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Rb Inhibits E2F-1-induced Cell Death in a LXCXE-dependent Manner by Active Repression

2004; Elsevier BV; Volume: 279; Issue: 22 Linguagem: Inglês

10.1074/jbc.m309809200

ISSN

1083-351X

Autores

Vincent Pennaneach, Valérie Barbier, Karine Regazzoni, Rati Fotedar, Arun Fotedar,

Tópico(s)

RNA modifications and cancer

Resumo

Rb (retinoblastoma protein) inhibits E2F-1-induced cell death. We now show that the ability of Rb to inhibit E2F-1-induced cell death is dependent on a functional LXCXE-binding site in Rb, thereby suggesting that proteins that bind the LXCXE-binding site in Rb may regulate the anti-apoptotic activity of Rb. HDAC1, an LXCXE protein that plays a critical role in Rb-mediated transcription repression, abrogates the effect of Rb on E2F-1-induced cell death. In contrast, RF-Cp145, another LXCXE protein, cooperates with Rb to inhibit E2F-1-induced cell death. Both proteins exert their effect in an LXCXE-dependent manner. Rb regulates E2F-induced cell death by acting upstream of p73. Rb represses the p73 promoter. Our results further suggest a model in which Rb-E2F-1 complexes mediate the anti-apoptotic activity of Rb through active repression of target genes without recruiting HDAC1. Rb (retinoblastoma protein) inhibits E2F-1-induced cell death. We now show that the ability of Rb to inhibit E2F-1-induced cell death is dependent on a functional LXCXE-binding site in Rb, thereby suggesting that proteins that bind the LXCXE-binding site in Rb may regulate the anti-apoptotic activity of Rb. HDAC1, an LXCXE protein that plays a critical role in Rb-mediated transcription repression, abrogates the effect of Rb on E2F-1-induced cell death. In contrast, RF-Cp145, another LXCXE protein, cooperates with Rb to inhibit E2F-1-induced cell death. Both proteins exert their effect in an LXCXE-dependent manner. Rb regulates E2F-induced cell death by acting upstream of p73. Rb represses the p73 promoter. Our results further suggest a model in which Rb-E2F-1 complexes mediate the anti-apoptotic activity of Rb through active repression of target genes without recruiting HDAC1. The Rb tumor suppressor gene is frequently mutated in many kinds of tumors. Rb functions in part through interaction with E2F family of transcription factors. Rb represses the transcription of E2F-responsive genes by at least two mechanisms. Rb binds to E2F transcription factor family members such as E2F-1 and directly represses transcriptional activation by E2F. In addition, the Rb-E2F complex that forms at the promoter actively represses transcription by recruiting chromatin modeling enzymes (1Weintraub S.J. Prater C.A. Dean D.C. Nature. 1992; 358: 259-261Crossref PubMed Scopus (560) Google Scholar, 2Sellers W.R. Rodgers J.W. Kaelin Jr., W.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11544-11548Crossref PubMed Scopus (230) Google Scholar, 3Weintraub S.J. Chow K.N. Luo R.X. Zhang S.H. He S. Dean D.C. Nature. 1995; 375: 812-815Crossref PubMed Scopus (458) Google Scholar, 4Zhang H.S. Gavin M. Dahiya A. Postigo A.A. Ma D. Luo R.X. Harbour J.W. Dean D.C. Cell. 2000; 101: 79-89Abstract Full Text Full Text PDF PubMed Scopus (541) Google Scholar). The second mechanism is termed active repression and is invoked to explain transcriptional repression of activator proteins bound to the proximal promoter. Rb can repress transcription by recruiting class I histone deacteylases such as HDAC1 (4Zhang H.S. Gavin M. Dahiya A. Postigo A.A. Ma D. Luo R.X. Harbour J.W. Dean D.C. Cell. 2000; 101: 79-89Abstract Full Text Full Text PDF PubMed Scopus (541) Google Scholar, 5Brehm A. Miska E.A. McCance D.J. Reid J.L. Bannister A.J. Kouzarides T. Nature. 