The Adenovirus Oncoprotein E1a Stimulates Binding of Transcription Factor ETF to Transcriptionally Activate the p53 Gene
1999; Elsevier BV; Volume: 274; Issue: 34 Linguagem: Inglês
10.1074/jbc.274.34.23777
ISSN1083-351X
AutoresTracy K. Hale, Antony W. Braithwaite,
Tópico(s)Cancer Research and Treatments
ResumoExpression of the tumor suppressor protein p53 plays an important role in regulating the cellular response to DNA damage. During adenovirus infection, levels of p53 protein also increase. It has been shown that this increase is due not only to increased stability of the p53 protein but to the transcriptional activation of the p53 gene during infection. We demonstrate here that the E1a proteins of adenovirus are responsible for activating the mouse p53 gene and that both major E1a proteins, 243R and 289R, are required for complete activation. E1a brings about the binding of two cellular transcription factors to the mouse p53 promoter. One of these, ETF, binds to three upstream sites in the p53 promoter and one downstream site, whereas E2F binds to one upstream site in the presence of E1a. Our studies indicate that E2F binding is not essential for activation of the p53 promoter but that ETF is. Our data indicate the ETF site located downstream of the start site of transcription is the key site in conferring E1a responsiveness on the p53 promoter. Expression of the tumor suppressor protein p53 plays an important role in regulating the cellular response to DNA damage. During adenovirus infection, levels of p53 protein also increase. It has been shown that this increase is due not only to increased stability of the p53 protein but to the transcriptional activation of the p53 gene during infection. We demonstrate here that the E1a proteins of adenovirus are responsible for activating the mouse p53 gene and that both major E1a proteins, 243R and 289R, are required for complete activation. E1a brings about the binding of two cellular transcription factors to the mouse p53 promoter. One of these, ETF, binds to three upstream sites in the p53 promoter and one downstream site, whereas E2F binds to one upstream site in the presence of E1a. Our studies indicate that E2F binding is not essential for activation of the p53 promoter but that ETF is. Our data indicate the ETF site located downstream of the start site of transcription is the key site in conferring E1a responsiveness on the p53 promoter. conserved domain initiator chloramphenicol acetyltransferase cytomegalovirus normal rat kidney rat embryo fibroblast adenovirus 5 multiplicity of infection helix-loop-helix p53 factor electrophoretic mobility shift assay infectious units The tumor suppressor protein p53 plays an important role in maintaining the genomic integrity of a cell. Following exposure of a normal cell to genotoxic stress, with agents such as DNA-damaging drugs (1Hall P.A. McKee P.H. Menage H.D. Dover R. Lane D.P. Oncogene. 1993; 8: 203-207PubMed Google Scholar) and radiation (2Maltzman W. Czyzyk L. Mol. Cell. Biol. 1984; 4: 1689-1694Crossref PubMed Scopus (839) Google Scholar), levels of p53 protein increase. As the p53 protein is a sequence-specific DNA binding transcription factor (reviewed in Ref. 3Vogelstein B. Kinzler K.W. Cell. 1992; 70: 523-526Abstract Full Text PDF PubMed Scopus (1936) Google Scholar), this increase in p53 protein results in an increase in p53-dependent gene transcription, which in turn leads to cell cycle arrest or apoptosis (reviewed in Refs. 4Levine A. Annu. Rev. Biochem. 