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• W2003135979 abstract "The adenovirus E1A-associated protein p300 is a transcriptional cofactor that interacts with YY1 and mediates the relief of YY1 transcriptional repression by E1A. These observations raise the possibility that p300 may function as a bridging factor between E1A and cellular transcription factors. Here we show that p300, but not a mutant defective for binding to E1A, activated cAMP-responsive element-binding protein/activating transcription factor (CREB/ATF) binding site-mediated transcription in the presence of E1A. Among proteins that can recognize the CREB/ATF site, CREB appeared to be modulated by E1A in a p300 binding-dependent manner. This effect of E1A was correlated with a specific physical interaction between CREB and p300. These results suggest that p300 plays a crucial role in mediating the functional interplay between E1A and certain members of the CREB/ATF family. Two separate domains within p300 were identified that are capable of activating transcription. One of the domains interacted with the basal factor TFIIB, suggesting that p300 may function as a coactivator by making contacts with both sequence-specific transcription factors and the basal transcriptional machinery. This pivotal role of p300 may make it a prime target for viral proteins such as E1A in programming the cellular transcription machinery. The adenovirus E1A-associated protein p300 is a transcriptional cofactor that interacts with YY1 and mediates the relief of YY1 transcriptional repression by E1A. These observations raise the possibility that p300 may function as a bridging factor between E1A and cellular transcription factors. Here we show that p300, but not a mutant defective for binding to E1A, activated cAMP-responsive element-binding protein/activating transcription factor (CREB/ATF) binding site-mediated transcription in the presence of E1A. Among proteins that can recognize the CREB/ATF site, CREB appeared to be modulated by E1A in a p300 binding-dependent manner. This effect of E1A was correlated with a specific physical interaction between CREB and p300. These results suggest that p300 plays a crucial role in mediating the functional interplay between E1A and certain members of the CREB/ATF family. Two separate domains within p300 were identified that are capable of activating transcription. One of the domains interacted with the basal factor TFIIB, suggesting that p300 may function as a coactivator by making contacts with both sequence-specific transcription factors and the basal transcriptional machinery. This pivotal role of p300 may make it a prime target for viral proteins such as E1A in programming the cellular transcription machinery. The E1A region of human adenoviruses gives rise to two major alternatively spliced products, 12 S and 13 S mRNAs (1Chow L.T. Broker T.R. Lewis J.B. J. Mol. Biol. 1979; 134: 265-303Google Scholar, 2Berk A.J. Sharp P.A. Cell. 1978; 14: 695-711Google Scholar, 3Perricaudet M. Akusjarvi G. Virtanen A. Pettersson U. Nature. 1979; 281: 694-696Google Scholar). The corresponding protein products are nuclear phosphoproteins of 243 and 289 amino acids, respectively (3Perricaudet M. Akusjarvi G. Virtanen A. Pettersson U. Nature. 1979; 281: 694-696Google Scholar, 4Yee S.P. Rowe D.T. Tremblay M.L. McDermott M. Branton P.E. J. Virol. 1983; 46: 1003-1013Google Scholar, 5Ferguson B. Krippl B. Andrisani O. Jones N. Westphal H. Rosenberg M. Mol. Cell. Biol. 1985; 5: 2653-2661Google Scholar, 6Harlow E. Franza B.R. Schley C. J. Virol. 1985; 55: 533-546Google Scholar). Both polypeptides have identical amino- and carboxyl-terminal ends, the only difference being a region of 46 internal amino acids unique to the 13 S product (3Perricaudet M. Akusjarvi G. Virtanen A. Pettersson U. Nature. 