1998; 391: 597-601Crossref PubMed Scopus (1078) Google Scholar, 6Luo R.X. Postigo A.A. Dean D.C. Cell. 1998; 92: 463-473Abstract Full Text Full Text PDF PubMed Scopus (838) Google Scholar, 7Magnaghi-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 (804) Google Scholar) and other proteins like RBP1 (8Lai A. Lee J.M. Yang W.M. DeCaprio J.A. Kaelin Jr., W.G. Seto E. Branton P.E. Mol. Cell. Biol. 1999; 19: 6632-6641Crossref PubMed Scopus (142) Google Scholar, 9Lai A. Kennedy B.K. Barbie D.A. Bertos N.R. Yang X.J. Theberge M.C. Tsai S.C. Seto E. Zhang Y. Kuzmichev A. Lane W.S. Reinberg D. Harlow E. Branton P.E. Mol. Cell. Biol. 2001; 21: 2918-2932Crossref PubMed Scopus (165) Google Scholar). Histone deacetylases facilitate condensation of chromatin, thereby conferring transcriptional repression. Rb regulates cell cycle progression from the G1 to S phase and in the S phase (Refs. 10Knudsen E.S. Buckmaster C. Chen T.T. Feramisco J.R. Wang J.Y. Genes Dev. 1998; 12: 2278-2292Crossref PubMed Scopus (190) Google Scholar and 11Niculescu III, A.B. Chen X. Smeets M. Hengst L. Prives C. Reed S.I. Mol. Cell. Biol. 1998; 18: 629-643Crossref PubMed Google Scholar; and reviewed in Ref. 12Weinberg R.A. Cell. 1995; 81: 323-330Abstract Full Text PDF PubMed Scopus (4319) Google Scholar). In addition, Rb inhibits cell death induced by DNA-damaging agents (13Haas-Kogan D.A. Kogan S.C. Levi D. Dazin P. T'Ang A. Fung Y.K. Israel M.A. EMBO J. 1995; 14: 461-472Crossref PubMed Scopus (292) Google Scholar, 14Pennaneach V. Salles-Passador I. Munshi A. Brickner H. Regazzoni K. Dick F. Dyson N. Chen T.T. Wang J.Y. Fotedar R. Fotedar A. Mol. Cell. 2001; 7: 715-727Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar) or E2F-1 (15Hsieh J.K. Fredersdorf S. Kouzarides T. Martin K. Lu X. Genes Dev. 1997; 11: 1840-1852Crossref PubMed Scopus (263) Google Scholar). The demonstration that Rb null mice die at 13–15 days of gestation accompanied by increased cell death in multiple organs (16Clarke A.R. Maandag E.R. van Roon M. van der Lugt N.M. van der Valk M. Hooper M.L. Berns A. te Riele H. Nature. 1992; 359: 328-330Crossref PubMed Scopus (896) Google Scholar, 17Jacks T. Fazeli A. Schmitt E.M. Bronson R.T. Goodell M.A. Weinberg R.A. Nature. 1992; 359: 295-300Crossref PubMed Scopus (1520) Google Scholar, 18Lee E.Y. Chang C.Y. Hu N. Wang Y.C. Lai C.C. Herrup K. Lee W.H. Bradley A. Nature. 1992; 359: 288-294Crossref PubMed Scopus (1126) Google Scholar) suggests that Rb plays a critical role in regulating apoptosis. Inactivation of the E2F-1 gene in Rb null embryos partially rescues the apoptosis seen in Rb -/- embryos (19Tsai K.Y. Hu Y. Macleod K.F. Crowley D. Yamasaki L. Jacks T. Mol. Cell. 1998; 2: 293-304Abstract Full Text Full Text PDF PubMed Scopus (319) Google Scholar) and implicates E2F-1 as a critical mediator of apoptosis observed in Rb -/- embryos. The AB pocket residues 379–772 of Rb are highly conserved through evolution. The majority of germ line mutations found in hereditary retinoblastoma and most tumor-derived mutations in the Rb gene map to the AB pocket. Viral oncoproteins also bind to the AB pocket and inactivate Rb (20Templeton D.J. Park S.H. Lanier L. Weinberg R.A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3033-3037Crossref PubMed Scopus (171) Google Scholar, 21Hu Q.J. Dyson N. Harlow E. 