Title: The Proapoptotic Gene SIVA Is a Direct Transcriptional Target for the Tumor Suppressors p53 and E2F1
Abstract: The p53 tumor suppressor gene is believed to play an important role in neuronal cell death in acute neurological disease and in neurodegeneration. The p53 signaling cascade is complex, and the mechanism by which p53 induces apoptosis is cell type-dependent. Using DNA microarray analysis, we have found a striking induction of the proapoptotic gene, SIVA. SIVA is a proapoptotic protein containing a death domain and interacts with members of the tumor necrosis factor receptor family as well as anti-apoptotic Bcl-2 family proteins. SIVA is induced following direct p53 gene delivery, treatment with a DNA-damaging agent camptothecin, and stroke injury in vivo. SIVA up-regulation is sufficient to initiate the apoptotic cascade in neurons. Through isolation and analysis of the SIVA promoter, we have identified response elements for both p53 and E2F1. Like p53, E2F1 is another tumor suppressor gene involved in the regulation of apoptosis, including neuronal injury models. We have identified E2F consensus sites in the promoter region, whereas p53 recognition sequences were found in intron1. Sequence analysis has shown that these consensus sites are also conserved between mouse and human SIVA genes. Electrophoretic mobility shift assays reveal that both transcription factors are capable of binding to putative consensus sites, and luciferase reporter assays reveal that E2F1 and p53 can activate transcription from the SIVA promoter. Here, we report that the proapoptotic gene, SIVA, which functions in a broad spectrum of cell types, is a direct transcriptional target for both tumor suppressors, p53 and E2F1. The p53 tumor suppressor gene is believed to play an important role in neuronal cell death in acute neurological disease and in neurodegeneration. The p53 signaling cascade is complex, and the mechanism by which p53 induces apoptosis is cell type-dependent. Using DNA microarray analysis, we have found a striking induction of the proapoptotic gene, SIVA. SIVA is a proapoptotic protein containing a death domain and interacts with members of the tumor necrosis factor receptor family as well as anti-apoptotic Bcl-2 family proteins. SIVA is induced following direct p53 gene delivery, treatment with a DNA-damaging agent camptothecin, and stroke injury in vivo. SIVA up-regulation is sufficient to initiate the apoptotic cascade in neurons. Through isolation and analysis of the SIVA promoter, we have identified response elements for both p53 and E2F1. Like p53, E2F1 is another tumor suppressor gene involved in the regulation of apoptosis, including neuronal injury models. We have identified E2F consensus sites in the promoter region, whereas p53 recognition sequences were found in intron1. Sequence analysis has shown that these consensus sites are also conserved between mouse and human SIVA genes. Electrophoretic mobility shift assays reveal that both transcription factors are capable of binding to putative consensus sites, and luciferase reporter assays reveal that E2F1 and p53 can activate transcription from the SIVA promoter. Here, we report that the proapoptotic gene, SIVA, which functions in a broad spectrum of cell types, is a direct transcriptional target for both tumor suppressors, p53 and E2F1. The p53 tumor suppressor gene is believed to play an important role in neuronal cell death in acute neurological disease (1Li Y. Chop M. Zhang Z.G. Zaloga C. Niewenhuis L. Gautam S. Stroke. 1994; 25 (discussion 855-846): 849-855Crossref PubMed Scopus (173) Google Scholar, 2Sakhi S. Bruce A. Sun N. Tocco G. Baudry M. Schreiber S.S. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7525-7529Crossref PubMed Scopus (248) Google Scholar, 3McGahan L. Hakim A.M. Robertson G.S. Brain Res. Mol. Brain Res. 1998; 56: 133-145Crossref PubMed Scopus (81) Google Scholar, 4Banasiak K.J. Haddad G.G. 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Neurosci. 1999; 19: 6818-6824Crossref PubMed Google Scholar) and that forced up-regulation of p53 alone is sufficient to cause neuronal cell death (5Xiang H. Hochman D.W. Saya H. Fujiwara T. Schwartzkroin P.A. Morrison R.S. J. Neurosci. 1996; 16: 6753-6765Crossref PubMed Google Scholar, 9Slack R.S. Belliveau D.J. Rosenberg M. Atwal J. Lochmuller H. Aloyz R. Haghighi A. Lach B. Seth P. Cooper E. Miller F.D. J. Cell Biol. 1996; 135: 1085-1096Crossref PubMed Scopus (114) Google Scholar, 10Miller F.D. Pozniak C.D. Walsh G.S. Cell Death Differ. 2000; 7: 880-888Crossref PubMed Scopus (174) Google Scholar, 11Johnson M.D. Kinoshita Y. Xiang H. Ghatan S. Morrison R.S. J. Neurosci. 1999; 19: 2996-3006Crossref PubMed Google Scholar). Consistent with this, mice carrying a p53 null mutation exhibit reduced brain damage following excitotoxicity and stroke (5Xiang H. Hochman D.W. Saya H. Fujiwara T. Schwartzkroin P.A. Morrison R.S. J. Neurosci. 1996; 16: 6753-6765Crossref PubMed Google Scholar, 6Morrison R.S. Wenzel H.J. Kinoshita Y. Robbins C.A. Donehower L.A. Schwartzkroin P.A. J. Neurosci. 1996; 16: 1337-1345Crossref PubMed Google Scholar, 12Crumrine R.C. Thomas A.L. Morgan P.F. J. Cereb. Blood Flow Metab. 1994; 14: 887-891Crossref PubMed Scopus (275) Google Scholar). Finally, recent studies have shown that p53-blocking peptides are neuroprotective following acute brain injury and may serve as potential therapeutic agents (13Culmsee C. Zhu X. Yu Q.S. Chan S.L. Camandola S. Guo Z. Greig N.H. Mattson M.P. J. Neurochem. 2001; 77: 220-228Crossref PubMed Scopus (293) Google Scholar). Taken together, these studies demonstrate the importance of p53 as a key apoptotic factor following neuronal injury and underscores the necessity to uncover the mechanisms by which p53 induces the death of postmitotic neurons.The p53 signaling cascade is complex, and it has become increasingly clear that the mechanisms by which p53 induces apoptosis vary depending on the tissue type (reviewed by Prives and Hall (14Prives C. Hall P.A. J. Pathol. 1999; 187: 112-126Crossref PubMed Scopus (1225) Google Scholar)). For example, recent studies have demonstrated that, in certain cell types, p53 can induce apoptosis exclusively at the mitochondrial level through direct interaction with Bcl-2 family proteins (15Mihara M. Erster S. Zaika A. Petrenko O. Chittenden T. Pancoska P. Moll U.M. Mol. Cell. 2003; 11: 577-590Abstract Full Text Full Text PDF PubMed Scopus (1450) Google Scholar). In contrast, other cell types such as postmitotic neurons exposed to DNA-damaging agents require the transcriptional activation domain of p53 for death to occur. 1S. P. Cregan, N. A. Arbour, J. G. MacLaurin, S. M. Callaghan, A. Fortin, E. C. C. Cheung, D. S. Park, and R. S. Slack, unpublished observation. 1S. P. Cregan, N. A. Arbour, J. G. MacLaurin, S. M. Callaghan, A. Fortin, E. C. C. Cheung, D. S. Park, and R. S. Slack, unpublished observation. In postmitotic neurons, we and others (16Xiang H. Kinoshita Y. Knudson C.M. Korsmeyer S.J. Schwartzkroin P.A. Morrison R.S. J. Neurosci. 1998; 18: 1363-1373Crossref PubMed Google Scholar, 17Cregan S.P. MacLaurin J.G. Craig C.G. Robertson G.S. Nicholson D.W. Park D.S. Slack R.S. J. Neurosci. 1999; 19: 7860-7869Crossref PubMed Google Scholar, 18Keramaris E. Stefanis L. MacLaurin J. Harada N. Takaku K. Ishikawa T. Taketo M.M. Robertson G.S. Nicholson D.W. Slack R.S. Park D.S. Mol. Cell. Neurosci. 