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• W2017471019 abstract "Activation of the transcription unit early region 2 (E2) promoter of the oncogenic adenovirus serotype 12 (Ad12), which regulates the expression of proteins essential for viral replication, requires the assembly of a ternary complex consisting of cAMP response element-binding protein (CREB)-1/activating transcription factor (ATF)-1, the Ad12 12S oncogene product of early region 1A (E1A12S), and the co-activator p300/CBP on the E2Ad12 cAMP response element (E2-CRE). Here we show that the active E2Ad12 promoter is associated with acetylated histone H4 whereas an E2-CRE point-mutated promoter which is transcriptionally inactive due to its inability to assemble this ternary complex is not bound by acetylated histone H4. The histone deacetylase 1 as well as Roscovitine, which blocks the activation of the histone acetyltransferase (HAT) activity of CBP by cyclin E-Cdk2, prevents E2Ad12 promoter activation through E1A12S. p300/CBP counteracts the repressive function of histone deacetylase 1 in a HAT domain-dependent manner whereas the p300/CBP-associated factor PCAF failed to rescue E2Ad12 promoter activity. E1A12S bound p300/CBP displays strong HAT activity. Most interestingly, E1A12S-mediated activation of the E2Ad12promoter correlates well with the ability of the viral protein to associate with the HAT activity of p300/CBP in vivo. Taken together these data indicate that the recruitment of the HAT activity of p300/CBP by E1A12S plays an important role in E2Ad12 promoter activation. Activation of the transcription unit early region 2 (E2) promoter of the oncogenic adenovirus serotype 12 (Ad12), which regulates the expression of proteins essential for viral replication, requires the assembly of a ternary complex consisting of cAMP response element-binding protein (CREB)-1/activating transcription factor (ATF)-1, the Ad12 12S oncogene product of early region 1A (E1A12S), and the co-activator p300/CBP on the E2Ad12 cAMP response element (E2-CRE). Here we show that the active E2Ad12 promoter is associated with acetylated histone H4 whereas an E2-CRE point-mutated promoter which is transcriptionally inactive due to its inability to assemble this ternary complex is not bound by acetylated histone H4. The histone deacetylase 1 as well as Roscovitine, which blocks the activation of the histone acetyltransferase (HAT) activity of CBP by cyclin E-Cdk2, prevents E2Ad12 promoter activation through E1A12S. p300/CBP counteracts the repressive function of histone deacetylase 1 in a HAT domain-dependent manner whereas the p300/CBP-associated factor PCAF failed to rescue E2Ad12 promoter activity. E1A12S bound p300/CBP displays strong HAT activity. Most interestingly, E1A12S-mediated activation of the E2Ad12promoter correlates well with the ability of the viral protein to associate with the HAT activity of p300/CBP in vivo. Taken together these data indicate that the recruitment of the HAT activity of p300/CBP by E1A12S plays an important role in E2Ad12 promoter activation. CREB-binding protein amino acid adenovirus chloramphenicol acetyltransferase cAMP response element-binding protein early region 1A oncogene early region 1A 12S oncoprotein early region 1A 13S oncoprotein transcription unit early region 2 Ad12 E2 promoter E2Ad12 promoter cAMP response element glutathione S-transferase histone acetyltransferase histone deacetylase 1 p300/CREB-binding protein associated factor p300/CBP/co-integrator-associated protein polymerase chain reaction steroid receptor coactivator-1 activating transcription factor Nuclear integrators of diverse signaling pathways like p300 and CREB-binding protein (CBP)1play crucial roles in the coordinated regulation of gene expression (1Shikama N. Lyon J. La Thangue N.B. Trends Cell Biol. 1997; 7: 230-236Abstract Full Text PDF PubMed Scopus (432) Google Scholar). Although it turns out recently that p300 and CBP have partially different biological properties (2Kawasaki H. Eckner R. Yao T.P. Taira K. Chiu R. Livingston D.M. Yokoyama K.K. Nature. 