1993; 62: 623-651Crossref PubMed Scopus (488) Google Scholar, 5Gottlieb M.T. Oren M. Biochim. Biophys. Acta. 1996; 1287: 77-102Crossref PubMed Scopus (511) Google Scholar, 6Ko L.J. Prives C. Genes Dev. 1996; 10: 1054-1072Crossref PubMed Scopus (2301) Google Scholar). Cell cycle arrest is thought to be predominantly due to p53 transcriptionally activating the cyclin-dependent kinase inhibitor p21/WAF1 (7El-Deiry W.S. Tokino T. Velculescu V.E. Levy D.B. Parsons R. Trent J.M. Lin D. Mercer W.E. Kinzler K.W. Vogelstein B. Cell. 1993; 75: 817-825Abstract Full Text PDF PubMed Scopus (8054) Google Scholar), which inhibits the protein kinase activities of G1 cyclin/cyclin-dependent kinase complexes, preventing phosphorylation of the retinoblastoma protein (8Slobos R.J.C. Lee M.H. Plunkett B.S. Kessi T.D. Williams B.O. Jacks T. Hedrick L. Kastan M.B. Cho K.R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5320-5324Crossref PubMed Scopus (358) Google Scholar), thereby blocking cell cycle progression. It is less clear how p53 induces apoptosis, although several genes that play a role in regulating apoptotic pathways are transcriptionally regulated by p53 (9Buckbinder L. Talbott R. Velasco-Miguel S. Takenaka I. Faha B. Seizinger B.R. Kley N. Nature. 1995; 377: 646-649Crossref PubMed Scopus (812) Google Scholar, 10Miyashita T. Reed J.C. Cell. 1995; 80: 293-299Abstract Full Text PDF PubMed Scopus (305) Google Scholar). For example, p53 activates the bax gene (10Miyashita T. Reed J.C. Cell. 1995; 80: 293-299Abstract Full Text PDF PubMed Scopus (305) Google Scholar), the product of which binds to and prevents the ability of Bcl-2 to block apoptosis (11Oltvai Z.N. Milman C.L. Korsmeyer S.J. Cell. 1993; 74: 609-619Abstract Full Text PDF PubMed Scopus (5941) Google Scholar). It appears that, in part, the level of p53 determines whether a cell enters a cell cycle arrest or apoptotic pathway (12Chen X. Ko L.J. Jayaraman L. Prives C. Genes Dev. 1996; 10: 2438-2451Crossref PubMed Scopus (663) Google Scholar), although transcription-independent apoptosis induced by p53 has been reported (13Caelles C. Helmberg A. Karin M. Nature. 1994; 370: 220-223Crossref PubMed Scopus (844) Google Scholar, 14Wagner A.J. Kokontis J.M. Hay N. Genes Dev. 1994; 8: 2817-2830Crossref PubMed Scopus (518) Google Scholar). The increase in p53 levels is due in part to increased stabilization of the protein (1Hall P.A. McKee P.H. Menage H.D. Dover R. Lane D.P. Oncogene. 1993; 8: 203-207PubMed Google Scholar). Although the mechanism by which the p53 protein is stabilized is still unclear (15Lane D. Nature. 1998; 394: 616-617Crossref PubMed Scopus (52) Google Scholar), phosphorylation of p53 by the DNA-dependent protein kinase (16Woo R.A. McLure K.G. Lees-Miller S.P. Rancourt D.E. Lee P.W.K. Nature. 1998; 394: 700-704Crossref PubMed Scopus (295) Google Scholar) or ATM kinase (17Banin S. Moyal L. Shieh S.-Y. Taya Y. Anderson C.W. Chessa L. Smorodinsky N.I. Prives C. Reiss Y. Shiloh Y. Ziv Y. Science. 1998; 281: 1674-1677Crossref PubMed Scopus (1722) Google Scholar) may be responsible for activating the p53 protein. This phosphorylation may reduce the ability of p53 to interact with MDM2 (18Shieh S.-Y. Ikeda M. Taya Y. Prives C. Cell. 1997; 91: 325-334Abstract Full Text Full Text PDF PubMed Scopus (1783) Google Scholar) a negative regulator of p53 function (19Momand J. Zambetti G.P. Olson D.C. George D. Levine A.J. Cell. 1992; 69: 1237-1245Abstract Full Text PDF PubMed Scopus (2859) Google Scholar), thereby preventing ubiquitin-mediated degradation of p53 (20Haupt Y. Maya R. Kazaz A. Oren M. Nature. 1997; 387: 296-299Crossref PubMed Scopus (3790) Google Scholar). This in turn would enhance the ability of p53 to act as a transcriptional regulator (21Blaydes J.P. Gire V. Rowson J.M. Wynford-Thomas D. Oncogene. 1997; 14: 1859-1868Crossref PubMed Scopus (75) Google Scholar) to bring about growth arrest or apoptosis. However, two reports have raised the possibility that the p53 response to genotoxic stress may also be regulated at the transcriptional level (22Sun X. Shimizu H. Yamamoto K.