2000; 15: 368-379Crossref PubMed Scopus (84) Google Scholar) have demonstrated that p53-mediated cell death involves a Bax-dependent, caspase3 activation involving induction of APAF1. In proliferating cells, Bax was shown to be a direct target for p53 (19Miyashita T. Reed J.C. Cell. 1995; 80: 293-299Abstract Full Text PDF PubMed Scopus (303) Google Scholar), however no significant p53-mediated Bax up-regulation was found in neurons (16Xiang H. Kinoshita Y. Knudson C.M. Korsmeyer S.J. Schwartzkroin P.A. Morrison R.S. J. Neurosci. 1998; 18: 1363-1373Crossref PubMed Google Scholar, 17Cregan S.P. MacLaurin J.G. Craig C.G. Robertson G.S. Nicholson D.W. Park D.S. Slack R.S. J. Neurosci. 1999; 19: 7860-7869Crossref PubMed Google Scholar, 20Johnson M.D. Xiang H. London S. Kinoshita Y. Knudson M. Mayberg M. Korsmeyer S.J. Morrison R.S. J. Neurosci. Res. 1998; 54: 721-733Crossref PubMed Scopus (99) Google Scholar). Although we have found that p53 transcriptional activity is essential for the induction of neuronal cell death, the downstream targets responsible for Bax activation remain unknown. To identify the regulatory targets by which p53 induces neuronal cell death, we conducted DNA microarray analysis using postmitotic neurons undergoing p53-mediated apoptosis. Using this approach, we and others (21Moroni M.C. Hickman E.S. Denchi E.L. Caprara G. Colli E. Cecconi F. Muller H. Helin K. Nat. Cell Biol. 2001; 3: 552-558Crossref PubMed Scopus (530) Google Scholar, 22Cregan S.P. Fortin A. MacLaurin J.G. Callaghan S.M. Cecconi F. Yu S.W. Dawson T.M. Dawson V.L. Park D.S. Kroemer G. Slack R.S. J. Cell Biol. 2002; 158: 507-517Crossref PubMed Scopus (424) Google Scholar, 23Fortin A. Cregan S.P. MacLaurin J.G. Kushwaha N. Hickman E.S. Thompson C.S. Hakim A. Albert P.R. Cecconi F. Helin K. Park D.S. Slack R.S. J. Cell Biol. 2001; 155: 207-216Crossref PubMed Scopus (184) Google Scholar) have previously identified APAF1 as a key intermediate in the apoptotic signaling cascade that is directly activated by p53. We have now used DNA microarray analysis to identify p53 target genes involved in neuronal injury and have consistently found a striking induction of the proapoptotic gene, SIVA.SIVA is a proapoptotic protein that was originally identified through its association with the cytoplasmic tail of CD27, a member of the tumor necrosis factor receptor (TNFR) 2The abbreviations used are: TNFR, tumor necrosis factor receptor; GFP, green fluorescent protein; m.o.i., multiplicity of infection; Ad, adenovirus; RT, reverse transcription; EMSA, electrophoretic mobility shift assay; CMV, cytomegalovirus; DM, double transactivation mutant. 2The abbreviations used are: TNFR, tumor necrosis factor receptor; GFP, green fluorescent protein; m.o.i., multiplicity of infection; Ad, adenovirus; RT, reverse transcription; EMSA, electrophoretic mobility shift assay; CMV, cytomegalovirus; DM, double transactivation mutant. superfamily (24Gravestein L.A. Blom B. Nolten L.A. de Vries E. van der Horst G. 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A. 1997; 94: 6346-6351Crossref PubMed Scopus (250) Google Scholar).In addition to its interaction with TNFR superfamily members, SIVA has also been shown to interact with anti-apoptotic Bcl-2 family members (33Xue L. Chu F. Cheng Y. Sun X. Borthakur A. Ramarao M. Pandey P. Wu M. Schlossman S.F. Prasad K.V. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 6925-6930Crossref PubMed Scopus (67) Google Scholar). There are two SIVA splice variants, SIVA-1 and SIVA-2, of which SIVA-1 retains exon2, which is believed to be critical for apoptotic activity (34Yoon Y. Ao Z. Cheng Y. Schlossman S.F. Prasad K.V. Oncogene. 1999; 18: 7174-7179Crossref PubMed Scopus (44) Google Scholar), although a recent study suggests that both splice forms can induce apoptosis in a similar fashion (35Py B. Slomianny C. Auberger P. Petit P.X. Benichou S. J. Immunol. 