ZBTB2, a Novel Master Regulator of the p53 Pathway
2009; Elsevier BV; Volume: 284; Issue: 27 Linguagem: Inglês
10.1074/jbc.m809559200
ISSN1083-351X
AutoresBu‐Nam Jeon, Won‐Il Choi, Mi-Young Yu, A‐Rum Yoon, Myung‐Hwa Kim, Chae‐Ok Yun, Man‐Wook Hur,
Tópico(s)Epigenetics and DNA Methylation
ResumoWe found that ZBTB2, a POK family transcription factor, is a potent repressor of the ARF-HDM2-p53-p21 pathway important in cell cycle regulation. ZBTB2 repressed transcription of the ARF, p53, and p21 genes, but activated the HDM2 gene. In particular, ZBTB2 repressed transcription of the p21 gene by acting on the two distal p53 binding elements and the proximal Sp1 binding GC-box 5/6 elements. ZBTB2 directly interacted with Sp1 via its POZ domain and zinc fingers, which was important in the repression of transcription activation by Sp1. ZBTB2 and Sp1 competed with each other in binding to the GC-box 5/6 elements and the two p53 binding elements. ZBTB2 directly interacted with p53 via its zinc fingers, inhibiting p53 binding and repressing transcription activation by p53. The POZ domain, required for transcription repression, interacted with corepressors such as BCoR, NCoR, and SMRT. The interactions deacetylated histones Ac-H3 and -H4 at the proximal promoter. Although ectopic ZBTB2 stimulated cell proliferation, knock-down of ZBTB2 expression decreased cell proliferation and DNA synthesis. Overall, our data suggest that ZBTB2 is a potential proto-oncogenic master control gene of the p53 pathway and, in particular, is a potent transcription repressor of the cell cycle arrest gene p21 by inhibiting p53 and Sp1. We found that ZBTB2, a POK family transcription factor, is a potent repressor of the ARF-HDM2-p53-p21 pathway important in cell cycle regulation. ZBTB2 repressed transcription of the ARF, p53, and p21 genes, but activated the HDM2 gene. In particular, ZBTB2 repressed transcription of the p21 gene by acting on the two distal p53 binding elements and the proximal Sp1 binding GC-box 5/6 elements. ZBTB2 directly interacted with Sp1 via its POZ domain and zinc fingers, which was important in the repression of transcription activation by Sp1. ZBTB2 and Sp1 competed with each other in binding to the GC-box 5/6 elements and the two p53 binding elements. ZBTB2 directly interacted with p53 via its zinc fingers, inhibiting p53 binding and repressing transcription activation by p53. The POZ domain, required for transcription repression, interacted with corepressors such as BCoR, NCoR, and SMRT. The interactions deacetylated histones Ac-H3 and -H4 at the proximal promoter. Although ectopic ZBTB2 stimulated cell proliferation, knock-down of ZBTB2 expression decreased cell proliferation and DNA synthesis. Overall, our data suggest that ZBTB2 is a potential proto-oncogenic master control gene of the p53 pathway and, in particular, is a potent transcription repressor of the cell cycle arrest gene p21 by inhibiting p53 and Sp1. The POZ domain is an evolutionarily conserved protein-protein interaction motif found in many cellular regulatory proteins (1.Bardwell V.J. Treisman R. 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In particular, some of the POZ domain Krüppel-like zinc finger (POK) 3The abbreviations used are: POKPOZ domain Krüppel-like zinc fingerARFalternative reading frame geneBCL-6B-cell lymphoma-6BCoRBCL-6 interacting corepressorBTBbric-a-brac tramtrack broad complexPOZpoxvirus and zinc fingerChIPchromatin immunoprecipitationCV-1African green monkey kidney cellEMSAelectromobility shift assayFACSfluorescence-activated cell sorterFBI-1factor that binds to the inducer of short transcripts of human immunodeficiency virus-1GAPDHglyceraldehyde-3-phosphate dehydrogenaseGSTglutathione S-transferaseHDM2human analogue of mouse double minute oncogeneLacZβ-galactosidase geneLucluciferaseNCoRnuclear receptor corepressorPLZFpromyelocytic leukemia zinc finger proteinRbretinoblastomaSMRTsilencing mediator for retinoid and thyroid receptorsSp1specificity protein 1RTreverse transcriptionsiRNAsmall interfering RNAMTT3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. proteins are the major determinants of development, differentiation, and oncogenesis. 