1998; 393: 284-289Crossref PubMed Scopus (302) Google Scholar, 3Kung A.L. Rebel V.I. Bronson R.T. Ch′ng L.E. Sieff C.A. Livingston D.M. Yao T.P. Genes Dev. 2000; 14: 272-277PubMed Google Scholar, 4Yao T.P. Oh S.P. Fuchs M. Zhou N.D. Ch'ng L.E. Newsome D. Bronson T. Li E. Livingston D.M. Eckner R. Cell. 1998; 93: 361-372Abstract Full Text Full Text PDF PubMed Scopus (827) Google Scholar) they are referred here to as p300/CBP unless otherwise specified due to their indistinguishable function on the activation of CREB-dependent gene expression (5Goldman P.S. van Tran K. Goodman R.H. Recent Prog. Horm. Res. 1997; 52: 103-120PubMed Google Scholar). p300/CBP binds to and potentiates the function of a large number of transcription factors, including CREB (5Goldman P.S. van Tran K. Goodman R.H. Recent Prog. Horm. Res. 1997; 52: 103-120PubMed Google Scholar) and Ad E1A (6Giordano A. Avantaggiati M.L. J. Cell. Physiol. 1999; 181: 218-230Crossref PubMed Scopus (255) Google Scholar). In addition, it interacts with other co-factors like PCAF (7Yang X.J. Ogryzko V.V. Nishikawa J. Howard B.H. Nakatani Y. Nature. 1996; 382: 319-324Crossref PubMed Scopus (1320) Google Scholar), SRC-1 (8Yao T.P. Ku G. Zhou N. Scully R. Livingston D.M. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 10626-10631Crossref PubMed Scopus (395) Google Scholar), or p/CIP (9Torchia J. Rose D.W. Inostroza J. Kamei Y. Westin S. Glass C.K. Rosenfeld G. Nature. 1997; 387: 677-684Crossref PubMed Scopus (1108) Google Scholar) for promoter activation. Moreover, at least CBP was shown to associate directly with the RNA polymerase II holoenzyme via RNA helicase A (10Nakajima T. Uchida C. Anderson S.F. Lee C.G. Hurwitz J. Parvin J.D. Montminy M. Cell. 1997; 90: 1107-1112Abstract Full Text Full Text PDF PubMed Scopus (461) Google Scholar). Latter interaction is thought to induce local changes in chromatin structure that promote access of the transcriptional machinery on responsive promoters. The finding that p300/CBP carries intrinsic HAT activity (11Bannister A.J. Kouzarides T. Nature. 1996; 384: 641-643Crossref PubMed Scopus (1535) Google Scholar, 12Ogryzko V.V. Schilz R.L. Russanova V. Howard B.H. Nakatani Y. Cell. 1996; 87: 953-959Abstract Full Text Full Text PDF PubMed Scopus (2409) Google Scholar) was a major step forward in unraveling one mechanism how co-factors could function in transcriptional regulation. As nucleosomes are potent repressors of transcription in vitro and in vivoit is assumed that acetylation of histones disrupts the nucleosomal structure thereby facilitating access of DNA-binding factors and the transcriptional apparatus (13Struhl K. Genes Dev. 1998; 12: 599-606Crossref PubMed Scopus (1557) Google Scholar). p300/CBP has also been shown to acetylate non-histone proteins, like general as well as sequence-specific transcription factors (14Gu W. Roeder R.G. Cell. 1997; 90: 595-606Abstract Full Text Full Text PDF PubMed Scopus (2188) Google Scholar, 15Imhof A. Yang X.J. Ogryzko V.V. Nakatani Y. Wolffe A.P. Ge H. Curr. Biol. 1997; 7: 689-692Abstract Full Text Full Text PDF PubMed Scopus (536) Google Scholar, 16Boyes J. Byfield P. Nakatani Y. Orgryzko V. Nature. 