2004; 172: 4008-4017Crossref PubMed Scopus (67) Google Scholar). Exon2 is comprised of an amphipathic helical structure, known as the SAH domain, which is structurally similar to the BH3 domain of Bcl-2 family proteins (33Xue L. Chu F. Cheng Y. Sun X. Borthakur A. Ramarao M. Pandey P. Wu M. Schlossman S.F. Prasad K.V. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 6925-6930Crossref PubMed Scopus (67) Google Scholar). Consistent with an endogenous interaction with Bcl-2 family proteins, SIVA has been localized to the cytoplasm and mitochondria and was recently shown to directly bind to Bcl-XL through this amphipathic domain. Mutation of the SAH domain prevents interaction with anti-apoptotic Bcl-2 family proteins and abrogates its ability to induce apoptosis (33Xue L. Chu F. Cheng Y. Sun X. Borthakur A. Ramarao M. Pandey P. Wu M. Schlossman S.F. Prasad K.V. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 6925-6930Crossref PubMed Scopus (67) Google Scholar). This, as well as studies demonstrating interactions with TNFR family proteins, suggests that SIVA may function through multiple mechanisms that may be dependent on the cell type and apoptotic stimulus. Consistent with a key role in regulating apoptosis in tumor cells, SIVA is up-regulated in response to UV radiation and oxidative stress in a number of cell types (33Xue L. Chu F. Cheng Y. Sun X. Borthakur A. Ramarao M. Pandey P. Wu M. Schlossman S.F. Prasad K.V. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 6925-6930Crossref PubMed Scopus (67) Google Scholar, 36Cao C. Ren X. Kharbanda S. Koleske A. Prasad K.V. Kufe D. J. Biol. Chem. 2001; 276: 11465-11468Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Recently, SIVA was identified through DNA microarray analysis as a p53-induced and DNA damage-induced gene following treatment of colon carcinoma cells with a topoisomerase I inhibitor (37Daoud S.S. Munson P.J. Reinhold W. Young L. Prabhu V.V. Yu Q. LaRose J. Kohn K.W. Weinstein J.N. Pommier Y. Cancer Res. 2003; 63: 2782-2793PubMed Google Scholar). In addition, examination of certain types of cancer has revealed a down-regulation of the SIVA gene along with p53 suggesting that SIVA itself may have a potential role as a tumor suppressor due to its proapoptotic function (38Okuno K. Yasutomi M. Nishimura N. Arakawa T. Shiomi M. Hida J. Ueda K. Minami K. Dis. Colon Rectum. 2001; 44: 295-299Crossref PubMed Scopus (29) Google Scholar).The role of the proapoptotic gene, SIVA, in regulating the death of neuronal cells has not yet been explored. Using DNA microarray analysis to identify p53 target genes involved in neuronal cell death, we have identified SIVA as a p53 target. In addition to its proapoptotic role in the immune system and in tumor cells, we now demonstrate that SIVA also functions in injury-induced apoptosis of postmitotic neurons. By isolation and analysis of the SIVA promoter, we have identified response elements for both E2F1 and p53. Like p53, E2F1 is a tumor suppressor gene (39Yamasaki L. Jacks T. Bronson R. Goillot E. Harlow E. Dyson N.J. Cell. 1996; 85: 537-548Abstract Full Text Full Text PDF PubMed Scopus (639) Google Scholar, 40Field S.J. Tsai F.Y. Kuo F. Zubiaga A.M. Kaelin Jr., W.G. Livingston D.M. Orkin S.H. Greenberg M.E. Cell. 1996; 85: 549-561Abstract Full Text Full Text PDF PubMed Scopus (687) Google Scholar) involved in the regulation of apoptosis, and its involvement has been demonstrated in a number of neuronal injury models (41Hou S.T. Callaghan D. Fournier M.C. Hill I. Kang L. Massie B. Morley P. Murray C. Rasquinha I. Slack R. MacManus J.P. J. Neurochem. 