Specific Role for p300/CREB-binding Protein-associated Factor Activity in E2F1 Stabilization in Response to DNA Damage

E2F1, a member of the E2F family of transcription factors, plays a pivotal role in controlling both physiological cell-cycle progression and apoptotic cell death in response to DNA damage and oncogene activation. In response to genotoxic stresses, E2F1 is stabilized by signals that include ATM-dependent phosphorylation. We recently demonstrated that DNA damage induces also E2F1 acetylation, which is required for its recruitment onto apoptotic gene promoters. Here we show that E2F1 is stabilized in response to doxorubicin and cisplatin treatments even in the absence of either ATM-dependent phosphorylation or p53 and cAbl, two major transducers of DNA damage signaling. We found that acetylation of E2F1 is, instead, required to stabilize the protein in response to doxorubicin. Finally, we report that the formation of E2F1-p300/CREB-binding protein-associated factor (P/CAF) complexes is preferentially induced in doxorubicin-treated cells, and that P/CAF acetyltransferase (HAT), but not p300 HAT activity, is required for a significant E2F1 stabilization and accumulation. Our results unveil a differential role of P/CAF and p300 in acetylation-induced stabilization of E2F1, thus supporting a specific role for P/CAF HAT activity in E2F1-dependent apoptosis in response to DNA damage. E2F1, a member of the E2F family of transcription factors, plays a pivotal role in controlling both physiological cell-cycle progression and apoptotic cell death in response to DNA damage and oncogene activation. In response to genotoxic stresses, E2F1 is stabilized by signals that include ATM-dependent phosphorylation. We recently demonstrated that DNA damage induces also E2F1 acetylation, which is required for its recruitment onto apoptotic gene promoters. Here we show that E2F1 is stabilized in response to doxorubicin and cisplatin treatments even in the absence of either ATM-dependent phosphorylation or p53 and cAbl, two major transducers of DNA damage signaling. We found that acetylation of E2F1 is, instead, required to stabilize the protein in response to doxorubicin. Finally, we report that the formation of E2F1-p300/CREB-binding protein-associated factor (P/CAF) complexes is preferentially induced in doxorubicin-treated cells, and that P/CAF acetyltransferase (HAT), but not p300 HAT activity, is required for a significant E2F1 stabilization and accumulation. Our results unveil a differential role of P/CAF and p300 in acetylation-induced stabilization of E2F1, thus supporting a specific role for P/CAF HAT activity in E2F1-dependent apoptosis in response to DNA damage. E2F1 belongs to the E2F family of transcription factors. Seven different E2F members (E2F1 through E2F7) (1Di Stefano L. Jensen M.R. Helin K. EMBO J. 2003; 22: 6289-6298Crossref PubMed Scopus (215) Google Scholar, 2Johnson D.G. Mol. Carcinog. 2000; 27: 151-157Crossref PubMed Scopus (83) Google Scholar, 3Mundle S.D. Saberwal G. FASEB J. 2003; 17: 569-574Crossref PubMed Scopus (72) Google Scholar, 4Sears R.C. Nevins J.R. J. Biol. Chem. 2002; 277: 11617-11620Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar) and three DRTF protein (DP) 1The abbreviations used are: DP, DRTF protein; Rb, retinoblastoma; ATM, ataxia-telangiectasia mutated; ATR, ATM- and RAD3-related kinase; P/CAF, p300/CREB-binding protein-associated factor; CREB, cAMP-response element-binding protein; MEF, mouse embryo fibroblast; HA, hemagglutinin; GFP, green fluorescent protein; AT, ataxiatelangiectasia; TERT, telomerase reverse transcriptase. proteins have been identified so far. As a heterodimeric complex with the DP-1 protein, E2F1 binds to promoters containing E2F-responsive elements and activates transcription through its C-terminal activation domain. Initial