Artigo Acesso aberto Revisado por pares

Proto-oncogene FBI-1 (Pokemon/ZBTB7A) Represses Transcription of the Tumor Suppressor Rb Gene via Binding Competition with Sp1 and Recruitment of Co-repressors

2008; Elsevier BV; Volume: 283; Issue: 48 Linguagem: Inglês

10.1074/jbc.m802935200

ISSN

1083-351X

Autores

Bu‐Nam Jeon, Jung‐Yoon Yoo, Won‐Il Choi, Choong‐Eun Lee, Ho‐Geun Yoon, Man‐Wook Hur,

Tópico(s)

Hippo pathway signaling and YAP/TAZ

Resumo

FBI-1 (also called Pokemon/ZBTB7A) is a BTB/POZ-domain Krüppel-like zinc-finger transcription factor. Recently, FBI-1 was characterized as a proto-oncogenic protein, which represses tumor suppressor ARF gene transcription. The expression of FBI-1 is increased in many cancer tissues. We found that FBI-1 potently represses transcription of the Rb gene, a tumor suppressor gene important in cell cycle arrest. FBI-1 binds to four GC-rich promoter elements (FREs) located at bp –308 to –188 of the Rb promoter region. The Rb promoter also contains two Sp1 binding sites: GC-box 1 (bp –65 to –56) and GC-box 2 (bp –18 to –9), the latter of which is also bound by FBI-1. We found that FRE3 (bp –244 to –236) is also a Sp1 binding element. FBI-1 represses transcription of the Rb gene not only by binding to the FREs, but also by competing with Sp1 at the GC-box 2 and the FRE3. By binding to the FREs and/or the GC-box, FBI-1 represses transcription of the Rb gene through its POZ-domain, which recruits a co-repressor-histone deacetylase complex and deacetylates histones H3 and H4 at the Rb gene promoter. FBI-1 inhibits C2C12 myoblast cell differentiation by repressing Rb gene expression. FBI-1 (also called Pokemon/ZBTB7A) is a BTB/POZ-domain Krüppel-like zinc-finger transcription factor. Recently, FBI-1 was characterized as a proto-oncogenic protein, which represses tumor suppressor ARF gene transcription. The expression of FBI-1 is increased in many cancer tissues. We found that FBI-1 potently represses transcription of the Rb gene, a tumor suppressor gene important in cell cycle arrest. FBI-1 binds to four GC-rich promoter elements (FREs) located at bp –308 to –188 of the Rb promoter region. The Rb promoter also contains two Sp1 binding sites: GC-box 1 (bp –65 to –56) and GC-box 2 (bp –18 to –9), the latter of which is also bound by FBI-1. We found that FRE3 (bp –244 to –236) is also a Sp1 binding element. FBI-1 represses transcription of the Rb gene not only by binding to the FREs, but also by competing with Sp1 at the GC-box 2 and the FRE3. By binding to the FREs and/or the GC-box, FBI-1 represses transcription of the Rb gene through its POZ-domain, which recruits a co-repressor-histone deacetylase complex and deacetylates histones H3 and H4 at the Rb gene promoter. FBI-1 inhibits C2C12 myoblast cell differentiation by repressing Rb gene expression. The importance of the tumor suppressor retinoblastoma (Rb) 3The abbreviations used are: Rb, retinoblastoma; ARF, alternative reading frame gene; Bcl-6, B-cell lymphoma-6; BCoR, BCL-6 interacting co-repressor; BTB/POZ, bric-a-brac tramtrack broad complex/poxvirus and zinc finger; ChIP, chromatin immunoprecipitation; CV-1, African green monkey kidney cell; DBD, DNA binding domain; DMEM, Dulbecco's modified eagle medium; FBI-1, factor that binds to the inducer of short transcripts of human immunodeficiency virus-1; FRE, FBI-1 response element; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GST, glutathione S-transferase; LacZ, β-galactosidase gene; Luc, luciferase gene; LRF, leukemia/lymphoma-related factor; NCoR, nuclear receptor co-repressor; Pokemon, POK erythroid myeloid ontogenic factor; SMRT, silencing mediator for retinoid and thyroid receptors; Sp1, specificity protein 1; RT, reverse transcription; Wt, wild type; ZF, zinc finger; ZFDBD, zinc finger DNA binding domain; aa, amino acid(s); siRNA, small interference RNA; CMV, cytomegalovirus; CREB, cAMP-response element-binding protein; HDAC, histone deacetylase. protein in regulating key cellular events was first suggested by the identification of a tumor, retinoblastoma, in which the Rb locus was invariably deleted (1Friend S.H. Bernards R. Rogelj S. Weinberg R.A. Rapaport J.M. Albert D.M. Dryja T.P. Nature. 