Proto-oncogene FBI-1 Represses Transcription of p21CIP1 by Inhibition of Transcription Activation by p53 and Sp1
2009; Elsevier BV; Volume: 284; Issue: 19 Linguagem: Inglês
10.1074/jbc.m809794200
ISSN1083-351X
AutoresWon‐Il Choi, Bu-Nam Jeon, Chae‐Ok Yun, Pyung-Hwan Kim, Sung‐Eun Kim, Kang‐Yell Choi, Se Hoon Kim, Man‐Wook Hur,
Tópico(s)Epigenetics and DNA Methylation
ResumoAberrant transcriptional repression through chromatin remodeling and histone deacetylation has been postulated as the driving force for tumorigenesis. FBI-1 (formerly called Pokemon) is a member of the POK family of transcriptional repressors. Recently, FBI-1 was characterized as a critical oncogenic factor that specifically represses transcription of the tumor suppressor gene ARF, potentially leading indirectly to p53 inactivation. Our investigations on transcriptional repression of the p53 pathway revealed that FBI-1 represses transcription of ARF, Hdm2 (human analogue of mouse double minute oncogene), and p21CIP1 (hereafter indicated as p21) but not of p53. FBI-1 showed a more potent repressive effect on p21 than on p53. Our data suggested that FBI-1 is a master controller of the ARF-Hdm2-p53-p21 pathway, ultimately impinging on cell cycle arrest factor p21, by inhibiting upstream regulators at the transcriptional and protein levels. FBI-1 acted as a competitive transcriptional repressor of p53 and Sp1 and was shown to bind the proximal Sp1–3 GC-box and the distal p53-responsive elements of p21. Repression involved direct binding competition of FBI-1 with Sp1 and p53. FBI-1 also interacted with corepressors, such as mSin3A, NCoR, and SMRT, thereby deacetylating Ac-H3 and Ac-H4 histones at the promoter. FBI-1 caused cellular transformation, promoted cell cycle proliferation, and significantly increased the number of cells in S phase. FBI-1 is aberrantly overexpressed in many human solid tumors, particularly in adenocarcinomas and squamous carcinomas. The role of FBI-1 as a master controller of the p53 pathway therefore makes it an attractive therapeutic target. Aberrant transcriptional repression through chromatin remodeling and histone deacetylation has been postulated as the driving force for tumorigenesis. FBI-1 (formerly called Pokemon) is a member of the POK family of transcriptional repressors. Recently, FBI-1 was characterized as a critical oncogenic factor that specifically represses transcription of the tumor suppressor gene ARF, potentially leading indirectly to p53 inactivation. Our investigations on transcriptional repression of the p53 pathway revealed that FBI-1 represses transcription of ARF, Hdm2 (human analogue of mouse double minute oncogene), and p21CIP1 (hereafter indicated as p21) but not of p53. FBI-1 showed a more potent repressive effect on p21 than on p53. Our data suggested that FBI-1 is a master controller of the ARF-Hdm2-p53-p21 pathway, ultimately impinging on cell cycle arrest factor p21, by inhibiting upstream regulators at the transcriptional and protein levels. FBI-1 acted as a competitive transcriptional repressor of p53 and Sp1 and was shown to bind the proximal Sp1–3 GC-box and the distal p53-responsive elements of p21. Repression involved direct binding competition of FBI-1 with Sp1 and p53. FBI-1 also interacted with corepressors, such as mSin3A, NCoR, and SMRT, thereby deacetylating Ac-H3 and Ac-H4 histones at the promoter. FBI-1 caused cellular transformation, promoted cell cycle proliferation, and significantly increased the number of cells in S phase. FBI-1 is aberrantly overexpressed in many human solid tumors, particularly in adenocarcinomas and squamous carcinomas. The role of FBI-1 as a master controller of the p53 pathway therefore makes it an attractive therapeutic target. The BTB/POZ 2The abbreviations used are: BTB/POZ, bric-à-brac, tramtrack, broad complex/poxvirus and zinc finger; ARF, alternative reading frame; ChIP, chromatin immunoprecipitation; EMSA, electromobility shift assay; FACS, fluorescence-activated cell sorter; FBI-1, factor that binds to the inducer of short transcripts of human immunodeficiency virus-1; FBS, fetal bovine serum; FRE, FBI-1 response element; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GST, glutathione S-transferase; NCoR, nuclear receptor corepressor; PLZF, promyelocytic leukemia zinc finger protein; SMRT, silencing mediator for retinoid and thyroid receptors; Sp1, specificity protein 1; WT, wild type; ZFDBD, zinc finger DNA binding domain; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; GFP, green fluorescent protein; siRNA, small interfering RNA; Rb, retinoblastoma; TSA, trichostatin A; HDAC, histone deacetylase. 