studies implicated E2F1 in the control of cell-cycle progression, because many E2F1 target genes encode S-phase regulatory proteins as well as proteins regulating cell-cycle progression and DNA synthesis. However, transcriptional activation of cell-cycle-related genes is only one facet of E2F1 activity. Recent data obtained both from mouse and cellular models and from microarray analysis demonstrate that E2F1 has important roles also in modulating antiproliferative processes such as senescence and apoptosis (5Ginsberg D. FEBS Lett. 2002; 529: 122-125Crossref PubMed Scopus (156) Google Scholar). E2F1 activity is regulated at different levels. First, the transactivation potential of E2F1 is negatively affected by the interaction with the retinoblastoma gene product pRb. The E2F1-bound Rb inhibits transcription actively by contact with promoter-bound proteins (6Weintraub S.J. Chow K.N. Luo R.X. Zhang S.H. He S. Dean D.C. Nature. 1995; 375: 812-815Crossref PubMed Scopus (459) Google Scholar, 7Zhang H.S. Postigo A.A. Dean D.C. Cell. 1999; 97: 53-61Abstract Full Text Full Text PDF PubMed Google Scholar) and by recruiting a histone deacetylase complex (8Brehm A. Miska E.A. McCance D.J. Reid J.L. Bannister A.J. Kouzarides T. Nature. 1998; 391: 597-601Crossref PubMed Scopus (1080) Google Scholar, 9Luo R.X. Postigo A.A. Dean D.C. Cell. 1998; 92: 463-473Abstract Full Text Full Text PDF PubMed Scopus (839) Google Scholar, 10Magnaghi-Jaulin L. Groisman R. Naguibneva I. Robin P. Lorain S. Le Villain J.P. Troalen F. Trouche D. Harel-Bellan A. Nature. 1998; 391: 601-605Crossref PubMed Scopus (805) Google Scholar). The E2F1-Rb interaction is limited by Rb phosphorylation, which is mainly mediated by the cyclin D/cyclin-dependent kinase-4 at the G1/S transition (11Nevins J.R. Hum. Mol. Genet. 2001; 10: 699-703Crossref PubMed Scopus (745) Google Scholar). A second level of control of E2F1 activity concerns the regulation of its DNA-binding ability. Indeed, E2F1 interacts directly with cyclin A, which results in phosphorylation of DP-1 in S phase, causing down-regulation of E2F binding to DNA (12Krek W. Ewen M.E. Shirodkar S. Arany Z. Kaelin Jr., W.G. Livingston D.M. Cell. 1994; 78: 161-172Abstract Full Text PDF PubMed Scopus (414) Google Scholar, 13Krek W. Xu G. Livingston D.M. Cell. 1995; 83: 1149-1158Abstract Full Text PDF PubMed Scopus (317) Google Scholar). A final level of regulation of E2F1 relates to the control of its abundance by a number of mechanisms. After mitogenic stimuli, one or more E2F species activate(s) the E2F1 promoter in late G1, resulting in an increase in E2F1 RNA and protein synthesis. Conversely, transcription of the E2F1 gene decreases in late S, because of the negative regulation of the DNA-binding activity of E2F1–3 by cyclin A/cyclin-dependent kinase-2 (14Hateboer G. Kerkhoven R.M. Shvarts A. Bernards R. Beijersbergen R.L. Genes Dev. 1996; 10: 2960-2970Crossref PubMed Scopus (191) Google Scholar, 15Martelli F. Livingston D.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2858-2863Crossref PubMed Scopus (34) Google Scholar). E2F1 protein levels are also regulated by the ubiquitination-dependent proteasome degradation. The epitope recognized by the ubiquitin-proteasome pathway is located in the carboxyl terminus, near the acidic activation domain of E2F1 (16Harper J.W. Elledge S.J. Nat. Cell Biol. 1999; 1: E5-E7Crossref PubMed Scopus (31) Google Scholar, 17Marti A. Wirbelauer C. Scheffner M. Krek W. Nat. Cell Biol. 1999; 1: 14-19Crossref PubMed Scopus (283) Google Scholar). Both post-translational modifications, such as phosphorylation by the transcription factor IIH (TFIIH) kinase and protein-protein interactions with pRb have been shown to increase E2F1 stability through the inhibition of the ubiquitination process (18Vandel L. Kouzarides T. EMBO J. 1999; 18: 