-I. Mol. Cell. Biol. 1995; 15: 4489-4496Crossref PubMed Scopus (94) Google Scholar, 23Hellin A.-C. Calmant P. Gielen J. Bours V. Merville M.-P. Oncogene. 1998; 16: 1187-1195Crossref PubMed Scopus (92) Google Scholar). These reports show an increase in transcription from the p53 gene in cells exposed to DNA-damaging drugs. During adenovirus infection, the adenovirus early 1a (E1a) region expresses two major proteins, 243R and 289R, that differ by 46 amino acids that are present in 289R. Comparison of the primary amino acid sequences of 243R and 289R between serotypes suggests the presence of three conserved domains 1, 2, and 3 (CD1, CD2, and CD3)1 (reviewed in Ref. 24Braithwaite A.W. Nelson C.C. Bellett A.J.D. New Biol. 1991; 3: 18-26PubMed Google Scholar). These E1a proteins interact with numerous cellular proteins to drive cells through their cell cycle, thereby facilitating virus production (reviewed in Ref. 25Moran E. Curr. Opin. Genet. Dev. 1993; 3: 63-70Crossref PubMed Scopus (239) Google Scholar). Expression of E1a has been shown to cause an increase in the level of p53 protein and induce p53-dependent apoptosis (26Lowe S.W. Ruley H.E. Genes Dev. 1993; 7: 535-545Crossref PubMed Scopus (620) Google Scholar, 27Teodoro J.G. Shore G.C. Branton P.E. Oncogene. 1995; 11: 467-474PubMed Google Scholar, 28Querido E. Teodoro J.G. Branton P.E. J. Virol. 1997; 71: 3526-3533Crossref PubMed Google Scholar). Stabilization of p53 requires the amino terminus or CD1 of E1a and occurs through modification of a ubiquitin-protease pathway (29Nakajima T. Morita K. Tsunoda H. Imajoh-Ohmi S. Tanaka H. Yasuda H. Oda K. J. Biol. Chem. 1998; 273: 20036-20045Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Like DNA-damaging agents (22Sun X. Shimizu H. Yamamoto K.-I. Mol. Cell. Biol. 1995; 15: 4489-4496Crossref PubMed Scopus (94) Google Scholar, 23Hellin A.-C. Calmant P. Gielen J. Bours V. Merville M.-P. Oncogene. 1998; 16: 1187-1195Crossref PubMed Scopus (92) Google Scholar), it has been shown that adenovirus E1a products not only stabilize p53 protein but also transcriptionally activate p53 expression. Early studies performed in normal rat kidney (NRK) cells using nuclear run-on assays demonstrated that stimulation of endogenous p53 expression by adenovirus was at the level of transcriptional initiation and that the E1a proteins were most likely responsible (30Braithwaite A. Nelson C. Skulimowski A. McGovern J. Pigott D. Jenkins J. Virology. 1990; 177: 595-605Crossref PubMed Scopus (44) Google Scholar). The adenovirus E1a proteins represent an extensively studied set of viral transactivators (31Flint J. Shenk T. Annu. Rev. Genet. 1989; 23: 141-161Crossref PubMed Scopus (173) Google Scholar, 32Shenk T. Flint J. Adv. Cancer Res. 1991; 57: 47-85Crossref PubMed Scopus (157) Google Scholar, 33Bayley S.T. Mymryk J.S. Intl. J. Oncology. 1994; 5: 425-444PubMed Google Scholar) that have been widely used in the study of regulatory systems that control cellular transcription. Neither of the major E1a proteins, 243R and 289R, is capable of binding to double stranded DNA in a sequence-specific manner (34Ferguson B. Krippl B. Andrisani O. Jones N. Westphal H. Rosenberg M. Mol. Cell. Biol. 1985; 5: 2653-2661Crossref PubMed Scopus (110) Google Scholar); therefore, E1a must act through preexisting cellular transcription factors that interact with E1a-inducible promoters. Several sequence-specific DNA binding transcription factors, such as E2F (35Bagchi S. Raychaudhuri P. Nevins J.R. Cell. 1990; 62: 659-669Abstract Full Text PDF PubMed Scopus (291) Google Scholar), AP1 (36Maguire K. Shi X.-P. Horikoshi N. Rappaport J. Rosenberg M. Weinmann R. Oncogene. 