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EMBO J. 1993; 12: 1161-1167Crossref PubMed Scopus (590) Google Scholar, 9.Kerckaert J.P. Deweindt C. Tilly H. Quief S. Lecocq G. Bastard C. Nat. Genet. 1993; 5: 66-70Crossref PubMed Scopus (408) Google Scholar, 19.Chen W. Cooper T.K. Zahnow C.A. Overholtzer M. Zhao Z. Ladanyi M. Karp J.E. Gokgoz N. Wunder J.S. Andrulis I.L. Levine A.J. Mankowski J.L. Baylin S.B. Cancer Cell. 2004; 6: 387-398Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). Recently, FBI-1 (also called Pokemon) has been shown to act as a proto-oncogene by repressing transcription of the ARF gene, causing down-regulation of p53 and promoting oncogenic cellular transformation (10.Maeda T. Hobbs R.M. Merghoub T. Guernah I. Zelent A. Cordon-Cardo C. Teruya-Feldstein J. Pandolfi P.P. Nature. 2005; 433: 278-285Crossref PubMed Scopus (288) Google Scholar). POZ domain Krüppel-like zinc finger alternative reading frame gene B-cell lymphoma-6 BCL-6 interacting corepressor bric-a-brac tramtrack broad complex poxvirus and zinc finger chromatin immunoprecipitation African green monkey kidney cell electromobility shift assay fluorescence-activated cell sorter factor that binds to the inducer of short transcripts of human immunodeficiency virus-1 glyceraldehyde-3-phosphate dehydrogenase glutathione S-transferase human analogue of mouse double minute oncogene β-galactosidase gene luciferase nuclear receptor corepressor promyelocytic leukemia zinc finger protein retinoblastoma silencing mediator for retinoid and thyroid receptors specificity protein 1 reverse transcription small interfering RNA 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. The most striking property of some POZ domain transcription factors is their ability to repress transcription via their POZ domains (10.Maeda T. Hobbs R.M. Merghoub T. Guernah I. Zelent A. Cordon-Cardo C. Teruya-Feldstein J. Pandolfi P.P. Nature. 2005; 433: 278-285Crossref PubMed Scopus (288) Google Scholar, 11.Deltour S. Guerardel C. Leprince D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14831-14836Crossref PubMed Scopus (102) Google Scholar, 12.Dhordain P. Albagli O. Lin R.J. Ansieau S. Quief S. Leutz A. Kerckaert J.P. Evans R.M. Leprince D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10762-10767Crossref PubMed Scopus (296) Google Scholar, 13.Lin R.J. Nagy L. Inoue S. Shao W. Miller Jr., W.H. Evans R.M. Nature. 1998; 391: 811-814Crossref PubMed Scopus (981) Google Scholar, 14.Dong S. Zhu J. Reid A. Strutt P. Guidez F. Zhong H.J. Wang Z.Y. Licht J. Waxman S. Chomienne C. Chen Z. Zelent A. Chen S.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3624-3629Crossref PubMed Scopus (152) Google Scholar, 15.Chang C.C. Ye B.H. Chaganti R.S. Dalla-Favera R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6947-6952Crossref PubMed Scopus (388) Google Scholar, 16.Huynh K.D. Fischle W. Verdin E. 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In particular, the POZ domains of human BCL-6, FBI-1, HIC-1, and PLZF interact with BCoR, histone deacetylase, mSin3A, and SMRT/N-CoR (12.Dhordain P. Albagli O. Lin R.J. Ansieau S. Quief S. Leutz A. Kerckaert J.P. Evans R.M. Leprince D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 10762-10767Crossref PubMed Scopus (296) Google Scholar, 13.Lin R.J. Nagy L. Inoue S. Shao W. Miller Jr., W.H. Evans R.M. Nature. 