4280-4291Crossref PubMed Scopus (55) Google Scholar). Distinct modalities of E2F1 regulation might be responsible for opposite outcomes of its activation, cell-cycle progression, or apoptosis. A prompt response to genotoxic stresses has been reported to induce stabilization of E2F1 through its phosphorylation by the ataxia-telangiectasia mutated (ATM) kinase, the ATM and RAD3-related (ATR) kinase (19Lin W.C. Lin F.T. Nevins J.R. Genes Dev. 2001; 15: 1833-1844PubMed Google Scholar) and the checkpoint kinase 2 (20Stevens C. Smith L. La Thangue N.B. Nat. Cell Biol. 2003; 5: 401-409Crossref PubMed Scopus (327) Google Scholar, 21Cam H. Dynlacht B.D. Cancer Cell. 2003; 3: 311-316Abstract Full Text Full Text PDF PubMed Scopus (288) Google Scholar). We recently demonstrated that DNA damage also causes the acetylation-dependent activation of E2F1 apoptotic potential by boosting E2F1-driven transcription of the proapoptotic target gene p73 (22Pediconi N. Ianari A. Costanzo A. Belloni L. Gallo R. Cimino L. Porcellini A. Screpanti I. Balsano C. Alesse E. Gulino A. Levrero M. Nat. Cell Biol. 2003; 5: 552-558Crossref PubMed Scopus (230) Google Scholar). Although E2F1 can be acetylated by both p300/CREB-binding protein (CBP)-associated factor (P/CAF) and CREB-binding protein/P300 acetylases in vitro at the same lysine residues (23Marzio G. Wagener C. Gutierrez M.I. Cartwright P. Helin K. Giacca M. J. Biol. Chem. 2000; 275: 10887-10892Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 24Martinez-Balbas M.A. Bauer U.M. Nielsen S.J. Brehm A. Kouzarides T. EMBO J. 2000; 19: 662-671Crossref PubMed Scopus (570) Google Scholar), the relationship between this post-translational modification and the stabilization of E2F1 in response to DNA damage has not been explored. We describe here a specific role for P/CAF in the acetylation-induced accumulation of E2F1 in response to DNA damage. Cell Culture and Transfections—Human fibroblast cell lines AT-TERT and HFF-TERT, mouse embryo fibroblasts (MEFs) 3T3 ABL-/-, 3T3 P53-/-, and 3T3 E2F1-/-, U2OS and HEK293 cells were cultured in Dulbecco's modified Eagle's medium with heat-inactivated 10% fetal bovine serum. Human glioblastoma cell line T98G was cultured in RPMI 1640 medium with heat-inactivated 10% fetal bovine serum, sodium pyruvate, and non-essential amino acids. MEFs 3T3 ABL-/- and 3T3 P53-/- were kindly provided by Dr. J. Y. J. Wang. AT-TERT and HFF-TERT were kindly provided by Dr. J. Y. J. Wang and Dr. L. Wood. MEFs 3T3 E2F1 -/- were kindly provided by Dr. L. Yamasaki. Transfections were performed either with LipofectAMINE Plus reagent (Invitrogen) according to the manufacturer's instructions or with the standard CaPO4 method. Plasmids—The wild-type E2F1 expression vector was generated by subcloning the PCR-generated full-length E2F1 cDNA into the HApCDNA3 vector. The expression vectors pCDNA-HA-E2F1-S31/A (HA-E2F1-S/A) and pCDNA-HA-E2F1-K117,12,125R (HA-E2F1-R) were generated by PCR-mediated site-directed mutagenesis and verified by sequencing. The PMCV E2F1, the wild-type and ΔHAT (Δ 579–608) PCI P/CAF and the wild type and ΔHAT (LY-RR) PCI p300 expression vectors were kindly provided by V. Sartorelli and P. L. Puri (25Sartorelli V. Puri P.L. Hamamori Y. Ogryzko V. Chung G. Nakatani Y. Wang J.Y. Kedes L. Mol. Cell. 1999; 4: 725-734Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar). Immunoprecipitations and Immunoblotting—The following antibodies were purchased from Santa Cruz Biotechnology: anti-E2F1 (C-20), anti-hemagglutinin (HA) monoclonal (F-7) and polyclonal (Y-11), anti-green fluorescent protein (GFP) (FL), anti-actin (I-19), and the agarose-conjugate anti-HA monoclonal (F7). Monoclonal anti-acetyl-lysines were purchased from Upstate Biotechnology. Cells were lysed with RIPA lysis buffer (50 mm Tris, pH 7.6, 1% Nonidet P-40, 140 mm NaCl, 0.1% SDS), and the insoluble pellet was discarded after centrifugation. Protein concentration was determined by the BCA protein assay reagent (Pierce). Extracts were immunoprecipitated with the indicated antibodies and protein A/G plus (Santa Cruz Biotechnology). Pulse-chase—Subconfluent T98G cells in 10-cm dishes were transfected with 4 μg of either HA-E2F1-WT or with HA-E2F1-R. 12 h after transfection, cells were trypsinized and replated to normalize for the efficiency of transfection. 