1986; 323: 643-646Crossref PubMed Scopus (2232) Google Scholar, 2Lee W.H. Bookstein R. Hong F. Young L.J. Shew J.Y. Lee E.Y. Science. 1987; 235: 1394-1399Crossref PubMed Scopus (1075) Google Scholar, 3Huang H.J. Yee J.K. Shew J.Y. Chen P.L. Bookstein R. Friedmann T. Lee E.Y. Lee W.H. Science. 1988; 242: 1563-1566Crossref PubMed Scopus (627) Google Scholar). Rb is implicated in the development of various cancers (4Scambia G. Lovergine S. Masciullo V. Oncogene. 2006; 25 (review): 5302-5308Crossref PubMed Scopus (34) Google Scholar and references therein). Rb suppresses tumorigenesis by inhibiting cell cycle progression at G1/S by preventing the transcription of several genes important in cell cycle control (5Goodrich D.W. Wang N.P. Qian Y.W. Lee E.Y. Lee W.H. Cell. 1991; 67: 293-302Abstract Full Text PDF PubMed Scopus (617) Google Scholar). Rb is phosphorylated in a cell cycle-dependent manner (6Khidr L. Chen P.L. Oncogene. 2006; 25 (review): 5210-5219Crossref PubMed Scopus (97) Google Scholar and references therein). When Rb is hypophosphorylated, it forms complexes with E2F family proteins and inhibits transcription by recruiting proteins involved in transcriptional repression (7Nevins J.R. Science. 1992; 258: 424-429Crossref PubMed Scopus (1364) Google Scholar). Once phosphorylated, Rb can no longer form complexes with E2F proteins. E2F proteins, upon dimerization with their differentiation-regulated transcription factor partners, are capable of activating the expression of a number of genes that are likely to regulate or promote entry into S phase (6Khidr L. Chen P.L. Oncogene. 2006; 25 (review): 5210-5219Crossref PubMed Scopus (97) Google Scholar and references therein). Investigations on how transcription of the Rb gene is regulated are important in elucidating the cellular regulatory mechanism of Rb gene expression (8Gill R.M. Hamel P.A. Zhe J. Zacksenhaus E. Gallie B.L. Phillips R.A. Cell Growth & Differ. 1994; 5: 467-474PubMed Google Scholar, 9Magenta A. Cenciarelli C. De Santa F. Fuschi P. Martelli F. Caruso M. Felsani A. Mol. Cell. Biol. 2003; 23: 2893-2906Crossref PubMed Scopus (67) Google Scholar, 10Deléhouzée S. Yoshikawa T. Sawa C. Sawada J. Ito T. Omori M. Wada T. Yamaguchi Y. Kabe Y. Handa H. Genes Cells. 2005; 10: 717-731Crossref PubMed Scopus (43) Google Scholar, 11Sowa Y. Shiio Y. Fujita T. Matsumoto T. Okuyama Y. Kato D. Inoue J. Sawada J. Goto M. Watanabe H. Handa H. Sakai T. Cancer Res. 1997; 57: 3145-3148PubMed Google Scholar, 12Sekimata M. Homma Y. Biochem. Biophys. Res. Commun. 2004; 325: 653-659Crossref PubMed Scopus (6) Google Scholar). For example, induction of Rb gene transcription by MyoD, via CREB, is a key event in muscle differentiation (9Magenta A. Cenciarelli C. De Santa F. Fuschi P. Martelli F. Caruso M. Felsani A. Mol. Cell. Biol. 2003; 23: 2893-2906Crossref PubMed Scopus (67) Google Scholar). Furthermore, transcriptional activation of the Rb gene by GABP and HCF-1 is also important in muscle differentiation (10Deléhouzée S. Yoshikawa T. Sawa C. Sawada J. Ito T. Omori M. Wada T. Yamaguchi Y. Kabe Y. Handa H. Genes Cells. 