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Brickner H. Regazzoni K. Dick F. Dyson N. Chen T.T. Wang J.Y. Fotedar R. Fotedar A. Mol. Cell. 2001; 7: 715-727Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar), contain an LXCXE motif. The three-dimensional crystal structure of the AB pocket of Rb interacting with the LXCXE peptide revealed residues in Rb that contact the LXCXE peptide (25Lee J.O. Russo A.A. Pavletich N.P. Nature. 1998; 391: 859-865Crossref PubMed Scopus (380) Google Scholar). Accordingly, mutation(s) of these contact residues suppressed the ability of Rb to bind LXCXE-containing viral proteins. These Rb mutants retain the ability to bind E2F, repress E2F-1-dependent transcription, and inhibit growth comparable with wild type Rb (26Chen T.-T. Wang J.Y.J. Mol. Cell. Biol. 2000; 20: 5571-5580Crossref PubMed Scopus (64) Google Scholar, 27Dick F.A. Sailhamer E. Dyson N.J. Mol. Cell. Biol. 2000; 20: 3715-3727Crossref PubMed Scopus (105) Google Scholar) but fail to protect cells from apoptosis (14Pennaneach V. Salles-Passador I. Munshi A. Brickner H. Regazzoni K. Dick F. Dyson N. Chen T.T. Wang J.Y. Fotedar R. Fotedar A. Mol. Cell. 2001; 7: 715-727Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). The transcription factor E2F-1 induces both cell cycle progression and apoptosis. E2F-1-induced cell death acts via two parallel pathways. In the p53-dependent pathway, E2F activates p19ARF transcription. p19ARF binds MDM2 and relieves p53 from MDM2-mediated degradation (28Zhang Y. Xiong Y. Yarbrough W.G. Cell. 1998; 92: 725-734Abstract Full Text Full Text PDF PubMed Scopus (1410) Google Scholar, 29Pomerantz J. Schreiber-Agus N. Liegeois N.J. Silverman A. Alland L. Chin L. Potes J. Chen K. Orlow I. Lee H.W. Cordon-Cardo C. DePinho R.A. Cell. 1998; 92: 713-723Abstract Full Text Full Text PDF PubMed Scopus (1339) Google Scholar). These events thus enable p53 to induce cell death. This pathway is therefore impaired by inactivation of either the INK4a or the p53 locus. The second pathway is p53-independent and has been suggested to act via the p53-related gene p73 (30Irwin M. Marin M.C. Phillips A.C. Seelan R.S. Smith D.I. Liu W. Flores E.R. Tsai K.Y. Jacks T. Vousden K.H. Kaelin Jr., W.G. Nature. 2000; 407: 645-648Crossref PubMed Scopus (537) Google Scholar). E2F-1 transactivates the p73 gene (31Jost C.A. Marin M.C. Kaelin Jr., W.G. Nature. 1997; 389: 191-194Crossref PubMed Scopus (903) Google Scholar). Disruption of p73 function impairs E2F-1-induced apoptosis in p53-/- cells (30Irwin M. Marin M.C. Phillips A.C. Seelan R.S. Smith D.I. Liu W. Flores E.R. Tsai K.Y. Jacks T. Vousden K.H. Kaelin Jr., W.G. Nature. 2000; 407: 645-648Crossref PubMed Scopus (537) Google Scholar), thus suggesting that activation of p73 is required for p53-independent E2F-1-induced cell death. We have previously shown that the LXCXE interaction is critical for the ability of Rb to inhibit cell death induced by a variety of DNA-damaging agents (14Pennaneach V. Salles-Passador I. Munshi A. Brickner H. Regazzoni K. Dick F. Dyson N. Chen T.T. Wang J.Y. Fotedar R. Fotedar A. Mol. Cell. 2001; 7: 715-727Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Proteins that bind the LXCXE-binding site in Rb would be expected to regulate the anti-apoptotic activity of Rb. Indeed, we have shown that RF-Cp145, an LXCXE-containing protein, cooperates with Rb to mediate cell survival after DNA damage. In this paper we have investigated the mechanism of Rb-mediated inhibition of E2F-1-induced cell death. We find that in contrast to the wild type Rb, which promotes cell survival, the LXCXE nonbinding N757F Rb mutant fails to inhibit E2F-1-induced cell death. Unlike RF-Cp145, HDAC1, which also interacts with the