1979; 281: 694-696Google Scholar). These proteins are the first viral polypeptides synthesized after adenovirus infection (7Lewis J.B. Mathews M.B. Cell. 1980; 21: 303-313Google Scholar, 8Nevins J.R. Cell. 1981; 26: 213-220Google Scholar). In addition to activating transcription of other adenoviral genes, E1A affects a whole array of host cell functions such as DNA synthesis and cell cycle progression (9Kaczmarek L. Ferguson B. Rosenberg M. Baserga R. Virology. 1986; 152: 1-10Google Scholar, 10Stabel S. Argos P. Philipson L. EMBO J. 1985; 4: 2329-2336Google Scholar) to facilitate viral replication and propagation. E1A is also capable of immortalizing cells (11van der Eb A.J. van Ormondt H. Schrier P.I. Cold Spring Harbor Symp. Quant. Biol. 1979; 44: 383-399Google Scholar, 12Houweling A. van der Elsen P.J. van der Eb A.J. Virology. 1980; 105: 537-550Google Scholar), inducing full morphological transformation in cooperation with several oncogenes including the ras, polyoma middle T, and adenovirus E1B (13van der Elsen P. de Parter S. Houweling A. van der Veer J. van der Eb A.J. Gene (Amst.). 1982; 18: 175-185Google Scholar, 14van der Elsen P.J. Houweling A. van der Eb A.J. Virology. 1983; 131: 242-246Google Scholar, 15Ruley H.E. Nature. 1983; 304: 602-606Google Scholar), and inhibiting terminal differentiation (16Maruyama K. Schiavi S.C. Huse W. Johnson G.C. Ruley H.E. Oncogene. 1987; 1: 361-367Google Scholar, 17Webster K.A. Muscat G.E.O. Kedes L. Nature. 1988; 344: 260-262Google Scholar). The diverse biological activities of E1A are attributable, at least in part, to its ability to modulate the cellular transcriptional machinery, since E1A has been shown to activate and repress a large number of cellular genes important for cell proliferation and differentiation (17Webster K.A. Muscat G.E.O. Kedes L. Nature. 1988; 344: 260-262Google Scholar, 18Borrelli E. Hen R. Chambon P. Nature. 1984; 312: 608-612Google Scholar, 19Hen R. Borrelli E. Chambon P. Science. 1985; 230: 1391-1394Google Scholar, 20Jelsma T.N. Howe J.A. Mymryk J.S. Evelegh C.M. Cunnif N.F. Bayley S.T. Virology. 1989; 171: 120-130Google Scholar, 21Lillie J.W. Green M. Green M.R. Cell. 1986; 46: 1043-1045Google Scholar, 22Stein R.W. Ziff E.B. Mol. Cell. Biol. 1987; 7: 1164-1170Google Scholar, 23Stein R.W. Corrigan M. Yaciuk P. Whelan J. Moran E. J. Virol. 1990; 64: 4421-4427Google Scholar, 24Simon M.C. Kitchener K. Kao H.T. Hickey E. Weber L. Voellmy R. Heintz N. Nevins J.R. Mol. Cell. Biol. 1987; 7: 2884-2890Google Scholar, 25Winberg G. Shenk T. EMBO J. 1984; 3: 1907-1912Google Scholar, 26Zerler B. Roberts R.J. Mathews M.B. Moran E. Mol. Cell. Biol. 1987; 7: 821-829Google Scholar, 27Kraus V.B. Moran E. Nevins J.R. Mol. Cell. Biol. 1992; 12: 4391-4399Google Scholar). Unlike conventional transcription factors, E1A does not recognize specific DNA sequences (5Ferguson B. Krippl B. Andrisani O. Jones N. Westphal H. Rosenberg M. Mol. Cell. Biol. 1985; 5: 2653-2661Google Scholar, 28Chatterjee P.K. Bruner M. Flint S.J. Harter M.L. EMBO J. 1988; 7: 835-841Google Scholar), and the E1A-responsive promoters do not share common sequence elements (reviewed in 29Flint S.J. Shenk T. Annu. Rev. Genet. 1989; 23: 141-161Google Scholar). Therefore, it has been proposed that E1A must exert its transcriptional effects via multiple mechanisms that are likely to involve protein/protein interactions (29Flint S.J. Shenk T. Annu. Rev. Genet. 1989; 23: 141-161Google Scholar). Studies in the past several years have provided evidence that supports such an hypothesis. It has been shown that in some cases, a direct interaction between E1A and certain transcription factors targets E1A to the promoters for transcriptional activation (30Lee W.S. Kao C.C. Bryant G.O. Liu X. Berk A.J. Cell. 1991; 67: 365-376Google Scholar, 31Horikoshi N. Maguire K. Krelli A. Maldanado E. Reinberg D. Weinmann R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5124-5128Google Scholar, 32Liu F. Green M.R. Nature. 