2005; 10: 717-731Crossref PubMed Scopus (43) Google Scholar, 11Sowa Y. Shiio Y. Fujita T. Matsumoto T. Okuyama Y. Kato D. Inoue J. Sawada J. Goto M. Watanabe H. Handa H. Sakai T. Cancer Res. 1997; 57: 3145-3148PubMed Google Scholar). In contrast, YY1 and MIZF repress transcription of Rb, and this repression is important for inhibiting myogenesis (10Deléhouzée S. Yoshikawa T. Sawa C. Sawada J. Ito T. Omori M. Wada T. Yamaguchi Y. Kabe Y. Handa H. Genes Cells. 2005; 10: 717-731Crossref PubMed Scopus (43) Google Scholar, 12Sekimata M. Homma Y. Biochem. Biophys. Res. Commun. 2004; 325: 653-659Crossref PubMed Scopus (6) Google Scholar). All these data suggest that multiple transcription factors act on the transcriptional regulation of Rb gene. We have been investigating the biological functions of FBI-1 (also called Pokemon/ZBTB7A), which contains a BTB/POZ-domain at its N terminus and Krüppel-like zinc fingers at its C terminus (13Morrison D.J. Pendergrast P.S. Stavropoulos P. Colmenares S.U. Kobayashi R. Hernandez N. Nucleic Acids Res. 1999; 27: 1251-1262Crossref PubMed Scopus (47) Google Scholar, 14Lee D.K. Suh D. Edenberg H.J. Hur M.W. J. Biol. Chem. 2002; 277: 26761-26768Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Recently, there have been several reports on the function of FBI-1. FBI-1 stimulates the Tat activity of human immunodeficiency virus, type 1 long terminal repeat and represses human ADH5/FDH gene expression by interacting with Sp1 zinc fingers (14Lee D.K. Suh D. Edenberg H.J. Hur M.W. J. Biol. Chem. 2002; 277: 26761-26768Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 15Pendergrast P.S. Wang C. Hernandez N. Huang S. Mol. Biol. Cell. 2002; 13: 915-929Crossref PubMed Scopus (46) Google Scholar). The mouse counterpart of FBI-1, LRF, co-immunoprecipitates and co-localizes with Bcl-6, and is involved in chondrogenesis and adipogenesis (16Davies J.M. Hawe N. Kabarowski J. Huang Q.H. Zhu J. Brand N.J. Leprince D. Dhordain P. Cook M. Morriss-Kay G. Zelent A. Oncogene. 1999; 18: 365-375Crossref PubMed Scopus (127) Google Scholar, 17Liu C.J. Prazak L. Fajardo M. Yu S. Tyagi N. Di Cesare P.E. J. Biol. Chem. 2004; 279: 47081-47091Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 18Laudes M. Christodoulides C. Sewter C. Rochford J.J. Considine R.V. Sethi J.K. Vidal-Puig A. O'Rahilly S. J. Biol. Chem. 2004; 279: 11711-11718Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). The rat homolog of FBI-1, OCZF, is a transcriptional repressor and is involved in osteoclastogenesis (19Kukita A. Kukita T. Ouchida M. Maeda H. Yatsuki H. Kohashi O. Blood. 1999; 94: 1987-1997Crossref PubMed Google Scholar). FBI-1 enhances NF-κB-mediated transcription through an interaction between the POZ-domain of FBI-1 and the RHD of NF-κB (20Lee D.K. Kang J.E. Park H.J. Kim M.H. Yim T.H. Kim J.M. Heo M.K. Kim K.Y. Kwon H.J. Hur M.W. J. Biol. Chem. 2005; 280: 27783-27791Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Recently, FBI-1 was identified as a proto-oncogene (21Maeda 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 (291) Google Scholar). Serial analysis of gene expression analysis showed that the expression of FBI-1 is increased in cancer tissues. In transgenic mice overexpressing FBI-1, FBI-1 represses transcription of tumor suppressor gene, ARF, by binding to its promoter region. The product of ARF is a transcriptional activator of p53, another tumor suppressor. Thus, repression of ARF can eventually inhibit expression of p53, promoting oncogenesis in the thymus, liver, and spleen. In FBI-1 knockout mice, overexpression of ARF increases expression of p53, induces senescence, apoptosis, and eventually blocks cellular differentiation (21Maeda 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 (291) Google