1998; 391: 811-814Crossref PubMed Scopus (981) Google Scholar, 14.Dong S. Zhu J. Reid A. Strutt P. Guidez F. Zhong H.J. Wang Z.Y. Licht J. Waxman S. Chomienne C. Chen Z. Zelent A. Chen S.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 3624-3629Crossref PubMed Scopus (152) Google Scholar, 15.Chang C.C. Ye B.H. Chaganti R.S. Dalla-Favera R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6947-6952Crossref PubMed Scopus (388) Google Scholar, 16.Huynh K.D. Fischle W. Verdin E. Bardwell V.J. Genes Dev. 2000; 14: 1810-1823PubMed Google Scholar, 20.Jeon B.N. Yoo J.Y. Choi W.I. Lee C.E. Yoon H.G. 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Aside from p53, a variety of other factors, including Smads, AP2, STAT, BRCA1, E2F-1/E2F-3, and C/EBPα and -β, activate the transcription of p21. In addition to its role in responding to DNA damage, p21 has also been implicated in terminal differentiation, replicative senescence, and protection from p53-dependent and -independent apoptosis (Ref. 26.Gartel A.L. Radhakrishnan S.K. Cancer Res. 2005; 65: 3980-3985Crossref PubMed Scopus (695) Google Scholar and references therein). Sp1 family transcription factors that bind at the proximal promoter (bp −120 to −50) of the p21 gene represent another group of major regulators that affect p21 gene expression (Ref. 26.Gartel A.L. Radhakrishnan S.K. Cancer Res. 2005; 65: 3980-3985Crossref PubMed Scopus (695) Google Scholar and references therein). Sp1 is one of the best characterized transcription factors that bind to GC-rich DNA sequences in numerous cellular and viral genes (Refs. 30.Kadonaga J.T. Carner K.R. Masiarz F.R. Tjian R. 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In contrast, GC-boxes 1 and 2 mediate transcriptional activation by phorbol esters and okadaic acid, tumor suppressor protein BRCA1, and gut-enriched Krüppel-like factor (GKLF, KLF4). To date, no specific role has been attributed to the most proximal and overlapping GC-boxes 5 and 6 (Ref. 26.Gartel A.L. Radhakrishnan S.K. Cancer Res. 2005; 65: 3980-3985Crossref PubMed Scopus (695) Google Scholar and references therein). Together, these observations suggest that the specificity of utilizing different proximal GC-boxes under different p21 gene regulation conditions is important. In this article, we investigated whether a novel POK family protein, ZBTB2, could regulate any components of the ARF-HDM2-p53-p21 pathway, and examined the mechanisms and physiological consequences of ZBTB2 action. ZBTB2 repressed transcription of the ARF, p53, and p21 genes, and potently activated the HDM2 gene, which overall down-regulates the p53 pathway significantly. ZBTB2 increased cell proliferation significantly. Our data suggest that ZBTB2 may be a master regulator of the p53 pathway and may play a critical role in important biological processes controlled by p21 and other genes of the p53 pathway. The p21-Luc plasmid was kindly provided by Dr. Yoshihiro Sowa of the Kyoto Perpetural University of Medicine (Kyoto, Japan). The various pGL2-p21-Luc, pGL2-p53-Luc, pGL2-ARF-Luc, pGL2-HDM2-Luc, pcDNA3.1-p53, pcDNA3.1-Sp1, pG5-5x(GC-box)-Luc, pPac-Sp1, expression vectors of corepressors, and VP16 corepressors we used have been reported elsewhere (20.Jeon B.N. Yoo J.Y. Choi W.I. Lee C.E. Yoon H.G. Hur M.W. J. Biol. Chem. 2008; 283: 33199-33210Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 22.Choi W.I. Jeon B.N. Park H. Yoo J.Y. Kim Y.S. Koh