fusion plasmids expressing GST-truncated Ets-2 fusion proteins, as well as full-length Ets-1 proteins, were constructed by a polymerase chain reaction-mediated cloning approach using pSVK3-Ets2 and pSG5-Ets-1 expression plasmids as templates. 3E. Ferreira and G. Buttice, manuscript in preparation. p300-Ets-2 interaction in yeast cells was assayed using the Matchmaker Two-hybrid system of CLONTECH (catalog number 1604-1), following the manufacturer's instructions. Briefly, DNA fragments coding for the p300 coding segments from a.a. 328 to 1000 and 962 to 1575 were fused in frame with Gal-4 DNA binding domain in the DNA binding domain plasmid pGBT9 (p300-1 and p300-2 respectively). cDNAs coding for the WT Ets-2, a mutant Ets-2 containing the T72A mutation and the AAAAA mutation (both created by using the Stratagene QuickChange site-directed mutagenesis kit), were cloned into the GAL-4 activation domain plasmid pACT-2. Yeast strain CG1945 was cotransformed with the two-hybrid vectors expressing segments of p300 and Ets-2 proteins, and the cells were selected for growth on SD medium lacking leucine, tryptophan, and histidine. The colonies were then amplified and qualitatively assayed for β-galactosidase activity usingo-nitrophenyl β-d-galactopyranoside as a substrate (42Himmelfarb H.J. Pearlberg J. Last D.H. Ptashne M. Cell. 1990; 63: 1299-1309Abstract Full Text PDF PubMed Scopus (112) Google Scholar). Transcription of the human stromelysin gene is driven by AP1/Fos and Ets-2 transcription factors and regulated by a variety of factors including 12-O-tetradecanoylphorbol-13-acetate, cytokines, growth factors, and protooncogenes (37Buttice G. Kurkinen M. J. Biol. Chem. 1993; 268: 7196-7204Abstract Full Text PDF PubMed Google Scholar, 43Matrisian L.M. Ganser G.L. Kerr L.D. Pelton R.W. Wood L.D. Mol. Reprod. Dev. 1992; 32: 111-120Crossref PubMed Scopus (60) Google Scholar, 44Wasylyk C. Gutman A. Nicholson R. Wasylyk B. EMBO J. 1991; 10: 1127-1134Crossref PubMed Scopus (326) Google Scholar, 45Frisch S.M. Ruley H.E. J. Biol. Chem. 1987; 262: 16300-16304Abstract Full Text PDF PubMed Google Scholar, 46Buttice G. Quinones S. Kurkinen M. Nucleic Acids Res. 1991; 19: 3723-3731Crossref PubMed Scopus (107) Google Scholar). The AP1/Fos and Ets elements of the stromelysin promoter map between −64 and −71 and −201 and −218 with respect to cap site, respectively (Fig.1 A) (37Buttice G. Kurkinen M. J. Biol. Chem. 1993; 268: 7196-7204Abstract Full Text PDF PubMed Google Scholar, 46Buttice G. Quinones S. Kurkinen M. Nucleic Acids Res. 1991; 19: 3723-3731Crossref PubMed Scopus (107) Google Scholar). The Ets site in this promoter consists of two copies of a motif similar to the polyoma virus enhancer A-binding protein-3 (PEA-3) site that binds to the Ets family transcription factors Ets-1 and -2 and stimulates transcription (44Wasylyk C. Gutman A. Nicholson R. Wasylyk B. EMBO J. 1991; 10: 1127-1134Crossref PubMed Scopus (326) Google Scholar). To determine whether p300 would cooperate with Ets-2 in the activation of the stromelysin promoter, HeLa cells were cotransfected with a stromelysin promoter-reporter construct that contained 542 bp upstream from the cap site fused to CAT reporter gene (pSK200) and expression vectors encoding p300 (p300CHA (27Eckner 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 (927) Google Scholar)) and mouseets-2 (pFNEts-2WT (16Galang C.K. Der C.J. Hauser C.A. Oncogene. 1994; 9: 2913-2921PubMed Google Scholar)), in combinations as shown in Fig.1 B. These 542-bp promoter sequences were shown to be sufficient for the basal, 12-O-tetradecanoylphorbol-13-acetate, and Ets-2-stimulated transcription of this promoter (46Buttice G. Quinones S. Kurkinen M. Nucleic Acids Res. 1991; 19: 3723-3731Crossref PubMed Scopus (107) Google Scholar). CAT activity was assayed 48 h after transfection as described (40Somasundaram K. Jayaraman G. Williams T. Moran E. Frisch S. Thimmapaya B. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3088-3093Crossref PubMed Scopus (69) Google Scholar). By itself, p300 or Ets-2 did not stimulate the stromelysin promoter significantly (less than 2-fold stimulation; Fig. 1 B). However, cotransfection of p300 andets-2 expression vectors together stimulated the stromelysin promoter by about 20-fold, indicating that p300 cooperates with Ets-2 in the activation of the stromelysin promoter. To determine whether the Ets-2 activation of the stromelysin promoter is mediated by the Ets-binding site in the promoter, mutant promoters with mutations in the AP-1 site (pSK201) or with a deletion of the two Ets sites (pSK202) were studied using same methods described above. Together, Ets-2 and p300 stimulated the AP-1 mutant promoter about 8-fold (Fig.1 B, pSK201). In contrast, a mutant promoter lacking the two Ets-2 sites was inactive in these assays (Fig. 1 B, pSK202). These results suggest that the two Ets-2 sites can respond to transcriptional superactivation in the presence of exogenously provided Ets-2 and p300. It is interesting that the promoter mutant lacking the AP-1 site was not efficiently induced by p300 in the presence of Ets-2. To study further the cooperative activity of Ets-2 and p300, we tested a synthetic promoter-reporter construct in which two palindromic Ets sites were placed upstream of the c-fos minimal promoter containing −56 to +109 bp of the c-fos promoter fused to CAT reporter gene (Fig. 1 A, E18pal) (16Galang C.K. Der C.J. Hauser C.A. Oncogene. 1994; 9: 2913-2921PubMed Google Scholar). By itself, p300 did not stimulate E18pal significantly (about 2-fold), whereas Ets-2 stimulated this construct by about 6-fold. In contrast, the two effectors together stimulated E18pal by about 17-fold (Fig.1 B, E18pal). These results provide evidence that Ets-2 and p300 can cooperate in the transcriptional activation of Ets-2 site containing promoters even in the absence of other upstream elements. CBP also cooperated with Ets-2 and stimulated the E18pal and the stromelysin promoter by about 8–10-fold (data not shown; also see Fig. 5). We considered it possible that the dramatic increase in the reporter activity by p300 and Ets-1/Ets-2 proteins together in the promoter-reporter assays, described above, may be due to a significant increase in the levels of Ets-1/Ets-2 as a result of stimulation of the CMV promoter of the Ets expression vectors by p300/CBP. We found that in human cells, using identical transfection assay conditions described above, p300 increased the levels of Ets-1 and Ets-2 only marginally (1.5-fold, Fig. 1 C; data for Ets-1 not shown). U2OS cells were used in this experiment because of their higher transfection efficiency; p300/CBP cooperated with Ets-1/Ets2 in the stimulation of the stromelysin promoter in these cells as efficiently as HeLa cells (data not shown). Thus we conclude that p300/CBP increases the transcriptional activation activity of Ets-1 and Ets-2 and stimulates the Ets-2 site containing promoters. Previous studies have shown that Ets-2 activates the human stromelysin promoter (37Buttice G. Kurkinen M. J. Biol. Chem. 1993; 268: 7196-7204Abstract Full Text PDF PubMed Google Scholar), whereas the rat stromelysin promoter is activated by both Ets-1 and Ets-2 (44Wasylyk C. Gutman A. Nicholson R. Wasylyk B. EMBO J. 1991; 10: 1127-1134Crossref PubMed Scopus (326) Google Scholar). Both promoters contain palindromic Ets binding sites. Therefore, it was of interest to determine whether p300/CBP would cooperate with Ets-1 to activate the human stromelysin promoter. Ets-1 did not stimulate