1991; 6: 1417-1422PubMed Google Scholar), ATF (37Chatton B. Bocco J.L. Gaire M. Hauss C. Reimund B. Goetz J. Kedinger C. Mol. Cell. Biol. 1993; 13: 561-570Crossref PubMed Scopus (94) Google Scholar), Sp1, and USF (38Liu F. Green M.R. Nature. 1994; 368: 520-525Crossref PubMed Scopus (233) Google Scholar), and components of the basal transcription initiation complex (39Horikoshi N. Maguire K. Kralli A. Maldonado E. Reinberg D. Weinmann R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5124-5128Crossref PubMed Google Scholar, 40Geisberg J.V. Chen J.-L. Ricciardi R.P. Mol. Cell. Biol. 1995; 15: 6283-6290Crossref PubMed Scopus (69) Google Scholar, 41Mazzarelli J.M. Mengus G. Davidson I. Ricciardi R.P. J. Virol. 1997; 71: 7978-7983Crossref PubMed Google Scholar) have been shown to interact directly with E1a and/or confer E1a responsiveness. In addition, E1a also interacts with the transcriptional cofactors CBP/p300 (42Arany Z. Newsome D. Oldread E. Livingston D.M. Eckner R. Nature. 1995; 374: 81-84Crossref PubMed Scopus (497) Google Scholar, 43Lundblad J.R. Kwok R.P.S. Laurance M.E. Harter M.L. Goodman R.H. Nature. 1995; 374: 85-88Crossref PubMed Scopus (534) Google Scholar, 44Ait-Si-Ali S. Ramirez S. Barre F.-X. Dkhissi 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 (272) Google Scholar), P/CAF (45Reid J.L. Bannister A.J. Zegerman P. Martinez-Balbas M.A. Kouzarides T. EMBO J. 1998; 17: 4469-4477Crossref PubMed Scopus (108) Google Scholar), and the retinoblastoma protein (46Wang H.G. Moran E. Yaciuk P. J. Virol. 1995; 69: 7917-7924Crossref PubMed Google Scholar). In comparison to the studies on the stabilization of the p53 protein, relatively little is known about the signal transduction pathways and transcription factors that regulate transcription from the p53 gene. The mouse p53 gene has a TATA-less promoter (47Bienz-Tadmor B. Zakut-Houri R. Givol D. Oren M. EMBO J. 1985; 4: 3209-3213Crossref PubMed Scopus (110) Google Scholar) that contains a region extending from −2 to +5 (all numbering is relative to the start site of transcription), which has homology to an initiator (Inr) element (48Javahery R. Khachi A. Lo K. Zenzie-Gregory B. Smale S.T. Mol. Cell. Biol. 1994; 14: 116-127Crossref PubMed Scopus (602) Google Scholar). Inr elements have been shown to position the start site of transcription in TATA-less promoters (49O'Shea-Greenfield A. Smale S.T. J. Biol. Chem. 1992; 267: 1391-1402Abstract Full Text PDF PubMed Google Scholar). Adjacent to the Inr (Fig. 1 a), located between +5 and +17, is a NF1-like site (50Ginsberg D. Oren M. Yaniv M. Piette J. Oncogene. 1990; 5: 1285-1290PubMed Google Scholar, 51Hale T.K. Braithwaite A.W. Nucleic Acids Res. 1995; 23: 663-669Crossref PubMed Scopus (34) Google Scholar). Further downstream of the Inr is a helix-loop-helix (HLH) consensus binding motif (Fig.1 a). Several members of the HLH family, including USF, have been shown to bind to this site and enhance promoter activity (52Reisman D. Elkind N.B. Roy B. Beamon J. Rotter V. Cell Growth & Differ. 1993; 4: 57-65PubMed Google Scholar, 53Reisman D. Rotter V. Nucleic Acids Res. 1993; 21: 345-350Crossref PubMed Scopus (107) Google Scholar). Wu and Lozano (54Wu H. Lozano G. J. Biol. Chem. 1994; 269: 20067-20074Abstract Full Text PDF PubMed Google Scholar) have demonstrated that NF-κB also binds downstream of the Inr to a site located between +55 and +64 (Fig. 1 a). Binding of NF-κB in response to TNF-α, an inducer of NF-κB activity, was shown to activate the mouse p53 promoter (54Wu H. Lozano G. J. Biol. Chem. 1994; 269: 20067-20074Abstract Full Text PDF PubMed Google Scholar). The mouse p53 promoter also contains a consensus TRE-like AP1 binding site between −64 and −57 (Fig. 1 a) that binds an unidentified factor designated p53 factor 1 (PF1) (50Ginsberg D. Oren M. Yaniv M. Piette J. Oncogene. 