Scholar). FBI-1 is overexpressed in solid tumors, such as cancers of the colon and bladder, in which the normal function of the ARF/p53 pathway is frequently lost. It is likely that FBI-1 has multiple additional target genes by which it can exert oncogenic activity (22Maeda T. Hobbs R.M. Cancer Res. 2005; 65: 8575-8578Crossref PubMed Scopus (91) Google Scholar and references therein). We suspected that FBI-1 might be involved in the transcriptional regulation of the genes involved in differentiation, cell cycle control, and tumor suppression, such as Rb. We found that the Rb gene is the molecular target of proto-oncogene FBI-1, and we investigated the molecular mechanism of transcriptional regulation in detail. Plasmids Construction—pGL3-Enhancer-Rb-Luc plasmid was kindly provided by Dr. Masayuki Sekimata (Fukushima Medical University, Japan) (12Sekimata M. Homma Y. Biochem. Biophys. Res. Commun. 2004; 325: 653-659Crossref PubMed Scopus (6) Google Scholar, 23Sekimata M. Homma Y. Nucleic Acids Res. 2004; 32: 590-597Crossref PubMed Scopus (35) Google Scholar). The pGL2-Rb-Luc fusion plasmid was prepared by cloning the Rb promoter (bp –370 to +106) into the pGL2-Basic plasmid (Promega, Madison, WI). Various mutant Rb-Luc plasmids were prepared using a site-directed mutagenesis kit (Stratagene). Expression plasmid vectors for the VP16-co-repressors, BCoR (aa 112–753), NCoR (aa 1709–2215), and SMRT (aa 194–657) fusion proteins (pKH135EF-BCoR, pKH73/110EF-NCoR, and pCMX-SMRT) were reported elsewhere (24Lee J.A. Suh D.C. Kang J.E. Kim M.H. Park H. Lee M.N. Kim J.M. Jeon B.N. Roh H.E. Yu M.Y. Choi K.Y. Kim K.Y. Hur M.W. J. Biol. Chem. 2005; 280: 28061-28071Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Construction of pcDNA3-FBI-1, pcDNA3-FBI-1ΔPOZ, and pG5-Luc were reported elsewhere (14Lee D.K. Suh D. Edenberg H.J. Hur M.W. J. Biol. Chem. 2002; 277: 26761-26768Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). To prepare the recombinant GST-POZFBI-1 fusion protein expression vector, a cDNA fragment encoding the POZ-domain of FBI-1 was subcloned into pGEX4T3 (Amersham Biosciences) (14Lee D.K. Suh D. Edenberg H.J. Hur M.W. J. Biol. Chem. 2002; 277: 26761-26768Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). The expression vectors for Sp1ZFDBD (zinc finger DNA binding domain) (aa 624–718) and FBI-1ZFDBD (aa 366–495) was prepared by cloning the PCR-amplified cDNA fragments into pGEX4T1 (24Lee J.A. Suh D.C. Kang J.E. Kim M.H. Park H. Lee M.N. Kim J.M. Jeon B.N. Roh H.E. Yu M.Y. Choi K.Y. Kim K.Y. Hur M.W. J. Biol. Chem. 2005; 280: 28061-28071Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 25Kadonaga J.T. Carner K.R. Masiarz F.R. Tjian R. Cell. 1987; 51: 1079-1090Abstract Full Text PDF PubMed Scopus (1255) Google Scholar). The oligonucleotide primers used for PCR amplification of FBI-1ZFDBD are as follows: forward, 5′-GATCGGATCCGCCTGGTCGCAGAAGGTGGAG-3′ and reverse, 5′-GATCGTCGACCGAGGGGACGCCGTTGCAGCCGTC-3′. Mammalian expression vectors for the co-repressors used in GST-pulldown assays were prepared by subcloning the cDNA fragments encoding BCoR (aa 112–753), NCoR (aa 1709–2215), and SMRT (aa 194–657) into pcDNA3.0 (Invitrogen) as reported elsewhere (24Lee J.A. Suh D.C. Kang J.E. Kim M.H. Park H. Lee M.N. Kim J.M. Jeon B.N. Roh H.E. Yu M.Y. Choi K.Y. Kim K.Y. Hur M.W. J. Biol. Chem. 2005; 280: 28061-28071Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). The FBI-1 expression vector in Drosophila SL2 cells was prepared by cloning FBI-1 cDNA into pPac-PL provided by Dr. Carl Thummel (University of Utah). pPac-Sp1 was provided by Dr. Robert Tjian (University of California, Berkeley, CA). Cell Culture/Stable Cell Line—HeLa cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen). Stable HeLa cells overexpressing FBI-1 were prepared by transfecting HeLa cells with a recombinant Lenti virus, LentiM1.4-FBI-1-FLAG. Control stable cells were prepared using recombinant LentiM1.4-LacZ virus. Drosophila SL2 cells were cultured in Schneider's medium (Invitrogen) supplemented with 10% fetal bovine serum. Transcription Analysis of the Rb Gene Promoter—HeLa, HCT116, and HEK 293A cells were cultured with the medium recommended by the ATCC (Manassas, VA) on 6-well culture vessels and were transfected with 0.3 μg of pGL2-Rb-Luc Wt or mutants and 0.5 μg each of pcDNA3.0, pcDNA3.0-FBI-1, and pcDNA3.0-FBI-1ΔPOZ using Lipofectamine Plus reagent (Invitrogen). After 36 h of incubation, cells were harvested, lysed, and assayed for luciferase activities. Luciferase activity was normalized with the protein concentration of the lysate. Electrophoretic Mobility Shift Assays—EMSAs were carried out as described previously (24Lee J.A. Suh D.C. Kang J.E. Kim M.H. Park H. Lee M.N. Kim J.M. Jeon B.N. Roh H.E. Yu M.Y. Choi K.Y. Kim K.Y. Hur M.W. J. Biol. Chem. 2005; 280: 28061-28071Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Binding reactions were carried out in 20 μl of binding buffer containing 10 mm HEPES, pH 7.9, 60 mm KCl, 5 μm ZnCl2, 1 mm dithiothreitol, 1% bovine serum albumin, 7% glycerol, 0.1 μg of recombinant FBI-1ZFDBD or Sp1ZFDBD, and 10,000 cpm of probe at room temperature for 30 min. Antibodies against GST-Tag, FBI-1, or Xpress were added to binding reactions for supershift of the probe-protein complex. The sequences of FREs and Sp1-binding GC-box oligonucleotides were as follows (only the top strand sequences are shown): FRE1, 5′-GATCGGATGAGGCCCACAGTCACC-3′; FRE2, 5′-GATCCCACAGTCACCCACCAGACT-3′; FRE3, 5′-GATCAGGGGGTGGTTCTGGGTAGA-3′; FRE4, 5′-GATCCGCCTGGACCCACGCCAGGT-3′; GC-box 1, 5′-GATCACGGGCGGAAGTGAC-3′; and GC-box 2, 5′-GATCAGTTGCCGGGCGGGGGAGGG-3′. Site-directed Mutagenesis of the Rb Promoter—To investigate the role of FBI-1 binding sites on transcription, mutations were introduced into the promoter sequence using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA). The core GGG of the binding sequences (5′-GDGGGYYYY-3′) of the FREs and the GC-boxes was replaced with AAA using the following mutagenesis oligonucleotides primers (only the top strands are shown): mFRE1, 5′-CCCGGGATAGGGATGAAATTTACAGTCACCCACCAGA-3′; mFRE2, 5′-ATGAGGCCCA; CAGTCATTTACCAGACTCTTTGTAT-3′; mFRE3, 5′-CACCCCGGCCTGGAGGAAAAAATTCTGGGTAGAAGCAC-3′; mFRE4, 5′-CTGGAAGGCGCCTGGATTTACGCCAGGTTTC; CCAG-3′; mGC-Box 1, 5′-ACGTGACGCCGCGGGCAAAAGTGACGTTTTCCCG-3′; and mGC-Box 2, 5′-CGCTCAGTTGCCGGGCAAGGGAGGGCGCGTCCGG-3′. Mammalian Two-hybrid Assays—African green monkey kidney cells (CV-1) were grown in DMEM supplemented with 10% fetal bovine serum (Invitrogen). CV-1 cells were transiently transfected with pCMX-Gal4-POZFBI-1, pCMX-VP16-BCoR, pCMX-VP16-NCoR, pCMX-VP16-SMRT, and pG5-Luc using Lipofectamine Plus reagent (Invitrogen). Cells were cultured for 36 h. The cells were then harvested, lysed, and analyzed for luciferase activity on a luminometer (Microplate Luminometer LB 96V, EG&G Berthold, MD). Luciferase activity was normalized with the cotransfected β-galactosidase activity or protein concentration. GST Fusion Protein Pulldown Assays—Escherichia coli BL21(DE3), transformed with either GST or GST fusion protein expression vector, was induced with 0.5 mm isopropyl-1-thio-β-d-galactopyranoside for 4 h at 37°C. After E. coli pellets were lysed and sonicated in lysis buffer (1× phosphate-buffered saline, 1 mm phenylmethysulfonyl fluoride, 2 mm EDTA, and 0.2 mg/ml lysozyme), recombinant proteins were purified by affinity