1998; 396: 594-598Crossref PubMed Scopus (633) Google Scholar, 17Munshi N. Merika M. Yie J. Senger K. Chen G. Thanos D. Mol. Cell. 1998; 2: 457-467Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar). These acetylations increase DNA binding activity (14Gu W. Roeder R.G. Cell. 1997; 90: 595-606Abstract Full Text Full Text PDF PubMed Scopus (2188) Google Scholar, 16Boyes J. Byfield P. Nakatani Y. Orgryzko V. Nature. 1998; 396: 594-598Crossref PubMed Scopus (633) Google Scholar), decrease DNA binding activity (17Munshi N. Merika M. Yie J. Senger K. Chen G. Thanos D. Mol. Cell. 1998; 2: 457-467Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar), or prevent protein/protein interactions (18Waltzer L. Bienz M. Nature. 1998; 395: 521-525Crossref PubMed Scopus (327) Google Scholar). The Ad E1A oncoprotein is a potent transcriptional regulator (19Brockmann D. Esche H. Curr. Top. Microbiol. Immunol. 1995; 199: 81-112PubMed Google Scholar) which interferes with a variety of cellular processes such as modulation of gene expression, inhibition of cellular differentiation, promotion of cell-cycle progression, and transformation (20Moran E. Curr. Opin. Genet. Dev. 1993; 3: 63-70Crossref PubMed Scopus (234) Google Scholar, 21Baylay S.T. Mymryk J.S. Int. J. Oncol. 1994; 5: 425-444PubMed Google Scholar). Subsequent studies have clearly demonstrated a correlation between the modulation of these processes and the functional interaction of E1A with p300/CBP (Ref. 6Giordano A. Avantaggiati M.L. J. Cell. Physiol. 1999; 181: 218-230Crossref PubMed Scopus (255) Google Scholar, and references therein). Molecular analyses identified three binding domains within p300/CBP for E1A (22Kurokawa R. Kalafus D. Ogliastro M.-H. Kioussi C. Xu L. Torchia J. Rosenfeld M.G. Glass C.K. Science. 1998; 279: 700-703Crossref PubMed Scopus (199) Google Scholar, 23Lipinski K.S. Fax P. Wilker B. Hennemann H. Brockmann D. Esche H. Virology. 1999; 255: 94-105Crossref PubMed Scopus (21) Google Scholar): (i) aa 1–450, (ii) aa 1459–1891 (spanning the C/H3 domain), and (iii) aa 2058–2163. Interestingly, the C/H3-binding site is located immediately adjacent to the HAT domain of p300/CBP suggesting that E1A might affect cellular processes by interfering with this enzymatic activity. However, the modulation of the p300/CBP HAT activity by E1A remains unclear. On the one hand, several reports suggest that E1A inhibits the HAT activity of p300 (24Chakravarti D. Ogryzko V. Kao H-J. Nash A. Chen H. Nakatani Y. Evans R.M. Cell. 1999; 96: 393-403Abstract Full Text Full Text PDF PubMed Scopus (298) Google Scholar, 25Hamamori Y. Sartorelli V. Ogryzko V. Puri P.L. Wu H-Y. Wang J.Y.J. Nakatani Y. Kedes L. Cell. 1999; 96: 405-413Abstract Full Text Full Text PDF PubMed Scopus (325) Google Scholar), whereas on the other hand no modulation (11Bannister A.J. Kouzarides T. Nature. 1996; 384: 641-643Crossref PubMed Scopus (1535) Google Scholar) or even a stimulation of CBP's HAT activity through the adenoviral protein was reported (26Ait-Si-Ali S. Ramirez S. Barre F.-X. Dkhisse F. Magnaghi-Jaulin L. Girault J.A. Robin P. Knibiehler M. Pritchard L.L. Ducommun B. Trouche D. Harel-Bellan A. Nature. 1998; 396: 184-186Crossref PubMed Scopus (270) Google Scholar). The aim of this study was to analyze the functional relationship between the Ad E1A12S protein and the HAT activity of p300/CBP in the process of the E2Ad12 promoter activation. The Ad E2 gene encodes for proteins essential for viral replication and its expression is therefore of fundamental importance for the viral life cycle (27Swaminathan S. Thimmapaya B. Curr. Top. Microbiol. Immunol. 