1994; 368: 520-525Google Scholar, 33Chatton B. Bocco J.L. Gaire M. Hauss C. Reimund B. Goetz J. Kedinger C. Mol. Cell. Biol. 1993; 13: 561-570Google Scholar, 34Taylor D. Kraus V.B. Schwarz J.J. Olson E.N. Kraus W.E. Mol. Cell. Biol. 1993; 13: 4714-4727Google Scholar). In other cases, the interaction appears indirect and is mediated by E1A-associated proteins, such as the RB family of proteins. Through its physical interactions with RB, E1A disrupts the RB·E2F complex (35Bagchi S. Raychaudhuri P. Nevins J.R. Cell. 1990; 62: 659-669Google Scholar), releasing free, active form of E2F for transcriptional activation (reviewed in 36Nevins J.R. Science. 1992; 258: 424-429Google Scholar). Another protein that has been implicated in mediating the transcriptional effect of E1A is its associated protein p300 (37Yee S.P. Branton P.E. Virology. 1985; 147: 142-153Google Scholar, 38Harlow E. Whyte P. Franza Jr., B.R. Schely C. Mol. Cell. Biol. 1986; 6: 1579-1589Google Scholar). Genetic studies suggested that the ability of E1A to repress viral and cellular enhancers is dependent on the p300-binding domain of E1A (18Borrelli E. Hen R. Chambon P. Nature. 1984; 312: 608-612Google Scholar, 19Hen R. Borrelli E. Chambon P. Science. 1985; 230: 1391-1394Google Scholar, 22Stein R.W. Ziff E.B. Mol. Cell. Biol. 1987; 7: 1164-1170Google Scholar, 23Stein R.W. Corrigan M. Yaciuk P. Whelan J. Moran E. J. Virol. 1990; 64: 4421-4427Google Scholar, 39Velcich A. Ziff E. Cell. 1985; 40: 705-716Google Scholar, 40Rochette-Egly C. Fromental C. Chambon P. Genes Dev. 1990; 4: 137-150Google Scholar, 41Wang H.-G.H. Rikitake Y. Carter M.C. Yaciuk P. Abraham S.E. Brad Z. Moran E. J. Virol. 1993; 67: 476-488Google Scholar). The cDNA that encodes the p300 protein was cloned, and direct evidence was obtained that demonstrated the involvement of p300 in E1A-mediated repression of the SV40 enhancer (42Eckner R. Ewen M.E. Newsome D. Gerdes M. DeCaprio J.A. Lawrence J.B. Livingston D.M. Genes Dev. 1994; 8: 869-884Google Scholar). P300 shares extensive sequence homology with the transcriptional coactivator CBP 1The abbreviations used are: CBPCREB binding proteinCREBcAMP-responsive element-binding proteinATFactivating transcription factorCATchloramphenicolaaamino acid(s)GSTglutathione S-transferasePKAprotein kinase A. (REB-inding rotein) (43Chrivia J.C. Kwok R.P.S. Lamb N. Hagiwara M. Montminy M.R. Goodman R.H. Nature. 1993; 265: 855-859Google Scholar, 44Kwok R.P.S. Lundblad J.R. Chrivia J.C. Richards J.P. Bachinger H.P. Brennan R.G. Roberts S.G.E. Green M.R. Goodman R.H. Nature. 1994; 370: 223-226Google Scholar, 45Arany Z. Sellers W.R. Livingston D.M. Eckner R. Cell. 1994; 77: 799-800Google Scholar). As predicted from the sequence comparison, p300 functions like CBP as a coactivator of CREB and is capable of mediating the effect of E1A on CREB (46Arany Z. Newsome D. Oldread E. Livingston D.M. Eckner R. Nature. 1995; 374: 81-84Google Scholar, 47Lundblad J.R. Kwok R.P.S. Laurance M.E. Harter M.L. Goodman R.H. Nature. 1995; 374: 85-88Google Scholar). Recently, by analyzing the ability of E1A and its mutant derivatives to convert the transcription factor YY1 from a repressor to an activator, we identified p300 as a bridging factor that mediates the functional interaction between YY1 and E1A (48Lee J.