chromatography using glutathione-agarose 4 beads (Peptron, Daejeon, Korea). Purified proteins were resolved on a 12% SDS-PAGE gel to quantitate them and assess their purity. The same volume of protein-agarose bead complex was used for all GST fusion protein pulldown assays. Co-repressor proteins were prepared in vitro by incubating 1 μg of the pcDNA3.0 co-repressor expression vector 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. Purified GST fusion proteins (5 μg) were incubated with GSH-agarose for 1 h at 4 °C in HEMG buffer (40 mm HEPES, pH 7.9, 100 mm KCl, 0.2 mm EDTA, 5 mm MgCl2, 0.1% Nonidet P-40, 10% glycerol, 1.5 mm dithiothreitol, protease inhibitor mixture (1 tablet/50 ml)). After agarose-GST protein complexes were washed, 10 μl of in vitro translated [35S]methionine-co-repressor was added, and this mixture was incubated in HEMG buffer for 4 h at 4°C. The reactions mixtures were centrifuged, and the pellets were washed thoroughly with cold binding buffer. Bound proteins were separated on a 12% SDS-PAGE gel, and the gels were exposed to x-ray film using an image-intensifying screen (Kodak). Chromatin Immunoprecipitation Assays—The binding of FBI-1 to the FRE elements on the Rb promoter in vivo was analyzed by ChIP assays. Sub-confluent HeLa cells on a 10-cm dish were transfected with 3μg of pcDNA3.0 or pcDNA3.0-FBI-1-FLAG using Lipofectamine Plus (Invitrogen) and grown for an additional 48 h. The HeLa cells were treated with formaldehyde (final 1%) to cross-link FBI-1totheRbpromoter. The remainder of the ChIP assay procedure was performed as reported previously (14Lee D.K. Suh D. Edenberg H.J. Hur M.W. J. Biol. Chem. 2002; 277: 26761-26768Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). To amplify the proximal and distal promoter regions of the Rb gene, PCR reactions of the immunoprecipitated DNA were carried out using the following sets of oligonucleotide primers: distal FRE region (bp –370 to –147: forward primer, 5′-CACTAGCCAGATATTCCCTGCGGGG-3′ and reverse primer, 5′-TAAGTCATGAGGAATTAAACTGGGA-3′), proximal GC-box/FRE region (bp –131 to +93: forward primer, 5′-CACCGACCAGCGCCCCAGTTCCCCA-3′andreverseprimer, 5′-GGGAGGACGCGCGCGCACGTCG-3′) and downstream 3′ UTR region (bp +176326 to +176626: forward primer, 5′-GGATCTCAGGACCTTGGTGG-3′ and reverse primer, 5′-AGGGCCATTCTTACTATCCA-3′). The binding competition between FBI-1 and Sp1 on the Rb promoter in vivo were analyzed by ChIP assays in the HEK293A cells transfected with pcDNA3.1-FBI-1 expression vector (1, 2, and 4 μg) or knockdown siRNA and also in Drosophila SL2 cells. Drosophila SL2 cells lacking mammalian transcription factors were transfected with pGL2-Rb-Luc, pPac-PL, pPac-Sp1 (2 μg), and pPac-FBI-1 (1, 2, 4 μg) using Cellfectin (Invitrogen) and grown for an additional 48 h. The cells were fixed with formaldehyde (final 1%) to cross-link FBI-1 and Sp1 protein onto the Rb promoter. The remaining ChIP procedure was performed as described previously (14Lee D.K. Suh D. Edenberg H.J. Hur M.W. J. Biol. Chem. 2002; 277: 26761-26768Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). PCR was performed using the following cycling conditions: denaturation at 94 °C for 5 min, followed by 35 cycles of 94 °C for 30 s, 55 °C for 30 s, 72 °C for 30 s, and a final extension reaction at 72 °C for 5 min. We investigated whether co-repressor (BCoR) binding to the Rb promoter is dependent on FBI-1 using ChIP. HEK293A cells grown on 10-cm dishes were transfected with pcDNA3-FLAG-FBI-1 (3 μg) and/or pCMV-BCoR (3 μg). Chromatins were immunoprecipitated using anti-Myc or anti-FLAG antibody; the remaining ChIP procedures were performed as described above. Oligonucleotide primers used