1990; 5: 1285-1290PubMed Google Scholar). Finally, the transcription factor ETF binds to a downstream region in the p53 promoter, and another unidentified factor PF2 binds to a site upstream (Fig. 1 a) and appears to be essential for promoter activity (51Hale T.K. Braithwaite A.W. Nucleic Acids Res. 1995; 23: 663-669Crossref PubMed Scopus (34) Google Scholar). From the literature, it appears that DNA-damaging drugs and expression of E1a during adenovirus infection result in both the transcriptional activation of the p53 gene and stabilization of the protein (1Hall P.A. McKee P.H. Menage H.D. Dover R. Lane D.P. Oncogene. 1993; 8: 203-207PubMed Google Scholar, 22Sun X. Shimizu H. Yamamoto K.-I. Mol. Cell. Biol. 1995; 15: 4489-4496Crossref PubMed Scopus (94) Google Scholar, 23Hellin A.-C. Calmant P. Gielen J. Bours V. Merville M.-P. Oncogene. 1998; 16: 1187-1195Crossref PubMed Scopus (92) Google Scholar, 26Lowe S.W. Ruley H.E. Genes Dev. 1993; 7: 535-545Crossref PubMed Scopus (620) Google Scholar, 30Braithwaite A. Nelson C. Skulimowski A. McGovern J. Pigott D. Jenkins J. Virology. 1990; 177: 595-605Crossref PubMed Scopus (44) Google Scholar). Because the level of p53 may determine the fate of a cell (12Chen X. Ko L.J. Jayaraman L. Prives C. Genes Dev. 1996; 10: 2438-2451Crossref PubMed Scopus (663) Google Scholar), understanding the transcriptional mechanisms that regulate p53 expression is of importance. This study utilizes the ability of the viral transactivator E1a to activate p53 expression to identify several cellular factors that are involved in transcriptionally activating the p53 promoter, which may have relevance to the activation of p53 expression during the cellular response to genotoxic stress. The plasmid pCAT3M contains the chloramphenicol acetyltransferase (CAT) gene but no eukaryotic promoter sequences upstream of this gene (55Laimins L.A. Gruss P. Pozatti R. Khoury G. J. Virol. 1984; 49: 183-189Crossref PubMed Google Scholar). pAACAT (47Bienz-Tadmor B. Zakut-Houri R. Givol D. Oren M. EMBO J. 1985; 4: 3209-3213Crossref PubMed Scopus (110) Google Scholar) contains −224 to +116 of the mouse p53 promoter blunt-end cloned in front of the CAT gene in pCAT3M (Fig. 1 b). The plasmid pARCAT (47Bienz-Tadmor B. Zakut-Houri R. Givol D. Oren M. EMBO J. 1985; 4: 3209-3213Crossref PubMed Scopus (110) Google Scholar) contains −320 to +116 of the mouse p53 promoter also blunt-end cloned in front of the CAT gene in pCAT3M (Fig. 1 b). In the cytomegalovirus (CMV)-based plasmids, transcription is controlled by the immediate early enhancer-promoter of human CMV. pCMVE1a (56Morris G.F. Mathews M.B. J. Virol. 1991; 65: 6397-6406Crossref PubMed Google Scholar) contains a genomic fragment of adenovirus early region 1 that encodes all of the E1a proteins. pCMV12S and pCMV13S encode the adenovirus E1a proteins 243R and 289R, respectively (56Morris G.F. Mathews M.B. J. Virol. 1991; 65: 6397-6406Crossref PubMed Google Scholar). In order to create the reporter plasmids with mutated ETF sites (see Fig. 6 a) within pAACAT, the technique of inverse polymerase chain reaction was used (57Imai Y. Matsushima Y. Sugimura T. Terada M. Nucleic Acids Res. 1991; 19: 2785Crossref PubMed Scopus (320) Google Scholar). The following primer pairs were synthesized: for pETF2CAT, 5′-GTTTCAATAC ATTTT GCCCT CACAG C-3′ and 5′-CGATT CGGAG GGCTC CTGCC T-3′; for pETF4CAT, 5′-CTCAATTAGA ATCCT GACTC TGCAA-3′ and 5′-ATGTT GCCCT CAGCA GGAAC G-3′; and for pETF7CAT, 5′-GTGCT CACCC TGGCT AAAGT TCTGT-3′ and 5′-GTGGTATGTT AAAGT CCCAA TCCCA GC-3′ (substitutions are underlined). For pETF2/4/7CAT, each mutated site was introduced by successive rounds of polymerase chain reaction/ligations using the above primers