24 h after transfection, cells were starved for 1 h in methionine-cysteine-free medium and subsequently incubated with 200 μCi/ml of Promix (containing [35S]methionine-cysteine, Promix-Amersham) for 30 min. Medium with an excess of unlabelled methionine-cysteine (1 mg/ml) with or without doxorubicin 2 μm (Sigma) was then added, and cells were collected at the indicated times. The 35S-labeled HA-E2F1 in the anti-HA immunoprecipitate from each time point was quantified by PhosphorImager and normalized to that of the zero time point. Cycloheximide Chase—Transfected U2OS cells were seeded in 35-mm dishes. Two days after transfection, cells were treated with 2 μm doxorubicin for 8 h, and then Me2SO-solubilized cycloheximide (Sigma) was added to the culture medium at a final concentration of 50 μg/ml for the indicated times. E2F1 Is Induced in Response to DNA Damage in the AT-Tert Human ATM-defective Cell Line—Genotoxic stress has been shown to induce both phosphorylation and acetylation of E2F1 protein (19Lin W.C. Lin F.T. Nevins J.R. Genes Dev. 2001; 15: 1833-1844PubMed Google Scholar, 22Pediconi N. Ianari A. Costanzo A. Belloni L. Gallo R. Cimino L. Porcellini A. Screpanti I. Balsano C. Alesse E. Gulino A. Levrero M. Nat. Cell Biol. 2003; 5: 552-558Crossref PubMed Scopus (230) Google Scholar). To investigate the relative contribution of these two post-translational events in E2F1 stabilization induced by genotoxic stresses, we first assessed the role of the ATM/ATR pathway in E2F1 activation in response to DNA damage. To this purpose, we analyzed ATM-defective primary fibroblasts derived from a patient affected with ataxia-telangiectasia (AT) and immortalized by the stable expression of the catalytic subunit of telomerase (AT-TERT) (26Wood L.D. Halvorsen T.L. Dhar S. Baur J.A. Pandita R.K. Wright W.E. Hande M.P. Calaf G. Hei T.K. Levine F. Shay J.W. Wang J.J. Pandita T.K. Oncogene. 2001; 20: 278-288Crossref PubMed Scopus (88) Google Scholar). We found that treatment of AT-TERT cells with doxorubicin still leads to stabilization of the E2F1 protein, although to a lesser extent and with a delayed kinetics, with respect to control HFF-TERT cells (Fig. 1a). To rule out the hypothesis that the ATM and RAD3-related kinase ATR might be responsible for the delayed accumulation of E2F1 observed in our cells, we compared the kinetics of stabilization of wild-type E2F1 versus the E2F1 mutant (HA-E2F1-S/A) that cannot be phosphorylated by both ATM and ATR. We observed that exogenously expressed wild-type E2F1 was already accumulated after 1 h of doxorubicin treatment, whereas induction of the exogenously expressed HA-E2F1-S/A mutant still occurred, although it was delayed (Fig. 1b). our data that post-translational phosphorylation, might to E2F1 stabilization in cells to the doxorubicin. E2F1 in Response to Is of and for in stabilization of E2F1, we analyzed the role of Indeed, that the of the Rb pathway the activity of p53 and E2F1 with p53 to induce apoptosis by transcription and 2 p53 J.W. D. J. Cell 1999; 10: Google Scholar). In it has been shown that p53 interacts directly with the complex through the cyclin domain located in the of A inhibits E2F1 binding to p53 in vitro and the of E2F1 to with p53 to induce apoptosis in in response to DNA damage D. D.J. V. L. F. S. Mol. Cell. Biol. 2002; 22: PubMed Scopus Google Scholar). p53 