LX-CXE-binding site in Rb, does not cooperate with Rb to inhibit E2F-1-induced cell death. In contrast, HDAC1 inhibits Rb-mediated cell survival, which is independent of its deacetylase activity but dependent on the IXCXE motif in HDAC1. Interestingly, Rb-E2F-1 complexes mediate the anti-apoptotic activity of Rb through active repression without recruiting HDAC1. Plasmids, Cell Culture, and Transfection—The mammalian expression vectors for wild type Rb, the N757F Rb mutant, RF-Cp145, BRG1, and HDAC1 have been described (14Pennaneach V. Salles-Passador I. Munshi A. Brickner H. Regazzoni K. Dick F. Dyson N. Chen T.T. Wang J.Y. Fotedar R. Fotedar A. Mol. Cell. 2001; 7: 715-727Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). The H141A HDAC1 mutant was generated by site-specific mutagenesis by standard methods, and the mutation was verified by sequencing of the entire coding sequence of the cDNA. The HDAC1 ΔIXCXE mutant has been described (7Magnaghi-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 (804) Google Scholar). The mammalian expression vectors of p73α, p73β, the p73 dominant negative mutant (p73 DNM) and point mutant of the p73 dominant negative mutant (p73 DNM L-P) were as described (30Irwin M. Marin M.C. Phillips A.C. Seelan R.S. Smith D.I. Liu W. Flores E.R. Tsai K.Y. Jacks T. Vousden K.H. Kaelin Jr., W.G. Nature. 2000; 407: 645-648Crossref PubMed Scopus (537) Google Scholar). The mammalian expression vector of N-terminal DNA-binding domain of E2F-1 (1–368) and the chimeric E2F-1/Rb protein (N-terminal DNA-binding domain of E2F-1 (1–368), and the pocket domain of Rb (379–792)) have been described (2Sellers W.R. Rodgers J.W. Kaelin Jr., W.G. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11544-11548Crossref PubMed Scopus (230) Google Scholar, 32Sellers W.R. Novitch B.G. Miyake S. Heith A. Otterson G.A. Kaye F.J. Lassar A.B. Kaelin Jr., W.G. Genes Dev. 1998; 12: 95-106Crossref PubMed Scopus (288) Google Scholar). The N757F mutant of the E2F-1/Rb chimeric protein was generated by PCR, and its sequence was verified by sequencing. C33A cells (Rb null human cervical carcinoma) from ATCC were maintained in Dulbecco's modified Eagle's medium complemented with 10% fetal bovine serum. C33A cells were transfected using the calcium phosphate transfection method. The cells co-transfected with puroB-ABE vector (where indicated) were selected with 2.5 μg/ml of puromycin for 3 days as described previously (14Pennaneach V. Salles-Passador I. Munshi A. Brickner H. Regazzoni K. Dick F. Dyson N. Chen T.T. Wang J.Y. Fotedar R. Fotedar A. Mol. Cell. 2001; 7: 715-727Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Survival Assay—The cells were transfected with E2F and other vectors. The calcium phosphate precipitates were washed off 24 h later, the cells were washed, and fresh medium was added, and cell survival was determined 17 h later. Cell survival was measured 17 h after UV irradiation. In all survival assays, the cells were co-transfected with β-galactosidase expression vector. The cells were then fixed with 0.1% glutaraldehyde and incubated for 12 h in the presence of 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-gal; 1 mg/ml) to detect β-galactosidase activity as described (14Pennaneach V. Salles-Passador I. Munshi A. Brickner H. Regazzoni K. Dick F. Dyson N. Chen T.T. Wang J.Y. Fotedar R. Fotedar A. Mol. Cell. 2001; 7: 715-727Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). The relative number of surviving cells in the presence of the appropriate transfected vector was determined by counting the number of blue cells