2000; 75: 91-100Crossref PubMed Scopus (103) Google Scholar, 42O'Hare M.J. Hou S.T. Morris E.J. Cregan S.P. Xu Q. Slack R.S. Park D.S. J. Biol. Chem. 2000; 275: 25358-25364Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 43Wang F. Corbett D. Osuga H. Osuga S. Ikeda J.E. Slack R.S. Hogan M.J. Hakim A.M. Park D.S. J. Cereb. 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Furthermore, these consensus sites are also conserved in the human SIVA gene where E2F sites were also located within the promoter and p53 sites were again found in the first intron. Electrophoretic mobility shift assays revealed that both transcription factors are capable of binding their putative consensus sites, and luciferase reporter assays revealed that E2F1 and p53 can activate transcription of the SIVA promoter at these sites. Here, we report that the proapoptotic gene SIVA is a direct transcriptional target for both tumor suppressors, p53 and E2F1.MATERIALS AND METHODSPrimary Neuronal Cultures and Cell Lines—Cortical and cerebellar granule neurons were cultured as described previously (17Cregan S.P. MacLaurin J.G. Craig C.G. Robertson G.S. Nicholson D.W. Park D.S. Slack R.S. J. Neurosci. 1999; 19: 7860-7869Crossref PubMed Google Scholar, 23Fortin A. Cregan S.P. MacLaurin J.G. Kushwaha N. Hickman E.S. Thompson C.S. Hakim A. Albert P.R. Cecconi F. Helin K. Park D.S. Slack R.S. J. Cell Biol. 2001; 155: 207-216Crossref PubMed Scopus (184) Google Scholar). Murine SN48 cells were maintained in Dulbecco's modified Eagle's medium (Wisent Inc., St. Bruno, Quebec, Canada) supplemented with 10% fetal calf serum (Wisent Inc.) at 37 °C in 5% CO2 (23Fortin A. Cregan S.P. MacLaurin J.G. Kushwaha N. Hickman E.S. Thompson C.S. Hakim A. Albert P.R. Cecconi F. Helin K. Park D.S. Slack R.S. J. Cell Biol. 2001; 155: 207-216Crossref PubMed Scopus (184) Google Scholar). To culture progenitor cells, the epidermal ectoderm was removed from E12.5 mouse embryos, and the telencephalic neuroepithelia was dissected and transferred to a 1.5-ml Eppendorf tube containing 400 μl of serum-free stem cell media with 10 ng/ml basic fibroblast growth factor and 2 μg/ml heparin as previously described (48Reynolds B.A. Tetzlaff W. Weiss S. J. Neurosci. 1992; 12: 4565-4574Crossref PubMed Google Scholar, 49Tropepe V. Sibilia M. Ciruna B.G. Rossant J. Wagner E.F. van der Kooy D. Dev. Biol. 1999; 208: 166-188Crossref PubMed Scopus (671) Google Scholar). Neuroepithelia were mechanically dissociated, and single cells were plated at a density of 50,000 cells/ml in uncoated 60-mm Nunclon plates (Invitrogen). Primary neurospheres were expanded for 3–4 days. 7 days post-plating, the neurospheres were pelleted and all but 1–2 ml of media was removed. Neurospheres were triturated to generate a single cell suspension that was centrifuged and resuspended in Neurobasal medium containing B-27 supplement, N-2 supplement, 0.5 mm l-glutamine, 20 ng/ml platelet-derived growth factor, 1% nondialyzed fetal bovine serum, and 50 units/ml penicillin/streptomycin (Invitrogen). Cells were plated in Nunc 4-well (2 × 105 cells/well) dishes (Invitrogen) coated with poly-l-ornithine (Sigma-Aldrich).Recombinant Adenovirus Infection and Camptothecin Treatment—cDNA for SIVA was a kind gift from Dr. Kanteti V. S. Prasad (27Prasad K.V. Ao Z. Yoon Y. Wu M.X. Rizk M. Jacquot S. Schlossman S.