the stromelysin promoter significantly. In contrast, Ets-1 and p300 together stimulated the stromelysin promoter by about 25-fold (Fig. 1 B). In the case of E18pal, p300 did not stimulate the promoter significantly, whereas it was stimulated by Ets-1 by about 5-fold. When the E18pal was tested in the combined presence of Ets-1 and p300, it was superactivated by about 38-fold (data not shown). Because p300/CBP can cooperate with Ets-2 in the transcriptional activation of Ets-binding site containing promoters, we reasoned that Ets-2 and p300/CBP might interact and exist as a complex in vivo. To determine this, proliferating human 293 cells were lysed in a lysis buffer, and the cell lysate equivalent to 2 mg of protein was first immunoprecipitated with a polyclonal antibody directed against the C-terminal region of Ets-2. The immunoprecipitated proteins were then separated on an SDS-8% polyacrylamide gel (Fig.2, lane 2). As a negative control, an equal amount of protein from the cell lysate was immunoprecipitated with a polyclonal antibody directed against the cellular double-stranded RNA-activated protein kinase (eIF-2 α-kinase, also called PKR (47Proud C.G. Trends Biochem. Sci. 1995; 20: 241-246Abstract Full Text PDF PubMed Scopus (200) Google Scholar)), and the immunoprecipitated proteins were loaded on the same gel (lane 3). Five hundred μg of protein from the same lysate was also immunoprecipitated with a polyclonal antibody which recognizes the N-terminal region of human CBP and the immunoprecipitated proteins were separated as above (lane 4). In lane 1, 60 μg of the protein from the same lysate was loaded directly. The gel was electrophoretically transferred to a nitrocellulose membrane and probed with an anti-CBP antibody. As shown in Fig. 2, anti-CBP antibody recognizes a band in all lanes except in lane 3, which corresponds to a 300-kDa protein. This band is absent in anti-PKR immunoprecipitates (lane 3). The molecular weight of the protein that was brought down by anti-Ets-2 antibody in immunoprecipitations and which reacted with anti-CBP antibody in Western blot is similar to that reacted with anti-CBP antibody (lanes 1 and 4). Thus we conclude that Ets-2 and CBP exist as a complex in vivo. To determine the regions of p300/CBP that bind to Ets-2 and to determine whether Ets-1 also binds to p300/CBP in similar manner, we employed GST pull-down, coimmunoprecipitation, and yeast two-hybrid assays. Fragments of p300 from a.a. 1 to 596 (GST-p300N), 744 to 1571 (GST-p300M), and 1572 to 2370 (GST-p300C, Fig.3 A) were expressed as GST fusion proteins. Fig. 3 B shows a Coomassie Blue-stained gel of the affinity purified GST-p300 fusion proteins; all three fusion proteins were overexpressed in Escherichia coli. Ets-1 and Ets-2 were labeled with [35S]methionine in a coupled transcription/translation system and were then incubated with equal quantities of GST fusion proteins immobilized on agarose beads. Agarose beads containing GST and luciferase labeled with [35S]methionine in vitro, as described above, were used as the negative controls. Quantitation of the radiolabeled bands indicated that about 6 and 8% of the input Ets-2 were bound to the N- and the C-terminal regions of p300, respectively (Fig.3 C). Similarly, about 4% of the Ets-1 bound to the N- and the C-terminal regions of p300. In contrast, binding of the radiolabeled luciferase to GST-p300M was negligible. Similarly, negligible amounts of Ets-1 and Ets-2 bound t" @default.
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• W2023948035 title "p300/cAMP-responsive Element-binding Protein Interactions with Ets-1 and Ets-2 in the Transcriptional Activation of the Human Stromelysin Promoter" @default.
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