pairs. Polymerase chain reaction was performed with 1 cycle at 96 °C for 3 min, 67 °C for 15 s, and 72 °C for 5 min, followed by 25 cycles at 96 °C for 1 min, 67 °C for 15 s, and 72 °C for 5 min, and then 1 cycle of 72 °C for 9 min. This was done in 50 μl of reaction mixture containing 20 mmTris-HCl, pH 8.8, 10 mm KCl, 10 mm(NH4)2SO4, 2–10 mmMgSO4, 0.1% Triton X-100, 100 mm each of dNTPs, 10 ng of the template pAACAT, 40 pmol each of the primers, and 0.5 unit of Vent™ DNA polymerase (New England Biolabs). The amplified linear DNA was agarose gel-purified and T4polynucleotide kinase-treated, and then a portion was self-ligated in 15 mm Tris-HCl, pH 7.8, 5 mm MgCl2, 5 mm dithiothreitol, 0.25 mm ATP, 30 mm KCl, 1 mm hexamine cobalt chloride, and 8 units of T4 DNA ligase at 14 °C for 16 h and then used to transform competent Escherichia coli DH5 cells. The required substitutions within each construct were confirmed by sequencing. NRK and HeLa cells were maintained at 37 °C, 10% CO2 in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal bovine serum. Rat embryo fibroblasts (REFs) were prepared as described previously (58Bellett A.J.D. Younghusband H.B. J. Cell. Physiol. 1979; 101: 33-48Crossref PubMed Scopus (37) Google Scholar) and routinely cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum at 37 °C and 10% CO2. REFs were used up to passage seven and then replaced with new cells. Two transfection methods were used in this study: For REFs, 106 cells growing in a 10-cm dish were transfected using the calcium phosphate method (59Wigler M. Silverstein S. Lee L.S. Pellicer A. Cheng Y.C. Axel R. Cell. 1977; 11: 223-232Abstract Full Text PDF PubMed Scopus (925) Google Scholar) with the Transfinity kit from Life Technologies, Inc. Ten μg of plasmid DNA and 10 μg of carrier were used to transfect each 10-cm dish of cells. For HeLa cells, 2.5 × 105 cells were seeded into 35-mm dishes and transfected with FuGENE™ 6 (Roche Molecular Biochemicals) after 18 h. For each dish transfected, the total amount of DNA was kept at 4 μg. The amounts of reporter and expression plasmids used are indicated in the figure legends. Sonicated salmon sperm DNA was used as carrier DNA to keep the total amount of DNA constant. The ratio of DNA (μg) to the volume of FuGENE™ 6 Reagent (μl) used was kept at 2:3 for each transfection. Wild-type human adenovirus serotype 5 (Ad5) was obtained from the American Type Culture Collection and was free from adenovirus-associated virus. The mutant adenovirus dl 312 (60Jones N. Shenk T. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 3665-3669Crossref PubMed Scopus (414) Google Scholar) expresses no E1a proteins, due to a deletion of base pairs 448–1349. dl 347 (61Hearing P. Shenk T. Mol. Cell. Biol. 1985; 5: 3214-3221Crossref PubMed Scopus (52) Google Scholar), which expresses the 243R protein but not 289R, was created when the E1a gene in dl 309 (60Jones N. Shenk T. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 3665-3669Crossref PubMed Scopus (414) Google Scholar) was replaced with the 12S cDNA. The mutant adenovirus dl 348 (61Hearing P. Shenk T. Mol. Cell. Biol. 1985; 5: 3214-3221Crossref PubMed Scopus (52) Google Scholar), which expresses the 289R protein but not 243R, was created when the E1a gene in dl 309 (60Jones N. Shenk T. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 3665-3669Crossref PubMed Scopus (414) Google Scholar) was replaced with the 13S cDNA. dl 337 was created when base pairs 1770–1915 were deleted from dl 309, so it expresses a truncated E1b 19-kDa protein (62Pilder S. Logan J. Shenk T. J. Virol. 