in >200 nonoverlapping fields in the presence or absence of transfected E2F-1. We compared the number of surviving cells in the absence or presence of E2F-1 for each transfection condition to determine the percentage of survival (Supplementary Figure III). The data shown (means ± S.D.) are derived from five different experiments done at different times. Antibodies, Immunoblotting, and Immunoprecipitation—Anti-pRb antibody (IF-8) (Santa Cruz Biotechnology), anti-HA 1The abbreviation used is: HA, hemagglutinin. antibody (BabCo), anti-E2F-1 antibody (Santa Cruz), and a polyclonal Rb specific antibody 851 (33Welch P.J. Wang J.Y. Cell. 1993; 75: 779-790Abstract Full Text PDF PubMed Scopus (368) Google Scholar) were used. Whole cell extracts were prepared in buffer containing 20 mm Tris, pH 7.5, 150 mm NaCl, 10 mm EDTA, 1% Nonidet P-40, and protease inhibitors. The proteins were separated on 7.5% polyacrylamide gels and detected by immunoblotting using the ECL detection reagent (Amersham Biosciences). The LXCXE-binding Site in Rb Is Essential for Rb-mediated Inhibition of E2F-1-induced Cell Death—To investigate the role of the LXCXE-binding site in Rb in regulating E2F-1-dependent cell death, we utilized the β-galactosidase assay (14Pennaneach V. Salles-Passador I. Munshi A. Brickner H. Regazzoni K. Dick F. Dyson N. Chen T.T. Wang J.Y. Fotedar R. Fotedar A. Mol. Cell. 2001; 7: 715-727Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). In this assay, the cells are co-transfected with a vector expressing β-galactosidase, and the percentage of surviving β-galactosidase-positive cells (blue cells) is determined (Fig. 1D). We compared the number of surviving cells in the absence or presence of E2F-1 for each transfection condition to determine the percentage of survival. The data shown (means ± S.D.) are derived from five different experiments done at different times. Transfection of the Rb-deficient C33A cell line with vectors expressing E2F-1 induces cell death. Increasing amounts of wild type Rb inhibit E2F-1-induced cell death in a dose-dependent manner (Fig. 1A). In contrast, the LXCXE nonbinding N757F Rb mutant (hereafter referred to as Rb N757F mutant) fails to inhibit E2F-1-induced cell death (Fig. 1A). Increasing amounts of E2F-1 were also less efficient at inducing cell death in the presence of Rb as compared with the Rb N757F mutant (Fig. 1B). The selective inhibition of E2F-1-induced cell death by wild type Rb but not the Rb N757F mutant is not due to an effect on E2F-1 protein levels (Fig. 1C). Both wild type Rb and the Rb N757F mutant stabilize E2F-1 protein levels with similar efficacy (Fig. 1C) as shown previously (34Campanero M.R. Flemington E.K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2221-2226Crossref PubMed Scopus (183) Google Scholar, 35Hofmann F. Martelli F. Livingston D.M. Wang Z. Genes Dev. 1996; 10: 2949-2959Crossref PubMed Scopus (214) Google Scholar, 36Hateboer G. Kerkhoven R.M. Shvarts A. Bernards R. Beijersbergen R.L. Genes Dev. 1996; 10: 2960-2970Crossref PubMed Scopus (191) Google Scholar). Rb Does Not Inhibit p73-induced Cell Death—p73 is an inducer of cell death in many cell lines (30Irwin M. Marin M.C. Phillips A.C. Seelan R.S. Smith D.I. Liu W. Flores E.R. Tsai K.Y. Jacks T. Vousden K.H. Kaelin Jr., W.G. Nature. 2000; 407: 645-648Crossref PubMed Scopus (537) Google Scholar). It has been suggested that E2F-1-induced cell death in p53 -/- cells is mediated in a p73-dependent manner (30Irwin M. Marin M.C. Phillips A.C. Seelan R.S. Smith D.I. Liu W. Flores E.R. Tsai K.Y. Jacks T. Vousden K.H. Kaelin Jr., W.G. Nature. 