D.I. Kim M.H. Kim Y.R. Lee C.E. Kim K.S. Osborne T.F. Hur M.W. J. Biol. Chem. 2008; 283: 29341-29354Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). The pcDNA3.1-ZBTB2 and pcDNA3.1-ZBTB2ΔPOZ plasmids were prepared by cloning cDNA fragments into pcDNA3.1 (Invitrogen). The GAL4-POZZBTB2 plasmid was prepared by cloning cDNA fragment encoding the POZ domain (amino acids 24–117) into pBIND (Promega). To prepare recombinant GST-POZZBTB2 and GST-ZFZBTB2 proteins, cDNA fragments encoding the POZ domain (amino acids 24–117) and zinc fingers (amino acids 254–468) were cloned into pGEX4T3 (Amersham Biosciences). The pPac-PL-ZBTB2 plasmid was prepared by cloning the cDNA fragments into pPac-PL. All plasmid constructs were verified by sequencing. Antibodies against p21, p53, Sp1, GAPDH, Myc tag, FLAG tag, Ac-H3, Ac-H4, and HDAC3 were purchased from Upstate (Charlottesville, VA), Chemicon (Temecula, CA), Calbiochem, and Santa Cruz Biotechnology (Santa Cruz, CA), respectively. Most of the chemical reagents were purchased from Sigma. HEK293A, HeLa, MB352, and CV-1 cells were cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen). Saos-2 cells were cultured in McCoy's 5A medium (Invitrogen) supplemented with 15% fetal bovine serum. Drosophila SL2 cells were cultured in Schneider's Drosophila medium (Invitrogen) supplemented with 10% fetal bovine serum. The pGL2-ARF-Luc, pGL2-HDM2-Luc, pGL2-p53-Luc, and various pGL2-p21-Luc promoter reporter fusion plasmids as well as pcDNA3.1-ZBTB2, pcDNA3.1-ZBTB2ΔPOZ, pcDNA3.1-p53, pcDNA3.1-Sp1, and pCMV-LacZ in various combinations were transiently transfected into several cell lines such as HEK293A, HeLa, MB352, Saos-2, and CV-1 using Lipofectamine Plus reagent (Invitrogen). After 24–36 h of incubation, cells were harvested and analyzed for luciferase activity. Reporter activity was normalized with cotransfected β-galactosidase activity for transfection efficiency. Total RNA was isolated from brain, heart, liver, muscle, kidney, spleen, brown adipose tissues, and white adipose tissues or cells using TRIzol reagent (Invitrogen). Synthesis of cDNA was done using 5 μg of total RNA, random hexamer (10 pmol), and Superscript reverse transcriptase II (200 units) in 20 μl using a reverse transcription kit (Invitrogen). The following oligonucleotide PCR primers were used: murine Zbtb2 (forward, 5′-CCAACCATGGACTTATTCTA-3′; reverse, 5′-TTCATCCTGGATGCCTGTGG-3′), β-actin (forward, 5′-ATGGATGACGATATCGCTGC-3′, reverse, 5′-CACACTGTGCCCATCTACGA-3′), Human ZBTB2 (forward, 5′-GATCGGATCCGATTTGGCCAACCATGGA-3′; reverse, 5′-GATCCTCGAGAGAAAAGGCTCCCTGGCT-3′), p21 (forward, 5′-ATGTCAGAACCGGCTGGGGATGTCC-3′, reverse, 5′-TTAGGGCTTCCTCTTGGAGAAGATC-3′), and GAPDH (forward, 5′-ACCACAGTCCATGCCATCAC-3′; reverse, 5′-TCCACCACCCTGTTGCTGTA-3′). Cells were harvested and lysed in RIPA buffer (50 mm Tris-HCl, pH 8.0, 1% Nonidet P-40, 0.25% sodium deoxycholic acid, 150 mm NaCl, 1 mm EGTA, complete Mini-Protease mixture). Cell extracts (40 μg) were separated using 12% SDS-PAGE gel electrophoresis, transferred onto Immun-BlotTM polyvinylidene difluoride membranes (Bio-Rad), and blocked with 5% skim milk (BD Biosciences). Blotted membranes were incubated with antibodies against FLAG tag (Sigma), GAPDH (Chemicon), p21, p53, Sp1, Myc tag, (Santa Cruz Biotechnology) and then incubated with horseradish peroxidase-conjugated anti-mouse and anti-rabbit secondary antibody (Vector Laboratory). Protein bands were visualized with ECL solution (PerkinElmer Life Sciences). Four siRNA against ZBTB2 mRNA were designed and purchased from Dharmacon (Lafayette, CO): siZBTB2-1, 5′-GAUCAUCAGUUGAGACAAGUU-3′, 5′-PCUUGUCUCAACUGAUGAUCUU-3′; siZBTB2-2, 