-S. Galvin K.M. See R.H. Eckner R. Livingston D. Moran E. Shi Y. Genes Dev. 1995; 9: 1188-1198Google Scholar). CREB binding protein cAMP-responsive element-binding protein activating transcription factor chloramphenicol amino acid(s) glutathione S-transferase protein kinase A. The observation that p300 mediates the ability of E1A to modulate YY1 activity led us to ask whether p300 is a common cofactor that mediates the transcriptional effects of E1A. Promoter elements that were previously shown to respond to E1A were examined. One of the cis elements through which E1A exerts its transcriptional effects is the recognition sequence for the CREB/ATF family of proteins (49Lee K.A.W. Hai T.-Y. SivaRaman L. Thimmappaya B. Hurst H.C. Jones N.C. Grenn M.R. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 8355-8359Google Scholar, 50Lin Y.-S. Green M.R. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 3396-3400Google Scholar). The consensus sequence of the CREB/ATF binding sites can serve as a recognition site for either homo- or heterodimers between members of the CREB/ATF and the AP-1 family of transcription factors (51Hai T. Liu F. Allegretto E.A. Karin M. Green M.R. Genes Dev. 1988; 2: 1216-1226Google Scholar, 52Hai T. Curran T. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3720-3724Google Scholar). One of the ATF family members, ATF2, has been shown previously to mediate E1A-induced transcriptional activation via a direct interaction with E1A (32Liu F. Green M.R. Nature. 1994; 368: 520-525Google Scholar). In this paper, evidence is presented that p300 is involved in mediating the E1A-induced transcriptional activation via an ATF site (abbreviated as ATFf hereafter) taken from the fibronectin promoter (53Dean D.C. Bowlus C.L. Bourgeois S. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 1876-1880Google Scholar, 57Weintraub S. Dean D.C. Mol. Cell. Biol. 1992; 12: 512-517Google Scholar). In contrast, p300 failed to activate transcription via an Sp1 site in the presence of E1A. By gel shift/antibody supershift experiments, two CREB/ATF family members, CREB and ATF-1, were found to bind the ATFf site. Using a GAL4 fusion protein-based assay, CREB, but not ATF-1 or Sp1, was shown to respond to E1A in a p300 binding-dependent manner. This suggests that CREB, but not ATF-1, participates in the response of the ATFf site to p300/E1A-induced transcriptional activation. Consistent with this hypothesis, CREB, but not ATF-1 or Sp1, was shown to physically interact with p300 in HeLa cells. Interestingly, E1A activated CREB-mediated transcription in HeLa cells but repressed it in U2OS cells. The activation and repression functions of E1A on CREB-dependent transcription in different cells both required an intact p300 binding domain. To better understand the role of p300 as a cofactor of CREB-mediated transcription, experiments were initiated to analyze how p300 regulates transcription. Two separate domains of p300 were identified that activated transcription when targeted to a promoter via the heterologous GAL4 DNA-binding domain. One of these activation domains was shown to interact with the basal transcription factor TFIIB. These results suggest that p300 may function as a coactivator by making contacts with both sequence-specific DNA-binding transcription factors and the basal transcription machinery. Cells were grown on 10-cm dishes in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated calf serum (HeLa) or fetal calf serum (293 and U2OS). Transfections were performed by the calcium-phosphate precipitation method as described (54Shi Y. Seto E. Chang L.S. Shenk T. Cell. 1991; 67: 377-388Google Scholar). The total amount of DNA was adjusted to be identical for each set of transfections. Cells were harvested 48 h after addition of the precipitates. All transfection assays were carried out with at least two independent DNA preparations and were repeated at least three times. Whole cell extracts were prepared from the transfected cells. CAT activity was assayed as described (54Shi Y. Seto E. Chang L.S. Shenk T. Cell. 1991; 67: 377-388Google Scholar) and quantitated with a Beckman LS6500 scintillation counter. To ensure that CAT assays were performed within linear range, the proper amount of cell extracts was used to measure CAT activity. For instance, less extracts from cells transfected with stronger transactivators, such as GAL4-VP16 or GAL4-Sp1, were used for CAT reactions. For all the data presented, at least three independent transfections and CAT assays have been performed. pATF-TA-CAT, pSp1-TA-CAT, pTA-CAT, and pTATAA-CAT are kind gifts of D. Dean (Washington University School of Medicine). Wild-type p300 expression plasmid and its parental vector were courtesy of R. Eckner and D. Livingston (Dana Farber Cancer Institute). The p300 mutant dl10 plasmid was described previously (48Lee J.-S. Galvin K.M. See R.H. Eckner R. Livingston D. Moran E. Shi Y. Genes Dev. 1995; 9: 1188-1198Google Scholar). pGAL4-p300 aa 1257-2414 was constructed by fusing a BglII/KpnI fragment from a pBluescript plasmid containing full-length p300 cDNA (42Eckner R. Ewen M.E. Newsome D. Gerdes M. DeCaprio J.A. Lawrence J.B. Livingston D.M. Genes Dev. 1994; 8: 869-884Google Scholar) into the pSG424 expression vector of the yeast transcription factor GAL4 DNA-binding domain aa 1-147 (55Sadowski I. Ptashne M. Nucleic Acids Res. 1989; 17: 7539Google Scholar). The same region was taken from the p300 mutant dl10 (48Lee J.-S. Galvin K.M. See R.H. Eckner R. Livingston D. Moran E. Shi Y. Genes Dev. 1995; 9: 1188-1198Google Scholar) and cloned into pSG424. pGAL4-CREB is a gift of J. Licht (Mt. Sinai Medical School). pGAL4-ATF1 and -ATF2 are kind gifts of M. Green (University of Massachusetts Medical Center). pGAL4-Sp1 was provided by R. Tjian (University of California, Berkeley). E1A expression plasmids and the mutant derivatives were described previously (48Lee J.-S. Galvin K.M. See R.H. Eckner R. Livingston D. Moran E. Shi Y. Genes Dev. 1995; 9: 1188-1198Google Scholar) as were pGAL4-YY1 and pGAL4-E1BCAT (54Shi Y. Seto E. Chang L.S. Shenk T. Cell. 1991; 67: 377-388Google Scholar). p300-VP16 was cloned into the RC/CMV expression vector (Invitrogen) with the activation domain of VP16 inserted into the NheI site (aa 2377) of p300 cDNA. pGST-p300 aa 1-596, aa 744-1571, and aa 1572-2414 were described before (48Lee J.-S. Galvin K.M. See R.H. Eckner R. Livingston D. Moran E. Shi Y. Genes Dev. 1995; 9: 1188-1198Google Scholar). pGAL4-Np300 (aa 1-596) and pGAL4-Mp300 (aa 744-1571) were constructed by cloning the p300 coding regions from the respective GST constructs into pSG424. Nuclear extracts were prepared from 293 cells as described (56Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Google Scholar). The sequences of ATFf (57Weintraub S. Dean D.C. Mol. Cell. Biol. 1992; 12: 512-517Google Scholar) and YY1 binding site (AAV P5 +1 site, 54Shi Y. Seto E. Chang L.S. Shenk T. Cell. 1991; 67: 377-388Google Scholar) were described before. A typical binding reaction mixture contained labeled oligonucleotides (ATFf, 7 × 104 cpm; or YY1 binding site, 4 × 104 cpm), 1 µg of poly(dI-dC), 15 mM Hepes (pH 7.6), 5 mM dithiothreitol, 0.5 mM EDTA, 5 mM MgCl2, 30 mM KCl, 10% glycerol, and 8 µg of nuclear extracts in a final volume of 15 µl. The reaction mixture was incubated at room temperature for 20 min and analyzed by 4% native polyacrylamide gel electrophoresis. The specificity of the protein-DNA complexes was demonstrated by competition using unlabeled competitor oligonucleotides. To identify individual transcription factors involved in the complex formation, various antibodies were incubated with the nuclear extracts for 4 h at 4°C before the addition of the probes. The results were visualized by autoradiography. Dr. M.