2000; 407: 645-648Crossref PubMed Scopus (537) Google Scholar). Because C33A cells lack p53, we determined whether Rb inhibits E2F-1-induced cell death by regulating the ability of p73 to induce cell death. We first determined whether E2F-1-induced cell death in C33A cells is p73-dependent. We used a dominant negative mutant of p73 (p73 DNM) that selectively inhibits p73-mediated transactivation but not p53-dependent transactivation (30Irwin M. Marin M.C. Phillips A.C. Seelan R.S. Smith D.I. Liu W. Flores E.R. Tsai K.Y. Jacks T. Vousden K.H. Kaelin Jr., W.G. Nature. 2000; 407: 645-648Crossref PubMed Scopus (537) Google Scholar). This p73 DNM inhibited E2F-1-induced cell death in C33A cells (Fig. 2A). A point mutant of the p73 dominant negative mutant (p73 DNM L-P) had no effect on E2F-1-induced cell death in C33A cells (Fig. 2A). The inhibition of E2F-1-induced cell death by p73 DNM is not due to its effect on E2F-1 protein levels (Fig. 2D). Because p73 has been shown to induce cell death (30Irwin M. Marin M.C. Phillips A.C. Seelan R.S. Smith D.I. Liu W. Flores E.R. Tsai K.Y. Jacks T. Vousden K.H. Kaelin Jr., W.G. Nature. 2000; 407: 645-648Crossref PubMed Scopus (537) Google Scholar), we determined whether Rb inhibits p73α- or p73β-induced cell death. Rb did not inhibit p73α-induced (Fig. 2B) or p73β-induced (Fig. 2C) cell death in C33A cells. Rb had no effect on p73 protein levels (Fig. 2E). Because E2F-1 activates the p73 promoter (Ref. 30Irwin M. Marin M.C. Phillips A.C. Seelan R.S. Smith D.I. Liu W. Flores E.R. Tsai K.Y. Jacks T. Vousden K.H. Kaelin Jr., W.G. Nature. 2000; 407: 645-648Crossref PubMed Scopus (537) Google Scholar and data not shown), our results suggest that Rb and E2F regulate cell death by acting upstream of p73. Effect of HDAC1, BRG1, and RF-Cp145 on Rb-mediated Inhibition of E2F-induced Cell Death—Our demonstration that a functional LXCXE-binding site is required for Rb to inhibit E2F-1-induced cell death prompted us to investigate whether cellular LXCXE proteins such as HDAC1 (7Magnaghi-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 (804) Google Scholar), BRG1 (37Muchardt C. Reyes J.C. Bourachot B. Leguoy E. Yaniv M. EMBO J. 1996; 15: 3394-3402Crossref PubMed Scopus (194) Google Scholar), or RF-Cp145 (14Pennaneach V. Salles-Passador I. Munshi A. Brickner H. Regazzoni K. Dick F. Dyson N. Chen T.T. Wang J.Y. Fotedar R. Fotedar A. Mol. Cell. 2001; 7: 715-727Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar) cooperate with Rb to inhibit E2F-1-induced cell death. HDAC1, a histone deacetylase, contains an LXCXE motif that is essential to bind and cooperate with Rb in mediating optimal transcriptional repression of promoters containing E2F-binding sites (7Magnaghi-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 (804) Google Scholar). BRG1, a member of the SW12/SNF2 family of chromatin remodeling ATPases contains an LXCXE motif, binds Rb (4Zhang H.S. Gavin M. Dahiya A. Postigo A.A. Ma D. Luo R.X. Harbour J.W. Dean D.C. Cell. 2000; 101: 79-89Abstract Full Text Full Text PDF PubMed Scopus (541) Google Scholar, 24Dunaief J.L. Strober B.E. Guha S. Khavari P.A. Alin K. Luban J. Begemann M. Crabtree G.R. Goff S.P. Cell. 1994; 79: 119-130Abstract Full Text PDF PubMed Scopus (557) Google Scholar), and is required for Rb to inhibit cell cycle progression. The large subunit of RF-C (RF-Cp145) enhances cell survival after DNA damage in Rb null cells in a strict Rb- and LXCXE-dependent manner (14Pennaneach V. Salles-Passador I. Munshi A. Brickner H. Regazzoni K. Dick F. Dyson N. Chen T.T. Wang J.Y. Fotedar