to amplify the distal FRE cluster region (bp –370 to –147) are described above. We also investigated whether the acetylation status of histones H3 and H4 of nucleosomes at the proximal Rb gene promoter are modified by ectopic FBI-1 using antibodies specific to Ac-Histone H3 and Ac-Histone H4 (Upstate). The oligonucleotide primers used to amplify the Rb gene promoter region (bp –133 to +167) flanking the transcription start site were as follows: forward, 5′-CACCGACCAGCGCCCCAGTTCCCCA-3′; reverse, 5′-CCTGACGAGAGGCAGGTCCT-3′. Knockdown of FBI-1 mRNA by siRNA—The siRNAs designed to knock down glyceraldehyde-3-phosphate dehydrogenase and FBI-1 were purchased from Ambion Inc. (Austin, TX). Their sequences were as follows: siFBI-1 #1, 5′-GCUGGACCUUGUAGAUCAAtt-3′ and 5′-UUGAUCUACAAGGUCCAGCtt-3′; siFBI-1 #2, 5′-AGUACCUCGAGUUCUUCCAtt-3 and 5′-UGGAAGAACUCGAGGUACUtt-3′. The siRNAs for FBI-1 (20 ng of each) were transfected into 6 × 106 HEK293A cells using Lipofectamine 2000 (Invitrogen). After culturing for 72 h, cells were harvested, and analyzed for protein and mRNA expression by Western blot and RT-PCR, respectively. Co-immunoprecipitation Assays—The FLAG-FBI-1 and Myc-BCoR expression vectors were co-transfected, and cells were washed, pelleted, and resuspended in lysis buffer supplemented with protease inhibitors (20 mm Tris-HCl, pH 7.5, 150 mm NaCl, 10% glycerol, 1% Triton X-100). Cell lysates were pre-cleared, and the supernatant was incubated with anti-Myc antibody on a rotating platform overnight at 4 °C, followed by incubation with protein A-agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA). Beads were collected, washed, and resuspended in equal volume of 5× SDS loading buffer (10% SDS, 10 mm dithiothreitol, 20% glycerol, 0.2 m Tris-HCl, pH 6.8, 0.05% Bromphenol Blue). Immunoprecipitated proteins were separated by 12% SDS-PAGE. Western blot analysis was performed as described under "Western Blot Analysis." Reverse Transcription and PCR—Total RNA was isolated from the cells using TRIzol® reagent (Invitrogen). The DNAs were synthesized in a 20-μl reaction tube containing 5 μg of total RNA, 10 pmol of random hexamer, and 200 units of superscript reverse transcriptase II. PCR cycling conditions were as follows: denaturation at 94 °C for 5 min, 23 cycles of at 94 °C for 30 s, 62 °C for 30 s, and 72 °C for 40 s, and a final extension at 72 °C for 7 min. The PCR primers used for FBI-1 cDNA were 5′-GGCCTGCTGTGCGACGTGGT-3′ and 5′-CAGCAGGCGGGCGGCGCTGA-3′. The primers for Rb were 5′-AAAGAAAAAGGAACTGTGGG-3′ and 5′-AACTGCTGGGTTGTGTCAAA-3′. The primers for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were 5′-ACCACAGTCCATGCCATCAC-3′ and 5′-TCCACCACCCTGTTGCTGTA-3′. Western Blot Analysis of Rb and FBI-1—Cells were harvested and lysed in TEN buffer (10 mm Tris-HCl, pH 8.0, 1 mm EDTA, and 0.1 m NaCl). Cell extracts (40 μg) were separated on a 10% SDS-PAGE gel, transferred to Immun-Blot™ polyvinylidene difluoride membrane (Bio-Rad), and blocked with 5% skim milk (BD Biosciences) in TBST (20 mm Tris-HCl, pH 7.5, 140 mm NaCl, and 0.001% Tween 20) for 10 min. Blotted membranes were incubated with Ab-FBI-1 overnight at 4 °C (Abcam), Ab-Rb (BD Biosciences), GAPDH (Calbiochem), and Ab-α-tubulin (Calbiochem). Membranes were further incubated with horseradish peroxidase-conjugated anti-mouse IgG (Vector) or anti-rabbit IgG (Vector) or anti-goat IgG (Santa Cruz Biotechnology), and developed with ECL reagents (PerkinElmer). C2C12 Myoblast Cell Culture, Differentiation, and Western Blot Analysis—Mouse C2C12 myoblast cells were maintained in DMEM (Invitrogen) supplemented with 10% fetal bovine serum. Stable C2C12 cells overexpressing FBI-1 were prepared by transfecting C2C12 cells with a recombinant LentiM1.4-FBI-1-FLAG virus. To induce differentiation of the C2C12 cells into myotubes, medium was switched to DMEM with 2% horse serum as described elsewhere (12Sekimata M. Homma Y. Biochem. Biophys. Res. Commun. 