-E. Lee (Harvard School of Public Health) kindly provided us with antibodies including α-ATF1 (C41-5.1, Santa Cruz, catalog sc-243), α-ATF2 (Upstate Biotechnology Inc., UBI, catalog 06-326), α-CREB (UBI, catalog 06-244), and α-c-Jun (UBI, catalog 06-115). The α-YY1 polyclonal antibodies are affinity-purified. GST fusion proteins were induced and purified as described (58Lee J.-S. See R.H. Galvin K.M. Wang J. Shi Y. Nucleic Acids Res. 1995; 23: 925-931Google Scholar). TFIIB proteins were 35S-labeled and synthesized by in vitro translation reactions using the TNT kit (Promega). Labeled proteins were incubated for 2 h with various GST-p300 fusion proteins coupled to glutathione agarose beads (Sigma). The beads were washed five times with 0.1% Nonidet P-40 in phosphate-buffered saline, and protein complexes were eluted with Laemmli sample buffer. Following SDS-polyacrylamide gel electrophoresis, bound proteins were visualized by autoradiography. Previously, we demonstrated that p300 activates YY1 binding site-mediated transcription in 293 cells (which constitutively express E1A proteins) and that this function is dependent on the ability of p300 to interact with both YY1 and E1A (48Lee J.-S. Galvin K.M. See R.H. Eckner R. Livingston D. Moran E. Shi Y. Genes Dev. 1995; 9: 1188-1198Google Scholar). Together with several other lines of evidence, we proposed that p300 mediated the modulatory effects of E1A on the transcriptional activity of YY1 (48Lee J.-S. Galvin K.M. See R.H. Eckner R. Livingston D. Moran E. Shi Y. Genes Dev. 1995; 9: 1188-1198Google Scholar). We then wished to determine whether p300 is a general mediator of the transcriptional effects of E1A. Since a number of transcription factor binding sites have been demonstrated to mediate E1A responsiveness (reviewed in 29Flint S.J. Shenk T. Annu. Rev. Genet. 1989; 23: 141-161Google Scholar), we first asked whether, in the presence of E1A, p300 is capable of activating transcription of the reporter constructs previously shown to respond to E1A. The reporter CAT plasmids contain either an ATF site (ATFf) which was taken from the fibronectin promoter (nucleotide −176 to −161, 59Dean D.C. Blakeley M.S. Newby R.F. Ghazal P. Hennighausen L. Bourgeois S. Mol. Cell. Biol. 1989; 9: 1498-1506Google Scholar) (pATF-TA-CAT) or an Sp1 consensus sequence (pSp1-TA-CAT) at −40 relative to the start site of transcription (57Weintraub S. Dean D.C. Mol. Cell. Biol. 1992; 12: 512-517Google Scholar, kind gifts of D. Dean). The parental vector pTA-CAT was derived from pTATAA-CAT which contains the fibronectin gene sequence from +8 to −36 (53Dean D.C. Bowlus C.L. Bourgeois S. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 1876-1880Google Scholar). pTA-CAT is essentially the same as pTATAA-CAT except that the TATA element extending from −20 to −24 of the fibronectin gene promoter (TATAA) was replaced by the simian virus 40 (SV40) early gene TATA box equivalent TATTTAT, which has been shown not to respond to E1A (60Simon M.C. Fisch T.M. Benecke B.J. Nevins J.R. Heintz N. Cell. 1988; 52: 723-729Google Scholar). It has been shown that all reporters except pTA-CAT responded to E1A in transfection assays (57Weintraub S. Dean D.C. Mol. Cell. Biol. 1992; 12: 512-517Google Scholar). To determine whether the E1A response of these reporters may be mediated by p300, each reporter plasmid was cotransfected with a CMV-p300 expression plasmid into 293 cells. As shown in Fig. 1A, p300 selectively activated CAT expression from pATF-TA-CAT but not from the parental vector pTA-CAT (lanes 1-2, and 7-8), suggesting that the ATFf site may be responsible for the p300-induced activation. Importantly, the p300 mutant, p300 dl10, which is deleted of the E1A-binding domain (42Eckner R. Ewen M.E. Newsome D. Gerdes M. DeCaprio J.A. Lawrence J.B. Livingston D.M. Genes Dev. 1994; 8: 869-884Google Scholar, 48Lee J.