of E2F1 in the apoptotic pathway induced by treatment in mouse E. D. Zhang W. M. H. D.G. Nat. Cell Biol. 2003; 5: PubMed Scopus Google Scholar). As shown in E2F1 to a extent in MEFs as compared with wild-type MEFs to either cisplatin or thus out the of p53 protein in this we analyzed the of the kinase for E2F1 stabilization in response to DNA damage. is in DNA damage In response to treatment, induces the phosphorylation of the proapoptotic p73 protein and its accumulation and apoptotic activity in response to cisplatin and doxorubicin Costanzo A. G. Kaelin Jr., W.G. Levrero M. Wang J.Y. Nature. 1999; PubMed Scopus Google Scholar, A. P. N. M. Sartorelli V. G. M. L. G. Balsano C. Levrero M. Mol. Cell. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, R. G. M. Y. Nature. 1999; PubMed Scopus Google Scholar, H. T. J. H. S. R. D. Nature. 1999; PubMed Scopus Google Scholar). As shown in E2F1 protein in response to both treatments in thus a role for this kinase in this E2F1 Is for in Response to DNA can be acetylated in vitro at lysine residues and by P/CAF to a lesser p300 (23Marzio G. Wagener C. Gutierrez M.I. Cartwright P. Helin K. Giacca M. J. Biol. Chem. 2000; 275: 10887-10892Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 24Martinez-Balbas M.A. Bauer U.M. Nielsen S.J. Brehm A. Kouzarides T. EMBO J. 2000; 19: 662-671Crossref PubMed Scopus (570) Google Scholar). of these residues leads to DNA-binding induction of its transcriptional activity, and accumulation of the doxorubicin treatment leads to the accumulation of acetylated E2F1 protein species (Fig. 22Pediconi N. Ianari A. Costanzo A. Belloni L. Gallo R. Cimino L. Porcellini A. Screpanti I. Balsano C. Alesse E. Gulino A. Levrero M. Nat. Cell Biol. 2003; 5: 552-558Crossref PubMed Scopus (230) Google Scholar), we that DNA acetylation might be responsible for the E2F1 stabilization we observed in the absence of phosphorylation. To E2F1 acetylation is required for its stabilization after genotoxic we first analyzed the of a E2F1 mutant protein (HA-E2F1-R) M.A. Bauer U.M. Nielsen S.J. Brehm A. Kouzarides T. EMBO J. 2000; 19: 662-671Crossref PubMed Scopus (570) Google Scholar). As shown in exogenously expressed wild-type E2F1 is accumulated h after doxorubicin treatment, whereas the mutant is not accumulated at at the time treatment not cellular levels of E2F1 not 24Martinez-Balbas M.A. Bauer U.M. Nielsen S.J. Brehm A. Kouzarides T. EMBO J. 2000; 19: 662-671Crossref PubMed Scopus (570) Google Scholar, C. A. D. Mol. Cell. Biol. 1999; 19: PubMed Scopus Google Scholar). we acetylation might protein in cells to The levels of exogenously expressed wild-type E2F1 and proteins were in a either in the or the absence of doxorubicin. that HA-E2F1-WT is in response to doxorubicin treatment, whereas the of the mutant these results that acetylation of E2F1 is required to stabilize the protein in response to DNA damage. for P/CAF and in E2F1 after DNA vitro studies that P/CAF is more in E2F1 with respect to However, the relative role of the two in E2F1 stability following genotoxic stress was not To this we performed to investigate the of P/CAF and p300 to complexes with E2F1 in response to DNA damage. As shown in of exogenously expressed P/CAF in MEFs treated with doxorubicin results in levels of HA-E2F1 exogenously expressed HA-E2F1 only in treated cells (Fig. P/CAF, p300 complex formation with E2F1 is not by doxorubicin treatment (Fig. To investigate the relative of P/CAF and of HAT activity in E2F1 stabilization in response to DNA we performed a cycloheximide transfected U2OS cells treated with doxorubicin. As shown in exogenously expressed P/CAF and p300 not E2F1 in Conversely, in doxorubicin-treated cells, the of E2F1 is in the of P/CAF to of E2F1 is to the activation of the P/CAF acetyltransferase activity, because the mutant versus