1984; 52: 664-671Crossref PubMed Google Scholar).dl 338 was created when base pairs 2805–3329 were deleted from dl 309 and does not express any E1b 55-kDa protein (63Pilder S. Moore M. Logan J. Shenk T. Mol. Cell. Biol. 1986; 6: 470-476Crossref PubMed Scopus (241) Google Scholar).dl 327 (referred to as dl 324 in Ref. 64Thimmappaya B. Weinberger C. Schneider R.J. Shenk T. Cell. 1982; 31: 543-551Abstract Full Text PDF PubMed Scopus (251) Google Scholar) has a deletion in the protein coding region of E3, so it expresses only the minor E3 proteins of 12.5 and 3.6 kDa. The mutant dl 808 has the open reading frames 2–7 of E4 in adenovirus 2 deleted, so it expresses no E4 proteins (65Weinberg D.H. Ketner G. J. Virol. 1986; 57: 833-838Crossref PubMed Google Scholar). Monolayers of cells in 10-cm dishes were infected with adenovirus in 1 ml of Dulbecco's modified Eagle's medium containing virus, 40 h postseeding or 18 h posttransfection. The multiplicity of infection (MOI) was dependent on the experiment and is indicated in the figure legends. Cells were incubated for 1 h at 37 °C and 10% CO2, after which, the medium was replaced with 10 ml of Dulbecco's modified Eagle's medium containing 2% fetal bovine serum. Sixty hours posttransfection or 48 h postinfection, the same number of cells were washed twice in ice-cold PBS and then resuspended in 100 μl of 0.25 m Tris-HCl, pH 7.5. Extracts of cells were then prepared by three rounds of freezing and thawing followed by centrifugation for 15 min at 12,000 rpm and 4 °C to remove cellular debris. The supernatant was then heated to 65 °C for 10 min to inactivate a CAT inhibitor previously reported (66Sleigh M. Anal. Biochem. 1986; 156: 251-256Crossref PubMed Scopus (346) Google Scholar). CAT activities from a standard amount of lysate were determined as described by Sleigh (66Sleigh M. Anal. Biochem. 1986; 156: 251-256Crossref PubMed Scopus (346) Google Scholar). Details of this procedure have been described previously (67Jackson P. Bos E. Braithwaite A.W. Oncogene. 1993; 8: 589-597PubMed Google Scholar). Forty-eight hours postinfection, NRK cells in 10-cm dishes were washed twice with cold PBS, harvested by scraping, and transferred to microcentrifuge tubes. Nuclear proteins were isolated using a modified small-scale preparation method (68Schreiber E. Matthias P. Muller M.M. Schaffner W. Nucleic Acids Res. 1989; 17: 6419Crossref PubMed Scopus (4013) Google Scholar), after which, the nuclear proteins were centrifuged for 5 min at 4 °C; the supernatant was then dialyzed against 100 volumes of Buffer D (20 mm HEPES, pH 7.9, 20% glycerol, 0.1m KCl, 0.2 mm EDTA, 0.5 mmdithiothreitol, 0.5 mm phenylmethylsulfonyl fluoride) for 18 h at 4 °C. Double stranded oligonucleotides containing the consensus binding sites for AP2, ATF and Sp1 were purchased from Promega Corp. The other competitors were synthesized with the following sequences: ETF, 5′-GTCCG GGCAG CCCCC GGCGC AGCGC GGCCG-3′ (69Kageyama R. Merlino G.T. Pastan I. J. Biol. Chem. 1989; 264: 15508-15514Abstract Full Text PDF PubMed Google Scholar); E2F, 5′-AGTTT TCGCG CTTAA ATTTG AGAAA GGGCG CGAAA CTA-3′ (70Hiebert S.W. Blake M. Azizkhan J. Nevins J.R. J. Virol. 1991; 65: 3547-3552Crossref PubMed Google Scholar); and PF1, 5′-CAATC CTGAC TCTGC AAG-3′ (50Ginsberg D. Oren M. Yaniv M. Piette J. Oncogene. 1990; 5: 1285-1290PubMed Google Scholar). Binding reactions were performed in a volume of 15 μl containing 15–35 μg of nuclear extract, 0.5–2 μg of poly(dI·dC)·poly(dI·dC), 10 mm Tris-HCl, pH 7.5, 50 mm NaCl, 1 mm MgCl2, 0.5 mm dithiothreitol, 0.5 mm EDTA, 4% glycerol. Reactions were allowed to proceed for 15 min at room temperature. 