1995; 199: 177-195PubMed Google Scholar). We have recently shown that the E1A12Sprotein of Ad12 activates the E2Ad12 promoter through the E2-CRE (28Fax P. Lipinski K.S. Esche H. Brockmann D. J. Biol. Chem. 2000; 275: 8911-8920Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Furthermore, our results indicate that transactivation of this viral promoter requires the assembly of a ternary complex consisting of CREB-1/ATF-1, E1A12S, and CBP on the E2-CRE. However, the viral genome is rapidly chromatinized after infection (Refs. 29Dery C.V. Toth M. Brown M. Horvath J. Allaire S. Weber J.M. J. Virol. 1985; 66: 2671-2684Crossref Scopus (37) Google Scholar and 30Luo R.X. Postigo A.A. Dean D.C. Cell. 1998; 92: 463-473Abstract Full Text Full Text PDF PubMed Scopus (839) Google Scholar, and references therein). Therefore the question arises whether E1A12S-mediated activation of the E2Ad12 promoter requires the HAT activity of p300/CBP. Here we show that HDAC-1 and Roscovitine, which blocks CBP HAT activation (26Ait-Si-Ali S. Ramirez S. Barre F.-X. Dkhisse F. Magnaghi-Jaulin L. Girault J.A. Robin P. Knibiehler M. Pritchard L.L. Ducommun B. Trouche D. Harel-Bellan A. Nature. 1998; 396: 184-186Crossref PubMed Scopus (270) Google Scholar), suppress the E1A12S-mediated activation of the E2Ad12 promoter and that p300/CBP counteracts the HDAC-1-mediated repression in a HAT domain-dependent manner. In contrast, the histone acetyltransferase PCAF failed to counteract the HDAC-1-mediated repression indicating the necessity of a specific HAT activity for promoter activation. The active E2Ad12 promoter is associated with acetylated histone H4 whereas an E2Ad12 promoter rendered inactive through a mutation in the E2-CRE is not associated with acetylated H4. Most interestingly, activation of the E2Ad12 promoter through E1A12S correlates well with an association of the adenoviral protein with the HAT activity of p300/CBP. Taken together our data suggest that recruitment of the HAT activity of p300/CBP by E1A12S and acetylation of promoter bound histones are important steps in the activation process of the E2Ad12promoter. The expression vectors pRc/RSV-235R and pRc/RSV-Gal4-E1A12S encoding the E1A12S protein of Ad12 E1A or a Gal4-E1A12S fusion protein, respectively, were described elsewhere (31Brockmann D. Tries B. Esche H. Virology. 1990; 179: 585-590Crossref PubMed Scopus (12) Google Scholar, 32Lipinski K.S. Kröner-Lux G. Esche H. Brockmann D. J. Gen. Virol. 1997; 78: 413-421Crossref PubMed Scopus (12) Google Scholar). Expression vectors encoding the E1A12S mutants as Myc/His-tagged fusions were constructed by cloning their respective cDNAs amplified by PCR into the EcoRI/BamHI sites of pcDNA3.1-A-Myc/His (Invitrogen). The bacterial and eukaryotic E1A expression vectors as well as the reporter constructs E2Ad12-CAT and E2Ad12pmCRE-CAT have been described (23Lipinski K.S. Fax P. Wilker B. Hennemann H. Brockmann D. Esche H. Virology. 1999; 255: 94-105Crossref PubMed Scopus (21) Google Scholar, 28Fax P. Lipinski K.S. Esche H. Brockmann D. J. Biol. Chem. 2000; 275: 8911-8920Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). KB (human oral epidermoid carcinoma cells), HeLa (human cervix carcinoma cells), and COS7 cells (SV40-transformed African green monkey kidney cells) were maintained in Dulbecco‘s modified Eagle's medium supplemented with 10% fetal calf serum, penicillin, and streptomycin at 37 °C and 5% CO2. For transient expression assays 2.5 × 105 KB or HeLa cells were co-transfected with 1 μg of the respective E2Ad12 reporter construct and different amounts of expression vectors as indicated in the Figure legends using Polyfectine according to the manufacturer's instructions (Biontex, Martinsried, Germany). Transfections were stopped after 6 h and cells were harvested 24 h later to determine CAT activity (33Gorman C. Merlino G. Willingham M. Pastan L. Howard B. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 6777-6781Crossref PubMed Scopus (881) Google Scholar). In some experiments Roscovitine was added to the medium 14 h before harvesting. GST fusion proteins were expressed in Escherichia coli BL21 bacteria and purified as described (34Brockmann D. Bury C. Kröner G. Kirch H.-C. Esche H. J. Biol. Chem. 1995; 270: 10754-10763Abstract Full Text Full Text PDF PubMed Scopus (18) Google Scholar). His6-E1A12S was purified from bacterial extracts according to the manufacturer's protocol (Qiagen). For GST pull-down assays 10 μg of the appropriate GST-E1A fusion protein immobilized on glutathione-Sepharose beads was incubated with 2 mg of whole cell extract prepared from KB cells in IP + 150 buffer (50 mm Hepes/KOH, pH 7.5, 150 mmKCl, 1 mm EDTA, 10 mm NaF, 1 mmNa3VO4, 10% glycerol, 0.1% Nonidet P-40, 0.5 mm Pefabloc, and 10 μg/ml aprotinin) for 1 h at 4 °C. After extensive washings bound proteins were assayed for HAT activity. Liquid HAT assays with either recombinant proteins or protein complexes obtained by immunoprecipitations were performed in 30 μl of HAT buffer (50 mm Tris, pH 8.0, 1 mm EDTA, 10% glycerol, 0.05% Tween 20, 10 mm Na-butyrate) for 45 min at 30 °C using 1 μl [14C]acetyl-CoA (40–60 mCi/mmol, ICN) and 2 μg of a biotinylated synthetic peptide corresponding to the first 24 aa of histone H4 (12Ogryzko V.V. Schilz R.L. Russanova V. Howard B.H. Nakatani Y. Cell. 1996; 87: 953-959Abstract Full Text Full Text PDF PubMed Scopus (2409) Google Scholar, 26Ait-Si-Ali S. Ramirez S. Barre F.-X. Dkhisse F. Magnaghi-Jaulin L. Girault J.A. Robin P. Knibiehler M. Pritchard L.L. Ducommun B. Trouche D. Harel-Bellan A. Nature. 1998; 396: 184-186Crossref PubMed Scopus (270) Google Scholar) or 20 μg of calf thymus histones (Sigma type IIA). Thereafter, radiolabeled peptide was captured on streptavidin-agarose beads, washed twice with washing buffer (50 mm Tris, pH 8.0, 500 mm KCl, 1 mm EDTA, 0.1% Nonidet P-40, 0.5% sodium deoxycholate) and incorporated [14C]acetyl was determined by liquid scintillation counting. For detection of acetylated histones, protein complexes obtained by GST pull-down assays were resolved by 18% SDS-polyacrylamide gel electrophoresis and analyzed by fluorography. For Western blot analyses one-third of the pulled down protein was resolved on 6% SDS-polyacrylamide gel electrophoresis and analyzed by Western blotting using antibodies specific to CBP (C-20, Santa Cruz Biotechnology) or p300 (C-20, Santa Cruz Biotechnology) as described (23Lipinski K.S. Fax P. Wilker B. Hennemann H. Brockmann D. Esche H. Virology. 1999; 255: 94-105Crossref PubMed Scopus (21) Google Scholar). 1.6 × 106 COS7 cells were transfected with expression vectors for Myc/His-tagged wild type E1A12S or E1A12S mutants as described above. Transfected cells were harvested 48 h after transfection and lysed in IP + 150 buffer. Precleared lysates were incubated with 2 μg of anti-Myc mouse monoclonal antibody (Invitrogen) or anti-CBP rabbit polyclonal antibody (A-22, Santa Cruz Biotechnology) overnight at 4 °C. Immune complexes were captured by adding Protein A/G-Sepharose (Amersham Pharmacia Biotech) for 2 h at 4 °C, washed three times in 1 ml of IP + 150 buffer and twice in HAT assay buffer. Afterward, immune complexes were used to determine HAT activity or to analyze E1A content by Western blotting using the Ad12 E1A antiserum as described previously (28Fax P. Lipinski K.S. Esche H. Brockmann D. J. Biol. Chem. 2000; 275: 8911-8920Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Chromatin immunoprecipitation assays were performed as described (30Luo R.X. Postigo A.A. Dean D.C. Cell. 1998; 92: 463-473Abstract Full Text Full Text PDF PubMed Scopus (839) Google Scholar, 35Dedon P.C. Soults J.A. Allis C.D. Gorovsky M.A. Anal. Biochem. 