2004; 325: 653-659Crossref PubMed Scopus (6) Google Scholar). For the Rb rescue experiment, recombinant adenovirus overexpression Rb was purchased from Vector Biolabs (Philadelphia, PA) and infected at 100 multiplicity of infection. FBI-1 Represses Transcription of the Rb Gene—FBI-1 shows proto-oncogenic activity by repressing the expression of the tumor suppressor ARF gene (21Maeda 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 (291) Google Scholar). We suspected that ARF might not be the only target gene of transcription repression and that FBI-1 may have multiple additional target genes by which it can exert oncogenic activity (22Maeda T. Hobbs R.M. Cancer Res. 2005; 65: 8575-8578Crossref PubMed Scopus (91) Google Scholar and references therein). We investigated whether other tumor suppressor genes, such as the Rb gene, which is critically involved in cell cycle control and tumor suppression, is the target of FBI-1. Using the recently characterized FBI-1 consensus sequence, we identified four potential FBI-1 binding sites (FRE) on the Rb promoter. The four potential FREs are GC-rich and have varying degrees of homology with the consensus FRE sequence with either perfect homology or with one or two mismatches (Fig. 1A) (21Maeda 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 (291) Google Scholar, 26Pessler F. Hernandez N. J. Biol. Chem. 2003; 278: 29327-29335Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). We transiently co-transfected HeLa cells with two FBI-1 expression plasmids (pcDNA3-FBI-1 and pcDNA3-FBI-1ΔPOZ) and the pGL2-Rb-Luc plasmid, and analyzed the luciferase activity of these transfected cells. FBI-1 repressed transcription of Rb by >50% relative to the control. FBI-1 lacking the POZ-domain had a much weaker effect, repressing the transcription of Rb by 18% relative to the control, suggesting that the POZ-domain is important in transcriptional repression of Rb gene (Fig. 1B). In HeLa cells, human papilloma virus E7 is known to influence the activity of Rb. One may argue that FBI-1 affects papilloma virus E7 and thereby affects Rb expression. Consequently, we carried out transcription assays in human colon cancer HCT116 cells and HEK293A cells, which do not have papilloma virus E7. In these two cell types, FBI-1 also repressed Rb gene transcription, suggesting that FBI-1 directly acts on the Rb promoter to repress transcription (Fig. 1B). We also investigated whether FBI-1 represses Rb gene expression in stable HeLa and HCT116 cells overexpressing FBI-1 due to transfection with the recombinant control Lentivirus, LentiM1.4-FBI-1-FLAG. RT-PCR and Western blot analysis showed that FBI-1 decreased the mRNA and protein levels of the Rb gene, respectively, compared with those of the control cells transfected with the control Lentivirus-LacZ virus (Fig. 1, C and D, and supplement Fig. S1). Alternatively, we tried to show whether knockdown of endogenous FBI-1 mRNA expression could increase transcription of Rb by removing the transcription repression by FBI-1. Successful knockdown of endogenous FBI-1 mRNA by RNA interference using two independent siRNAs resulted in an increase in Rb gene transcription (Fig. 1E). RT-PCR and Western blots showed that siRNA treatment resulted in a significant decrease in endogenous FBI-1 mRNA and FBI-1 protein and a concomitant increase in Rb mRNA and protein expression, respectively (Fig. 1, E–G). These data suggested that

Referência(s)