-S. Galvin K.M. See R.H. Eckner R. Livingston D. Moran E. Shi Y. Genes Dev. 1995; 9: 1188-1198Google Scholar) was unable to activate the ATFf site-mediated transcription (Fig. 1A, lane 3). The result indicated that the interaction between p300 and E1A is critical for the observed transcriptional activation. This is consistent with the observation that, in HeLa and U2OS cells which do not express E1A proteins, overexpression of p300 had little effect on the activity of pATF-TA-CAT under the same assay condition (data not shown). These results supported the hypothesis that p300 is involved in mediating E1A-induced transcriptional activation through the ATFf site. Consistent with this notion, without the ATFf site, the parental vector pTA-CAT did not respond to E1A (57Weintraub S. Dean D.C. Mol. Cell. Biol. 1992; 12: 512-517Google Scholar) and also failed to respond to p300 in this assay (Fig. 1A, lane 8). Interestingly, p300 also did not activate the other two reporters, pSp1-TA-CAT and pTATAA-CAT (Fig. 1A, lanes 5 and 11), which previously were shown to respond to E1A (57Weintraub S. Dean D.C. Mol. Cell. Biol. 1992; 12: 512-517Google Scholar). These results suggest that E1A transactivates these reporters through alternative mechanisms that do not involve the p300 protein. In the case of pTATAA-CAT, it is likely that E1A, in particular the 13 S gene product, may activate the reporter by directly targeting the basal transcription factor TATA-binding protein (30Lee W.S. Kao C.C. Bryant G.O. Liu X. Berk A.J. Cell. 1991; 67: 365-376Google Scholar, 31Horikoshi N. Maguire K. Krelli A. Maldanado E. Reinberg D. Weinmann R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5124-5128Google Scholar). How does p300 activate transcription through the ATFf site in 293 cells? Previously, it has been shown that p300 has specific DNA binding activity for NF-kB/H2TF1-like sites (61Rikitake Y. Moran E. Mol. Cell. Biol. 1992; 12: 2826-2836Google Scholar), which bear no resemblance to the YY1 (48Lee J.-S. Galvin K.M. See R.H. Eckner R. Livingston D. Moran E. Shi Y. Genes Dev. 1995; 9: 1188-1198Google Scholar) or the ATFf site (this study) shown to respond to p300. In fact, accumulating evidence suggests that p300 may function in a more indirect way, i.e. as a transcriptional cofactor (42Eckner R. Ewen M.E. Newsome D. Gerdes M. DeCaprio J.A. Lawrence J.B. Livingston D.M. Genes Dev. 1994; 8: 869-884Google Scholar, 46Arany Z. Newsome D. Oldread E. Livingston D.M. Eckner R. Nature. 1995; 374: 81-84Google Scholar, 47Lundblad J.R. Kwok R.P.S. Laurance M.E. Harter M.L. Goodman R.H. Nature. 1995; 374: 85-88Google Scholar). If this property of p300 is indeed the mechanism by which p300 activates transcription, the failure of p300 dl10 to activate pATF-TA-CAT could also be due to a defect in its ability to function as a transcriptional coactivator. To address this issue, the ability of the carboxyl-terminal half of p300 (aa 1257-2414) and its mutant derivative containing the same internal deletion (aa 1679-1812) as p300 dl10 to regulate transcription was analyzed. The reporter plasmid pGAL4-E1BCAT contains five GAL4 DNA binding sites immediately upstream of the minimal adenovirus E1B promoter. pGAL4-E1BCAT has been widely used for studies of transcriptional activation, including E1A-induced transcriptional activation. As shown in Fig. 1B, both forms of p300, when fused to the GAL4 DNA-binding domain, activated transcription of the target gene GAL4-E1BCAT, whereas the GAL4 DNA-binding domain alone had virtually no effects (data not shown, Fig. 5). These data suggest that the carboxyl-terminal half of p300 may contain a functional domain for its transcriptional coactivator function, which