R. Fotedar A. Mol. Cell. 2001; 7: 715-727Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). C33A cells, in addition to lacking Rb, lack BRG1 and functional Rb-HDAC1 complexes and have been used to clarify the role of BRG1 and HDAC1 in Rb function (4Zhang H.S. Gavin M. Dahiya A. Postigo A.A. Ma D. Luo R.X. Harbour J.W. Dean D.C. Cell. 2000; 101: 79-89Abstract Full Text Full Text PDF PubMed Scopus (541) Google Scholar). RF-Cp145, the large subunit of RF-C, enhances the ability of Rb to inhibit E2F-1-induced cell death in a dose-dependent manner (Fig. 3A). In the absence of Rb, RF-Cp145 by itself was unable to inhibit E2F-1-induced cell death (Fig. 3A), thus showing that RF-Cp145 enhances cell survival only in the presence of Rb. BRG1 on the other hand had no significant effect in either enhancing or inhibiting the ability of Rb to inhibit E2F-1-induced cell death (Fig. 3A). BRG1 also had no effect on E2F-1-induced cell death in the absence of Rb (Fig. 3A). In contrast, HDAC1 abrogates the ability of Rb to inhibit E2F-1-induced cell death in a dose-dependent fashion but had no effect on E2F-1-induced cell death in the absence of Rb (Fig. 3A). BRG1, HDAC1, and RF-Cp145 had no effect on E2F-1 or Rb protein levels (Fig. 3B). These data show that the three LXCXE containing cellular proteins we tested had distinct effects on Rb-mediated inhibition of E2F-1-induced apoptosis. We then determined whether these distinct effects of LXCXE-containing cellular proteins on Rb-mediated inhibition of E2F-1-induced cell death was due to their effect on transcriptional repression. Because E2F-1-induced cell death is p73-dependent (Fig. 2A), we used the p73 promoter (-883 to +77) driving the expression of a luciferase reporter to monitor repression for these studies. This p73 promoter has been used to show that E2F activates the p73 promoter (30Irwin M. Marin M.C. Phillips A.C. Seelan R.S. Smith D.I. Liu W. Flores E.R. Tsai K.Y. Jacks T. Vousden K.H. Kaelin Jr., W.G. Nature. 2000; 407: 645-648Crossref PubMed Scopus (537) Google Scholar). As expected Rb repressed p73 promoter activity (Fig. 2F). We find that RF-Cp145 cooperates with Rb to mediate transcriptional repression, whereas HDAC1 does not (Fig. 2F). We find that RF-Cp145 has no effect on reporter activity in the absence of Rb (Fig. 2F, right panel). These results suggest that the effects of HDAC1 and RF-Cp145 on cell death are probably mediated via effects on Rb-mediated transcriptional repression. HDAC1 Inhibits the Ability of Rb to Promote Cell Survival in a LXCXE Motif-dependent Manner—To further investigate the effect of HDAC1 on cell death, we compared the ability of wild type HDAC1 and its deacetylase mutant to abrogate Rb mediated inhibition of E2F-1-induced cell death in C33A cells. The removal of acetyl groups from the tails of histone octamers by HDAC1 facilitates condensation of nucleosomes into chromatin, which in turn blocks access of transcription factors, leading to gene repression. The three-dimensional crystal structure of the HDAC1 active site has been resolved (38Finnin M.S. Donigian J.R. Cohen A. Richon V.M. Rifkind R.A. Marks P.A. Breslow R. Pavletich N.P. Nature. 1999; 401: 188-193Crossref PubMed Scopus (1509) Google Scholar). Based on these studies the H141A HDAC1 mutant (H141A) was shown to lack deacetylase activity (39Hassig C.A. Tong J.K. Fleischer T.C. Owa T. Grable P.G. Ayer D.E. Schreiber S.