of the acetyltransferase is to stabilize E2F1, compared with wild-type the p300 versus only E2F1 in the of whereas its mutant versus to the control vector both in treated and transcription have been identified as for P/CAF and p300/CREB-binding protein T. EMBO J. 2000; 19: PubMed Scopus Google Scholar). acetylation the transcriptional activity of p73 and p53 in response to genotoxic stress Y. Cell 2003; 10: PubMed Scopus Google Scholar). We recently demonstrated that DNA damage induces E2F1 apoptotic potential (22Pediconi N. Ianari A. Costanzo A. Belloni L. Gallo R. Cimino L. Porcellini A. Screpanti I. Balsano C. Alesse E. Gulino A. Levrero M. Nat. Cell Biol. 2003; 5: 552-558Crossref PubMed Scopus (230) Google Scholar). is by the of E2F1 from cell-cycle progression genes to the promoter of the proapoptotic p73 E2F1 into complexes that include the acetyltransferase We found that E2F1 acetylation is required in this (22Pediconi N. Ianari A. Costanzo A. Belloni L. Gallo R. Cimino L. Porcellini A. Screpanti I. Balsano C. Alesse E. Gulino A. Levrero M. Nat. Cell Biol. 2003; 5: 552-558Crossref PubMed Scopus (230) Google Scholar). Here we report that in to its transcriptional DNA acetylation of E2F1 is also important for its accumulation. Indeed, the that E2F1 is stabilized in the ATM-defective cell line AT-TERT that phosphorylation, are required to stabilize E2F1 in response to genotoxic We out a role for p53 and cAbl, two of the major transducers of in E2F1 accumulation in this We found that DNA acetylation of E2F1 is the major of its accumulation. We assessed the relative role of the two P/CAF and both reported to be in E2F1 Although E2F1 binding to p300 is by DNA the between P/CAF and E2F1 is in doxorubicin-treated only the HAT activity of P/CAF, and not that of is required to induce a of E2F1 in the of genotoxic The that to the stability of the acetylated E2F1 protein still of p53 has also been reported to induce its stabilization by its ubiquitination A. Y. J.J. Y. E. EMBO J. 2002; PubMed Scopus Google Scholar). to E2F1 is through the ubiquitin-proteasome at the in S by the kinase E2F1 for its ubiquitination and (18Vandel L. Kouzarides T. EMBO J. 1999; 18: 4280-4291Crossref PubMed Scopus (55) Google Scholar). might be that acetylation of E2F1 with the phosphorylation, thus the of E2F1 for binding to Rb also E2F1 from its ubiquitination (14Hateboer G. Kerkhoven R.M. Shvarts A. Bernards R. Beijersbergen R.L. Genes Dev. 1996; 10: 2960-2970Crossref PubMed Scopus (191) Google Scholar, 15Martelli F. Livingston D.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2858-2863Crossref PubMed Scopus (34) Google Scholar). A differential interaction of E2F1 with Rb in response to DNA damage to the is also by the of an binding for E2F1 Rb, which and is regulated by DNA damage N. Mol. Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). and the results we reported with the that Rb are regulated by acetylation as well M. Smith L. C. La Thangue N.B. Nat. Cell Biol. 2001; 3: PubMed Scopus Google Scholar), that acetylation might control interaction in response to DNA damage in this E2F1 stability and apoptotic activity. In the reported here (22Pediconi N. Ianari A. Costanzo A. Belloni L. Gallo R. Cimino L. Porcellini A. Screpanti I. Balsano C. Alesse E. Gulino A. Levrero M. Nat. Cell Biol. 2003; 5: 552-558Crossref PubMed Scopus (230) Google Scholar), that E2F1 acetylation plays a pivotal role in the recruitment of its apoptotic potential and are with a of E2F1-driven apoptotic response to In this DNA damage first acetylation of E2F1, resulting in its stabilization and formation of stable protein complexes with The levels of acetylated E2F1 are then for its recruitment onto the p73 gene promoter to the apoptotic this is in to of the or might be to the of genotoxic the of our We M. E. and G. for and of the