is unaffected by the dl10 deletion mutation. Thus, the failure of p300 dl10 to activate pATF-TA-CAT in 293 cells is most likely due to its inability to interact with E1A. However, it is unclear at the present time why the p300 dl10 activated transcription better than the wild-type p300. Taken together, these results demonstrated that p300 is capable of mediating E1A-induced transcriptional activation via the ATFf but not the Sp1 site. Since multiple CREB/ATF-related proteins can bind an ATF consensus site, we wished to determine which members of the CREB/ATF family bind the ATFf site that responded to p300 to activate transcription in 293 cells (Fig. 1A). An oligonucleotide representing the ATFf site was labeled with 32P and used in gel shift assays. As shown in Fig. 2, when the ATFf oligonucleotides were incubated with nuclear extracts prepared from 293 cells, two predominant complexes (labeled as A and B) were formed that were competed by molar excess of unlabeled ATFf but not by an unrelated YY1 oligonucleotide (lanes 1-7). Addition of α-ATF1 antibodies supershifted both the A and the B complexes whereas the α-CREB antibody supershifted only the A complex (Fig. 2, lanes 8 and 10). This suggests that the A and B complexes both contain ATF-1 while only the A complex contains CREB. In contrast, addition of α-ATF2, α-c-Jun, and α-YY1 antibodies had no effect on either the A or the B complexes (Fig. 2, lanes 9, 11, and 12). As a control, only α-YY1 antibodies abolished a YY1 complex (Fig. 2, lower panel, lane 12). Taken together, these results suggest that ATF-1 and CREB are the main components of the DNA-protein complexes formed on the ATFf site in 293 cells. However, these data do not rule out the possibility that other untested ATFs may also bind the ATFf site. In addition, the results do not differentiate whether the A complex is composed of ATF-1/CREB heterodimers or comigrating ATF-1 and CREB homodimers. Since both ATF-1 and CREB, and possibly other untested ATFs may bind the ATFf site, it was important to determine which one (or both) is responsible for the response of the ATFf site to the E1A-induced transcriptional activation via p300 (Fig. 1A). To address this issue, individual ATF family members were fused to the GAL4 DNA-binding domain and assayed for their ability to respond to E1A using pGAL4-E1BCAT as a target plasmid (54Shi Y. Seto E. Chang L.S. Shenk T. Cell. 1991; 67: 377-388Google Scholar). As shown in Fig. 3, GAL4-CREB responded to E1A in a cell type-dependent manner. In U2OS cells, E1A, especially the 12 S gene product, repressed GAL4-CREB-mediated transcription (Fig. 3A, lanes 1 to 3). In contrast, in HeLa cells, 12 S E1A activated GAL4-CREB-mediated transcription (Fig. 3B, lane 4), albeit to a lesser extent compared with 13 S E1A (Fig. 3B, lanes 1 and 2). The quantitative difference between the effects of the 12 S and 13 S E1A is most likely attributable to the 46 amino acids (CR3) unique to the 13 S E1A, which is a known transcriptional activation domain (62Lillie J.W. Loewenstein P.M. Green M.R. Green M. Cell. 1987; 50: 1091-1100Google Scholar). With this activation domain, 13 S E1A is often found to be a more potent transactivator than the 12 S E1A (24Simon M.C. Kitchener K. Kao H.T. Hickey E. Weber L. Voellmy R. Heintz N. Nevins J.R. Mol. Cell. Biol. 1987; 7: 2884-2890Google Scholar, 25Winberg G. Shenk T. EMBO J. 1984; 3: 1907-1912Google Scholar, 26Zerler B. Roberts R.J. Mathews M.B. Moran E. Mol. Cell. Biol. 1987; 7: 821-829Google Scholar, 27Kraus" @default.
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• W2003135979 title "Differential Interactions of the CREB/ATF Family of Transcription Factors with p300 and Adenovirus E1A" @default.
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