1991; 197: 83-90Crossref PubMed Scopus (104) Google Scholar, 36Braunstein M. Rose A.B. Holmes S.G. Allis C.D. Broach J.R. Genes Dev. 1993; 7: 592-604Crossref PubMed Scopus (714) Google Scholar) with minor modifications. 2 × 106 KB cells were transfected with 5 μg of E2Ad12-CAT or E2Ad12pmCRE-CAT using Polyfectin as described above. After 40 h, formaldehyde was added (final concentration 1%) to the culture medium. After incubation for 10 min at 37 °C, cells were washed twice in phosphate-buffered saline and scraped. Cells were lysed in lysis buffer (50 mm Hepes/KOH, pH 7.5, 150 mm KCl, 1 mm EDTA, 0.5% Nonidet P-40, 0.1% sodium deoxycholate, 0.5 mm Pefabloc, and 10 μg/ml aprotinin) for 10 min at 4 °C. Lysates were sonicated 5 times for 10 s and cleared by centrifugation. One-third of the lysate was used to quantitate the total amount of the respective reporter construct. Two-thirds of the lysates were diluted 5-fold with lysis buffer followed by incubation with 5 μl of anti-acetylhistone H4 antibody (UBI) or normal rabbit IgG (Santa Cruz) as control overnight at 4 °C. Immune complexes were collected, washed, and eluted as described (36Braunstein M. Rose A.B. Holmes S.G. Allis C.D. Broach J.R. Genes Dev. 1993; 7: 592-604Crossref PubMed Scopus (714) Google Scholar). Cross-linking of protein-DNA complexes was reversed at 65 °C for 5 h, followed by treatment with Proteinase K. DNA was extracted with phenol/chloroform and precipitated. Pellets were analyzed by PCR using primers specific for the E2Ad12 promoter (5′-E2Ad125′-AGGCGTGGGAATATCTTTACT-3′; 3′-E2Ad125′-CGCAGAAGCTCTGTTCAATAA-3′). To analyze whether the activity of the E2Ad12 promoter is modulated in an acetylation/deacetylation-dependent manner we performed transient expression assays. The reporter construct E2Ad12-CAT which carries the minimal promoter inducible by E1A12S (Fig. 1A; Ref. 28Fax P. Lipinski K.S. Esche H. Brockmann D. J. Biol. Chem. 2000; 275: 8911-8920Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar), was co-transfected with expression vectors coding for E1A12S and/or HDAC-1 in KB cells. CAT activity was determined 24 h later. As shown in Fig. 1 A, HDAC-1 strongly represses the E1A12S-induced CAT gene expression indicating that histone acetylation plays an important role in the activation of the E2Ad12 promoter. To confirm this assumption we made use of the drug Roscovitine. It has been shown that Roscovitine blocks proliferation signals which mediate CBP HAT activation (26Ait-Si-Ali S. Ramirez S. Barre F.-X. Dkhisse F. Magnaghi-Jaulin L. Girault J.A. Robin P. Knibiehler M. Pritchard L.L. Ducommun B. Trouche D. Harel-Bellan A. Nature. 1998; 396: 184-186Crossref PubMed Scopus (270) Google Scholar). E2Ad12-CAT was transfected in KB cells and Roscovitine was added at the indicated concentrations (Fig. 1 B) 14 h before harvesting the cells. The E1A12S mediated activation of the E2Ad12promoter in the absence of Roscovitine was set as 100% in these experiments (Fig. 1 B). Roscovitine inhibits the E1A12S-mediated activation of the E2Ad12promoter in a dose-dependent manner and nearly completely blocks CAT-gene expression at a concentration of 25 μm(Fig. 1 B). To exclude that Roscovitine inhibits indirectly the activation of the E2Ad12 promoter by interfering with the expression of E1A12S we made use of the Gal4 expression system. The reporter construct G5-E1BTATA-CAT which contains five binding sites for the yeast transcription factor Gal4 was co-transfected with pRc/RSV-Gal4-E1A12S in KB cells in the presence or absence of Roscovitine. PRc/RSV-Gal4-E1A12Scodes for a fusion protein consisting of the Gal4 DNA-binding domain and the E1A12S protein. Of note, its expression is controlled by the same RSV promoter as E1A12S used in the experiments summarized in Fig. 1 A. Gal4-E1A12Sstrongly activates CAT gene expression from G5-E1BTATA-CAT (Fig. 1 C; Ref. 32Lipinski K.S. Kröner-Lux G. Esche H. Brockmann D. J. Gen. Virol. 