5′-CAGGUGAAUCGGACAAAUAUU-3′, 5′-PUAUUUGUCCGAUUCACCUGUU-3′; siZBTB2-3, 5′-CGACCCGGUUCGAUUAGAAUU-3′, 5′-PUUCUAAUCGAACCGGGUCGUU-3′; and siZBTB2-4, 5′-AGACGAAGGGCGAUCCAUUUU-3′, 5′-PAAUGGAUCGCCCUUCGUCUUU-3′. The siRNA (200 pmol) were transfected into HEK293A cells using Lipofectamine 2000 (Invitrogen). After transfection, cells were harvested, and total RNA was prepared. RT-PCR analysis of mRNA was performed as described above. The molecular interaction between ZBTB2 and p53 or Sp1 on the p21 gene promoter and histone modification at the p21 proximal promoter in HEK293A, Saos-2, and Drosophila SL2 cells were analyzed by following the standard ChIP assay protocol, as described elsewhere (20.Jeon B.N. Yoo J.Y. Choi W.I. Lee C.E. Yoon H.G. Hur M.W. J. Biol. Chem. 2008; 283: 33199-33210Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 22.Choi W.I. Jeon B.N. Park H. Yoo J.Y. Kim Y.S. Koh D.I. Kim M.H. Kim Y.R. Lee C.E. Kim K.S. Osborne T.F. Hur M.W. J. Biol. Chem. 2008; 283: 29341-29354Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). PCR of chromatin immunoprecipitated DNA was carried out using oligonucleotide primer sets designed to amplify the upstream regulatory regions and proximal promoter region of the p21 gene. p53 RE-1 binding primers (bp, −2307–1947), forward, 5′-TGCTTGGGCAGCAGGCTGTG-3′, reverse, 5′-GCAACCATGCACTTGAATGT-3′; p53 RE-2 binding primers (bp, −1462–1128), forward, 5′-TGTCCTCCCACCCCTACCTGG-3′, reverse, 5′-AGAAATGAGTGATGTGTC-3′; proximal GC-boxes ChIP primers (bp, −261 to approximately +39), forward, 5′-GGCTCACTTCGTGGGGAAAT-3′, reverse, 5′-CACAAGGAACTGACTTCGGC-3′. To analyze histone H3 and H4 modification at the proximal promoter (bp, −133 to +100), forward 5′-GCGCTGGGCAGCCAGGAGCC-3′ and reverse 5′-CGCTCTCTCACCTCCTCT-3′ primers were used. Cells were washed, pelleted, and resuspended in a lysis buffer supplemented with protease inhibitors (20 mm Tris-HCl, pH 7.5, 150 mm NaCl, 10% glycerol, 1% Triton X-100). Cell lysate was precleared, and the supernatant was incubated overnight with anti-FLAG antibody on a rotating platform at 4 °C, followed by incubation with protein A-Sepharose Fast Flow beads. Beads were collected, washed, and resuspended in equal volumes of 5× SDS loading buffer. Immunoprecipitated proteins were separated with 12% SDS-PAGE. Western blot assay was performed as described above. CV-1 cells were co-transfected with pG5-Luc, pGal4-POZZBTB2, pVP16-corepressors, and pCMV-LacZ. After 24 h of transfection with Lipofectamine Plus (Invitrogen), CV-1 cells were harvested and assayed for luciferase activity. Luciferase activity was then normalized with cotransfected β-galactosidase activity. Recombinant GST, GST-POZZBTB2, and GST-ZFZBTB2 fusion proteins were prepared from Escherichia coli BL21 (DE3) grown for 5 h at 37 °C in a medium containing 1 mm isopropyl 1-thio-β-d-galactopyranoside. E. coli were lysed and purified using glutathione-agarose 4 bead affinity chromatography (Peptron, Taejeon, Korea). The purified proteins were then resolved with 12% SDS-PAGE to quantitate and assess purity. Corepressor, p53, and Sp1 polypeptides were prepared by incubating 1 μg of pcDNA3.0-corepressor, pcDNA3.1- p53, and pcDNA3.1-Sp1 expression plasmid with TnT Quick-coupled Transcription/Translation Extract (Promega) containing 40 μl of TnT Quick Master Mix and 2 μl of [35S]methionine (1175.0 Ci/mol) (PerkinElmer Life Sciences) at 30 °C for 90 min. Polypeptide expression levels were then analyzed by running 1 μl of the total mixture through 12% SDS-PAGE and autoradiography. For GST fusion protein pull-down assays, GST fusion protein-agarose bead complexes were incubated with 10 μl of in vitro translated [35S]methionine-labeled corepressors, p53, and Sp1 polypeptides at 4 °C for 4 h in HEMG buffer. The reaction mixtures were centrifuged, pellets were rinsed, and the bound proteins were separated using 12% SDS-PAGE. Gels were then exposed to x-ray film using an image-intensifying screen (Kodak). ZBTB2 cDNA was cloned into the adenovirus E1 shuttle vector pCA14 (Microbix, Ontario, Canada), to generate pCA14-ZBTB2. The pCA14-ZBTB2 shuttle vector was linearized by XmnI digestion, and the adenovirus vector vmdl324Bst (from Dr. Verca at the University of Fribourg, Switzerland) containing the Ad5 genome deleted in the E1 and E3 regions was also linearized with BstBI digestion. The linearized pCA14-ZBTB2 and vmdl324Bst digested with BstBI were co-transformed into E. coli BJ518 for homologous recombination. Proper homologous recombinant adenoviral plasmid was digested with PacI and transfected into HEK293 cells to generate the adenovirus expressing ZBTB2 (dl324-ZBTB2). Propagation and titration of the recombinant virus were carried out by standard methods. PCR amplification and DNA sequencing using primers specific to ZBTB2 confirmed the adenovirus genotype. EMSAs were carried out as described previously (20.Jeon B.N. Yoo J.Y. Choi W.I. Lee C.E. Yoon H.G. Hur M.W. J. Biol. Chem. 2008; 283: 33199-33210Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 22.Choi W.I. Jeon B.N. Park H. Yoo J.Y. Kim Y.S. Koh D.I. Kim M.H. Kim Y.R. Lee C.E. Kim K.S. Osborne T.F. Hur M.W. J. Biol. Chem. 2008; 283: 29341-29354Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 33.Haupt Y. Maya R. Kazaz A. Oren M. Nature. 1997; 387: 296-299Crossref PubMed Scopus (3722) Google Scholar). The probe sequences of Sp1 response elements on the p21 gene proximal promoter or the sequences of p53 response elements on the p21 gene distal promoter used in EMSA were as follows (only top strands are shown): GC-box 1, 5′-GATCGGGAGGGCGGTCCCG-3′; GC-box 2, 5′-GATCTCCCGGGCGGCGCG-3′; GC-box 3, 5′-GATCCGAGCGCGGGTCCCGCCTC-3′; GC-box 4, 5′-GATCCTTGAGGCGGGCCCG-3′; GC-box 5/6, 5′-GATCGGGCGGGGCGGTTGTATATCA-3′; p53 RE-1, 5′-GATCCGTTAGAGGAAGAAGACTGGGCATGTCTG-3′; and p53 RE-2, 5′-GATCCATCAGGAACATGTCCCAACATGTTGAGCTC-3′. HEK293A cells were lysed in HKMG buffer (10 mm HEPES, pH 7.9, 100 mm KCl, 5 mm MgCl2, 10% glycerol, 1 mm dithiothreitol, and 0.5% Nonidet P-40). Cellular extracts were incubated with 1 μg of biotinylated double-stranded oligonucleotides (p53 RE-1, p53 RE-2, and Sp1 GC-box 5/6) for 16 h. The sequences of the oligonucleotides are as follows (only top strands are shown): Sp1 GC-box 5/6, 5′-CCTTGAGGCGGGCCCGGGCGGGGCGGTTGTATATCAGGGC-3′; p53 RE-1, 5′-GTCAGGAACATGTCCCAACATGTTGAGCTC-3′; p53 RE-2, 5′-TAGAGGAAGAAGACTGGGCATGTCTGGGCA-3′. To collect DNA-bound proteins, the mixtures were incubated with streptavidin-agarose beads for 2 h, washed with HKMG buffer, and precipitated by centrifugation. The precipitate was analyzed by Western blot assay as described above. HEK293A cells were transfected with pcDNA3.1-ZBTB2 expression vector or siZBTB2 RNA in the presence or absence of p53 expression vector. Cells were washed, fixed with methanol, and stained with 50 μg/ml of propidium iodide in 100 μg/ml of ribonuclease A for 30 min at 37 °C in the dark. DNA content, cell cycle profiles, and forward scatter were analyzed by FACSCalibur (BD Biosciences) with emission detection at 488 nm (excitation) and 575 nm (peak emission). Data were analyzed using ModFit LT 2.0 (Verity Software House, Inc., ME) and WindMDI 2.8 (Joseph Trotter, The Scripps Research Institute). Confluent HEK293A cells grown on 10-cm culture dishes were
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