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3519-3524Crossref PubMed Scopus (332) Google Scholar). We find that both wild type HDAC1 and the H141A HDAC1 deacetylase mutant are equally effective at abrogating the ability of Rb to inhibit E2F-1-mediated cell death in a dose-dependent manner (Fig. 4A). The ability of HDAC1 to regulate Rb-mediated inhibition of E2F1-induced cell death is therefore not dependent on its catalytic activity. We next determined whether the IXCXE motif in HDAC1 is required for HDAC1 to abrogate the ability of Rb to inhibit E2F1-induced apoptosis. An HDAC1 mutant in which the IXCXE motif has been deleted (termed HDAC1 ΔIXCXE mutant) has a dramatically reduced ability to bind Rb (7Magnaghi-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 (804) Google Scholar). We therefore compared the ability of wild type HDAC1 with the HDAC1 ΔIXCXE mutant to abrogate Rb-mediated inhibition of E2F-1-induced cell death. We find that although wild type HDAC1 abrogates the ability of Rb to inhibit E2F-1-induced cell death, the HDAC1 ΔIXCXE mutant does not (Fig. 4A). We show that RF-Cp145 cooperates with Rb to inhibit E2F-1-induced cell death (Fig. 3). We therefore ascertained whether HDAC1 also abrogates the ability of Rb+RF-p145 to inhibit E2F-1-induced cell death. Indeed both wild type HDAC1 and the H141A HDAC1 mutant abrogate the ability of Rb+RF-Cp145 to inhibit E2F-1-induced cell death in a dose-dependent manner (Fig. 4A). In contrast, the HDAC1 ΔIXCXE mutant does not influence cell survival by Rb+RF-Cp145 (Fig. 4A). From these studies we conclude that the IXCXE motif in HDAC1 is required for HDAC1 to abrogate the ability of Rb to inhibit E2F-1-induced cell death. RF-Cp145 Enhances Rb-mediated Inhibition of E2F-induced Death in a LXCXE Motif- and ATPase-dependent Manner—We find that RF-Cp145 did not inhibit E2F-1-induced death in the absence of transfected Rb in Rb -/- C33A cells but enhances in a dose-dependent manner the ability of co-transfected Rb to inhibit E2F-1-induced apoptosis (Fig. 5). RF-Cp145 cotransfected with Rb N757F mutant, does not inhibit E2F-1-induced cell death (Fig. 5). The LXGXK RF-Cp145 mutant, in which the LXCXE motif has been mutated to LXGXK, was unable to cooperate with Rb to inhibit E2F-1-induced cell death (Fig. 5). The LXGXK RF-Cp145 mutant as expected also did not inhibit E2F-1-induced cell death in the absence of co-transfected Rb or the Rb N757F mutant (Fig. 5). These results suggest that both the LXCXE motif in RF-Cp145 and the LXCXE-binding site in Rb are required for inhibition of E2F-1-induced cell death. The RF-C complex has been known to have ATPase activity that is stimulated in the presence of other co-factors like DNA (40Cai J. Gibbs E. Uhlmann F. Phillips B. Yao N. O'Donnell M. Hurwitz J. J. Biol. Chem. 1997; 272: 18974-18981Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 41Cai J. Yao N. Gibbs E. Finkelstein J. Phillips B. O'Donnell M. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11607-11612Crossref PubMed Scopus (44) Google Scholar, 42Podust V.N. Tiwari N. Ott R. Fanning E. J. Biol. Chem. 1998; 273: 12935-12942Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 43Ellison V. Stillman B. J. Biol. Chem. 1998; 273: 5979-5987Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). The ability to assemble recombinant RF-C subunits in vitro into active RF-C complexes has allowed a characterization of mutants that selectively affect ATPase activity of RF-C. The RF-Cp145 Lys657 residue, which maps to the conserved Walker A motif, is essential for the ATPase activity of the RF-C complex (41Cai J. Yao N. Gibbs E. Finkelstein J. Phillips B. O'Donnell M. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A.

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