1997; 78: 413-421Crossref PubMed Scopus (12) Google Scholar). Roscovitine does not repress the Gal4-E1A12S-dependent activation of the G5-E1BTATA promoter (Fig. 1 C) indicating that this drug inhibits the E2 promoter directly and not via an indirect mechanism. Furthermore, Western blot analyses demonstrated a comparable E1A12S protein concentration in transiently transfected KB cells in the presence or absence of Roscovitine (data not shown). Taken together these data suggest that the activity of the E2Ad12promoter is modulated in an acetylation/deacetylation-dependent mechanism.Figure 4p300/CBP bound to E1A12S of Ad12 displays HAT activity. Whole cell extract prepared from KB cells was incubated with GST-E1A12S, GST-Δ1–79/E1A12S, or the GST leader sequence immobilized on glutathione-Sepharose beads. After extensive washings bound complexes were analyzed in a liquid HAT assay using [14C]acetyl-CoA and either a biotinylated synthetic peptide corresponding to the first 24 aa of histone H4 (A) or calf thymus histones (B) as substrates. A,acetylated peptide was captured on streptavidin-agarose beads and incorporated radioactivity was determined by liquid scintillation counting. B, acetylated histones were analyzed by SDS-polyacrylamide gel electrophoresis followed by fluorography.C, aliquots of the GST pull-down experiments were used for Western blot analyses using an anti-CBP or anti-p300 antiserum.Lane 3 shows proteins bound to the wild type E1A12S protein (GST-E1A12S), lane 2represents the GST leader sequence, lane 4 represents the E1A12S mutant lacking the N terminus and CR1 (GST-Δ1–79/E1A12S). Lane 1 contains 2% of the KB cell extract used in GST pull down assays. The positions of CBP and p300 are indicated on the right.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 1HDAC-1 as well as the CBP inhibitor Roscovitine repress E1A12S-mediated activation of the E2Ad12 promoter. A, KB cells were co-transfected with 1 μg of the E2Ad12-CAT reporter construct, 1.5 μg of pCMV-HA-HDAC-1 and 0.3 μg of pRc/RSV-E1A12S as indicated. Empty expression vectors were added to keep the amount of transfected DNA constant. The results are the average of three independent experiments performed in duplicate with standard deviations indicated. The promoter activity of E2Ad12-CAT in the presence of empty vectors was set as 1. The E2Ad12 promoter is schematically shown at the top of the figure. B,KB cells were transiently transfected with 1 μg of the E2Ad12-CAT reporter construct and 0.5 μg of pRc/RSV-E1A12S. Roscovitine was added at the indicated concentrations 10 h after transfection. 14 h later cells were harvested and CAT activity was determined. The results are the average of three independent experiments performed in duplicate with standard deviations indicated. The promoter activity of E2Ad12-CAT in the presence of pRc/RSV-E1A12S without Roscovitine was set as 100%. C, KB cells were transiently transfected with 0.2 μg of G5-E1BTATA-CAT and 1 μg of Gal4-E1A12Sexpression vector or empty expression vector as indicated at thebottom of the figure. Roscovitine was added at a concentration of 25 μm 10 h after transfection (open bars). The promoter activity of G5-E1BTATA-CAT in the presence of empty vector was set as 1. The reporter construct is schematically drawn at the top of the figure. 