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6346-6351Crossref PubMed Scopus (250) Google Scholar). cDNA for wild type p53, p53-ΔV (deletion of conserved box V: residues 270–286), and p53-173L (point mutation at residue 173 to Leu) were kind gifts from Dr. Karen Vousden (50Rowan S. Ludwig R.L. Haupt Y. Bates S. Lu X. Oren M. Vousden K.H. EMBO J. 1996; 15: 827-838Crossref PubMed Scopus (294) Google Scholar, 51Crook T. Marston N.J. Sara E.A. Vousden K.H. Cell. 1994; 79: 817-827Abstract Full Text PDF PubMed Scopus (224) Google Scholar). The p53 double transactivation mutant p53-DM (mutations L22E, W23S, W53F, and E54S) was a kind gift from Dr. Xinbin Chen (52Zhu J. Zhang S. Jiang J. Chen X. J. Biol. Chem. 2000; 275: 39927-39934Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Recombinant adenoviral vectors carrying the SIVA-GFP, GFP, and wild type or mutant p53 expression cassettes were constructed, purified, and titered as described previously (53Cregan S.P. MacLaurin J. Gendron T.F. Callaghan S.M. Park D.S. Parks R.J. Graham F.L. Morley P. Slack R.S. Gene Ther. 2000; 7: 1200-1209Crossref PubMed Scopus (39) Google Scholar). Prior to use, all recombinant adenovirus vectors were tested and confirmed to be wild type-free. All experiments were performed at a multiplicity of infection (m.o.i.) of 50 plaque-forming units/cell. Recombinant adenoviral vectors were added to cell suspensions immediately before plating for primary neuronal cultures and 24 h following plating for progenitor cell cultures. Cortical neurons were treated with 10 μm camptothecin (Sigma-Aldrich) 2 days after plating. For viability tests the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide survival assay (Cell Titer Kit, Promega, Madison, WI) that measures the mitochondrial conversion of the tetrazolium salt to a blue formazan salt was used as described previously (9Slack R.S. Belliveau D.J. Rosenberg M. Atwal J. Lochmuller H. Aloyz R. Haghighi A. Lach B. Seth P. Cooper E. Miller F.D. J. Cell Biol. 1996; 135: 1085-1096Crossref PubMed Scopus (114) Google Scholar).DNA Microarray Analysis—For p53 microarray analysis, cortical neurons were infected at an m.o.i. of 20 with recombinant adenovirus vectors carrying an expression cassette for either a full-length human p53 (Ad-p53), a transcriptionally defective p53 (Ad-p53-DM), or a DNA-binding mutant p53 (Ad-p53-ΔV). For E2F1 microarray analysis, neural precursor cells were infected at 50 m.o.i. with adenoviral vectors carrying E2F1 (Ad-E2F1) or the control vector Ad-GFP. Total RNA was extracted at 48 (p53) or 72 (E2F1) h post-infection using Tripure isolation reagent according to the manufacturer's instruction (Roche Diagnostics). RNA was sent to the Ottawa Genome Centre Affymetrix GeneChip Facility for analysis.Surgical Procedures—All animal procedures conformed to guidelines endorsed by the Canadian Institutes of Health Research and were approved by the Animal Care Committee of the University of Ottawa. Male C57BL/6 mice (Jackson Laboratories, Bar Harbor, ME) weighing 20–22 g were subjected to 2 h of middle cerebral artery occlusion as previously described (23Fortin A. Cregan S.P. MacLaurin J.G. Kushwaha N. Hickman E.S. Thompson C.S. Hakim A. Albert P.R. Cecconi F. Helin K. Park D.S. Slack R.S. J. Cell Biol. 2001; 155: 207-216Crossref PubMed Scopus (184) Google Scholar). Following reperfusion for 48 or 72 h, brains were removed and the striatum and cortex from the ipsilateral and contralateral sides were dissected separately and tissue was flash frozen using liquid nitrogen prior to protein extraction.Semiquantitative RT-PCR Analysis—Total RNA was isolated from cells using Tripure isolation reagent according to the manufacturer's instructions (Roche Diagnostics). Pilot experiments were done to determine the linear range of amplification with respect to quantity of starting template and PCR cycles using mouse-specific primers: SIVA forward (5′-CGCCCATCGCTTGTTCATCGTG-3′) and SIVA reverse (5′-CCGCAGCCCCAGCAGGTGTAT-3′). 