1 × 104 cpm of target oligonucleotide (oligomers 1–7), end-labeled with [32P]dCTP, was added to the binding reaction and incubated at room temperature for a further 15 min. Following this, 1.5 μl of 10× DNA loading dye was added, and the binding reaction was immediately loaded on to a pre-electrophoresed 5% polyacrylamide gel. After electrophoresis, gels were fixed in 10% acetic acid for 10 min, dried for 30 min at 80 °C, and exposed to Kodak X-OMAT AR film at −70 °C, usually for 7–14 h. For competition EMSAs, unlabeled oligonucleotides were added to the binding reactions prior to addition of the radiolabeled oligonucleotide. Adenovirus infection has been shown to increase levels of p53 mRNA and protein in mouse Swiss 3T3, hamster tsAF8, and NRK cells (30Braithwaite A. Nelson C. Skulimowski A. McGovern J. Pigott D. Jenkins J. Virology. 1990; 177: 595-605Crossref PubMed Scopus (44) Google Scholar, 71Liu H.T. Baserga R. Mercer W.E. Mol. Cell. Biol. 1985; 5: 2936-2942Crossref PubMed Google Scholar). Braithwaite et al. (30Braithwaite A. Nelson C. Skulimowski A. McGovern J. Pigott D. Jenkins J. Virology. 1990; 177: 595-605Crossref PubMed Scopus (44) Google Scholar) demonstrated that Ad5 can transcriptionally activate the endogenous p53 gene in NRK cells. To determine the region of the p53 promoter required for activation by Ad5, two p53 promoter fragments were tested for their ability to respond to Ad5. The construct pAACAT contains the region of the p53 promoter from −224 to +116, whereas pARCAT contains the region from −320 to +116 cloned in front of the reporter CAT gene (Fig. 1 b). These constructs along with the promoterless CAT construct pCAT3M were transfected into REFs. Eighteen hours posttransfection, the REFs were infected with wild-type Ad5 at varying MOIs of 10, 20, or 30 infectious units (iu) per cell. Forty-eight hours after infection, cell lysates were prepared and assayed for CAT activity. As shown in Fig. 1 c, both pAACAT and pARCAT were activated by Ad5 in a dose-dependent manner. However, whereas at an MOI of 30 iu per cell, activity from pAACAT was enhanced 8-fold, pARCAT activity was enhanced only 3-fold. There was no enhancement of pCAT3M activity at the same MOI (Fig.1 c). The reduced level of activity from the longer construct (pARCAT) in response to Ad5 is reproducible and may be due to the presence of elements that “dampen” the activation of p53 in this construct. The region between −320 and −224, located in pARCAT, contains a putative negative transcriptional regulatory element (47Bienz-Tadmor B. Zakut-Houri R. Givol D. Oren M. EMBO J. 1985; 4: 3209-3213Crossref PubMed Scopus (110) Google Scholar), and although it is not involved in basal expression (51Hale T.K. Braithwaite A.W. Nucleic Acids Res. 1995; 23: 663-669Crossref PubMed Scopus (34) Google Scholar), it may interfere with activation of the p53 promoter by Ad5. Nonetheless, these results in REFs show that the elements required for transcriptional activation of p53 by Ad5 are located between −224 and +116 of the mouse p53 promoter. Similar results were obtained in L929 cells (data not shown). Although the E1a proteins of adenovirus are extensively studied transcriptional regulators (31Flint J. Shenk T. Annu. Rev. Genet. 1989; 23: 141-161Crossref PubMed Scopus (173) Google Scholar, 32Shenk T. Flint J. Adv. Cancer Res. 1991; 57: 47-85Crossref PubMed Scopus (157) Google Scholar, 33Bayley S.T. Mymryk J.S. Intl. J. Oncology. 1994; 5: 425-444PubMed Google Scholar), other early region genes have also been shown to be involved in regulating transcription. For example, the E4 19-kDa protein stabilizes the E2F complex on the adenovirus E2 promoter (72Neill S.D. Hemstrom C. Virtanen A. Nevins J.R. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2008-2012Crossref PubMed Scopus (77) Google Scholar). To identify involvement of any other early region proteins in transactivating the p53 promoter, REFs were transfected with pAACAT and then infected with a pane
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