5XGal4represents five binding sites for the yeast Gal4 transcription factor.View Large Image Figure ViewerDownload Hi-res image Download (PPT) As E1A12S recruits p300/CBP to the E2Ad12 promoter (28Fax P. Lipinski K.S. Esche H. Brockmann D. J. Biol. Chem. 2000; 275: 8911-8920Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar) we asked next if this co-factor might be able to counteract the inhibition of the E2Ad12 promoter mediated by the histone deacetylase HDAC-1. We therefore performed transient expression assays co-transfecting the E2Ad12-CAT reporter construct with expression vectors for HDAC-1, p300/CBP, and/or E1A12S. For this experiment the concentration of each expression vector was titrated to be able to measure the effect of E1A12S as well as p300/CBP. As already shown in Fig. 1 A, HDAC-1 represses the E1A12S-mediated activation of the E2Ad12promoter (Fig. 2, A andB). p300 as well as CBP restores E1A12S-dependent transactivation of the E2Ad12 promoter in the presence of HDAC-1 (Fig. 2,A and B). Most importantly, a CBP mutant which is completely defective in HAT activity due to a deletion of aa 1431–1569 in the HAT domain (37Ait-Si-Ali S. Polesskaya A. Filleur S. Ferreira R. Duquet A. Robin P. Vervish A. Trouche D. Cabon F. Harel-Bellan A. Oncogene. 2000; 19: 2430-2437Crossref PubMed Scopus (120) Google Scholar) failed to counteract the HDAC-1-mediated repression (Fig. 2 B). This finding demonstrates the necessity of the HAT activity of p300/CBP for promoter activation in the presence of HDAC-1 and excludes the possibility that the co-factor circumvents the HDAC-1-mediated repression by a HAT-domain independent mechanism. In the presence of the mutant Δ1–79/E1A12S, which lacks aa 1–79 essential for the activation of the E2Ad12 promoter (Fig. 2 A, Ref. 28Fax P. Lipinski K.S. Esche H. Brockmann D. J. Biol. Chem. 2000; 275: 8911-8920Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar) as well as for the binding to both co-factors (Fig. 6B; Ref. 28Fax P. Lipinski K.S. Esche H. Brockmann D. J. Biol. Chem. 2000; 275: 8911-8920Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar), p300/CBP failed to rescue the HDAC-1-mediated repression (Fig. 2 A) suggesting that the recruitment of their HAT activity through E1A12S to the E2Ad12 promoter is necessary to counteract the HDAC-1 effect. Interestingly, p300/CBP does not activate the E2Ad12 promoter in the absence of HDAC-1 indicating that the endogenous co-activator concentration is not limiting with respect to promoter activation in the absence of overexpressed HDAC-1 under the conditions used (Fig. 2 A). These data are consistent with observations of Chen and co-workers (38Chen H. Lin R.J. Xie W. Wilpitz D. Evans R.M. Cell. 1999; 98: 675-686Abstract Full Text Full Text PDF PubMed Scopus (563) Google Scholar) who analyzed the activation of hormone-dependent gene expression through p300. Finally, the HAT P/CAF, which binds to E1A12S (39Reid J.L. Bannister A.J. Zegerman P. Martı́nez-Balbás M.A. Kouzarides T. EMBO J. 1998; 17: 4469-4477Crossref PubMed Scopus (108) Google Scholar) as well as to p300/CBP (7Yang X.J. Ogryzko V.V. Nishikawa J. Howard B.H. Nakatani Y. Nature. 1996; 382: 319-324Crossref PubMed Scopus (1320) Google Scholar) failed to counteract the HDAC-1-mediated E2Ad12 promoter repression (Fig. 2 A). This result suggests that the activation of the E2Ad12 promoter depends on a specific HAT activity, namely on that of p300/CBP. Taken together these results support our hypothesis that the HAT activity of p300/CBP is necessary for the activation of the E2Ad12promoter. We have shown previously that a point mutation in the E2-CRE prevent" @default.
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