6–12 ng of total RNA was used for cDNA synthesis and targeted gene amplification using the Super-Script One-Step RT-PCR kit (Invitrogen). cDNA synthesis was carried out at 50 °C for 30 min followed by a 2-min initial denaturation step at 94 °C. This was followed by 35 cycles (SIVA) or 25 cycles (glyceraldehyde-3-phosphate dehydrogenase) at 94 °C for 30 s, 54 °C for 30 s, and 72 °C for 1 min. Primers were designed to amplify nucleotides 329–437 of the SIVA transcript and 139–740 of the glyceraldehyde-3-phosphate dehydrogenase transcript. The resulting product was sequenced and confirmed to be SIVA.Western Blot Analysis—Western blot analysis was performed as described previously (17Cregan S.P. MacLaurin J.G. Craig C.G. Robertson G.S. Nicholson D.W. Park D.S. Slack R.S. J. Neurosci. 1999; 19: 7860-7869Crossref PubMed Google Scholar) with antibodies against p53 (1C12, Cell Signaling Technology, Beverly, MA) and β-actin (SC-1616, Santa Cruz Biotechnologies, Santa Cruz, CA) as a loading control.Electrophoretic Mobility Shift Assay—EMSAs were performed on total protein extracts as described previously (23Fortin A. Cregan S.P. MacLaurin J.G. Kushwaha N. Hickman E.S. Thompson C.S. Hakim A. Albert P.R. Cecconi F. Helin K. Park D.S. Slack R.S. J. Cell Biol. 2001; 155: 207-216Crossref PubMed Scopus (184) Google Scholar), with the following modifications. In brief, cells were harvested, centrifuged, and extracted in lysis buffer and assayed by the method of Bradford (Bio-Rad Laboratories protein assay reagent). 10–20 μg of total cell lysate was incubated with an excess of indicated 32P-labeled double-stranded DNA probes (60,000 cpm/0.2 ng of DNA). Oligonucleotides used included 5′-AGTCTAGACATGGCCTGGCGTCGTGGCTTGTTT-3′ (p53-BS1) and 5′-GTCTATGCAAGCCTGGACATGAGT-3′ (p53-BS2) corresponding to the p53 binding consensus sequences located between +752 to +780 and +897 to +917, respectively, and 5′-CAGAGCCTTCAGGCTTTTCGCGCGCT-3′ (E2F-BS1) and 5′-CGCCCTTGGCCTTTTCCCGCGCC-3′ (E2F-BS2) corresponding to the E2F binding consensus sequences located between -392 to -372 and -296 to -280, respectively, from the transcription start site (+1) referenced from the longest published SIVA sequence (GenBank™ accession number AF033114). The binding reaction (25 μl) was carried out at room temperature for 20 min in binding buffer with 0.1 μg of sonicated herring sperm DNA, and, for p53 binding, 1 μl of p53 Ab-1 monoclonal antibody was added to the binding buffer (OP03L; Oncogene Research Products). To control for binding specificity, a 100-fold excess of unlabeled oligonucleotide for BS1 and BS2 (p53 and E2F) was added to the binding reaction, and the mixture was incubated for 20 min before the addition of labeled probe. Furthermore, supershifts were performed with a p53-specific antibody FL393 (Santa Cruz Biotechnology, Inc.) and an E2F1-specific antibody C20-X (Santa Cruz Biotechnology, Inc.). Complexes were resolved on a 5% polyacrylamide, 1× Tris-glycine gel, dried, and visualized by autoradiography.SIVA Promoter Luciferase Reporter Assays—The SIVA luciferase reporter construct (pGL3b-SIVA) was generated by subcloning a murine SIVA gene fragment (-440 to +1770) containing the putative promoter, exon1 and intron1 into the SmaI site of pGL-3 basic (Promega). P53 binding site deletion constructs were generated by excising p53-BS1 (+752 to +780) with BsmB1 and PvuII, p53-BS2 (+897 to +917) with PvuII and EcoR1, and p53-BS1 and p53-BS2 (+752 to +917) with BsmB1 and EcoR1. SN48 cells were transfected by calcium phosphate precipitation as previously described (23Fortin A. Cregan S.P. MacLaurin J.G. Kushwaha N. Hickman E.S. Thompson C.S. Hakim A. Albert P.R. Cecconi F. Helin K. Park D.S. Slack R.S. J. Cell Biol. 2001; 155: 207-216Crossref PubMed Scopus (184) Google Scholar) with some modifications. Briefly 15 μg/plate of luciferase construct, 3 μg/plate of either the pCMV-p53, the pCMV-E2F1, the DNA binding mutant pCMV-p53-173L, or the empty pCMV vectors, and 2 μg/plate of pPGK-LacZ vector as an internal standard. After 4 h,