2The abbreviations used are: BTB/POZ, bric-à-brac, tramtrack, broad complex/poxvirus and zinc finger; ARF, alternative reading frame; ChIP, chromatin immunoprecipitation; EMSA, electromobility shift assay; FACS, fluorescence-activated cell sorter; FBI-1, factor that binds to the inducer of short transcripts of human immunodeficiency virus-1; FBS, fetal bovine serum; FRE, FBI-1 response element; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GST, glutathione S-transferase; NCoR, nuclear receptor corepressor; PLZF, promyelocytic leukemia zinc finger protein; SMRT, silencing mediator for retinoid and thyroid receptors; Sp1, specificity protein 1; WT, wild type; ZFDBD, zinc finger DNA binding domain; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; GFP, green fluorescent protein; siRNA, small interfering RNA; Rb, retinoblastoma; TSA, trichostatin A; HDAC, histone deacetylase. domain, originally identified in Drosophila melanogaster bric-à-brac, tramtrack, and broad complex transcription regulators, and in pox virus zinc finger proteins (1Koonin E.V. 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They also demonstrated that LRF is a key for instructing early lymphoid progenitors in mice to develop into B lineage cells by repressing T cell-instructive signals produced by the cell-fate signal protein, Notch (30Maeda T. Merghoub T. Hobbs R.M. Dong L. Maeda M. Zakrzewski J. van den Brink M.R. Zelent A. Shigematsu H. Akashi K. Teruya-Feldstein J. Cattoretti G. Pandolfi P.P. Science. 2007; 316: 860-866Crossref PubMed Scopus (174) Google Scholar). Even more recently, we have shown that FBI-1 represses transcription of the tumor suppressor Rb gene, and we also demonstrated that FBI-1 blocked differentiation of mouse C2C12 myoblast cells into myotubes by repressing transcription of the Rb gene (19Jeon B.N. Yoo J.Y. Choi W.I. Lee C.E. Yoon H.G. Hur M. J. Biol. Chem. 2008; 283: 33199-33210Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Also, we have demonstrated that expression of FASN (fatty-acid synthase), which is important in palmitate synthesis and cell proliferation in cancer cells, is potently activated by FBI-1 in the presence of SREBP-1 (20Choi 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). FBI-1 is overexpressed in some human cancers, adipose tissues isolated from genetically obese mice, and diet-induced obese mice, and prostate LNCaP cancer cells treated with androgen (thus growing fast) (8Maeda 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 (287) Google Scholar, 20Choi 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. 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Other major regulators that affect p21 gene expression are the Sp1 family transcription factors, which bind to the proximal promoter (38Koutsodontis G. Tentes I. Papakosta P. Moustakas A. Kardassis D. J. Biol. Chem. 2001; 276: 29116-29125Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 39Kardassis D. Papakosta P. Pardali K. Moustakas A. J. Biol. Chem. 1999; 274: 29572-29581Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). The Sp1–3 GC-box bound by Sp1 has been shown to be particularly important; mutation of the site nearly eliminates transcription, and it also disrupts the synergistic transcriptional activation by Sp1 and p53 (38Koutsodontis G. Tentes I. Papakosta P. Moustakas A. Kardassis D. J. Biol. Chem. 2001; 276: 29116-29125Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar). 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Chem. 2005; 280: 28061-28071Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 41Kwon H.S. Kim M.S. Edenberg H.J. Hur M.W. J. Biol. Chem. 1999; 274: 20-28Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 42Kaczynski J. Cook T. Urrutia R. Genome Biol. 2004; 4: 206.1-206.8Google Scholar, 43Lomberk G. Urrutia R. Biochem. J. 2005; 392: 1-11Crossref PubMed Scopus (162) Google Scholar). Expression of proto-oncogenic FBI-1 is increased in multiple cancers (8Maeda 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 (287) Google Scholar, 44Maeda T. Hobbs R.M. Pandolfi P.P. Cancer Res. 2005; 65: 8575-8578Crossref PubMed Scopus (90) Google Scholar). FBI-1 was recently shown to repress the tumor suppressor gene ARF, which in turn lowers expression of the tumor suppressor gene p53. FBI-1 overexpression causes oncogenesis in the thymus, liver, and spleen (8Maeda 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 (287) Google Scholar). Because FBI-1 is also overexpressed in solid tumors, such as cancers of the colon and bladder, where the normal functions of ARF and p53 are lost (8Maeda 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 (287) Google Scholar, 44Maeda T. Hobbs R.M. Pandolfi P.P. Cancer Res. 2005; 65: 8575-8578Crossref PubMed Scopus (90) Google Scholar), it is likely that FBI-1 has additional target genes through which it exerts its oncogenic activity. In this study, we investigated whether the ARF-Hdm2-p53-p21 pathway (referred to as the p53 pathway) (45Toledo F. Wahl G.M. Nat. Rev. Cancer. 2006; 6: 909-923Crossref PubMed Scopus (1040) Google Scholar), an important element in cell cycle control and oncogenesis, is controlled by FBI-1; in particular, we focused on the cyclin-dependent kinase inhibitor p21 gene. Furthermore, we investigated the mechanism and physiological consequence of FBI-1 action. Our data suggest that FBI-1 is a master regulator of the p53 pathway and plays a critical role in regulating important biological processes controlled by p21 and other members of the p53 pathway, such as oncogenic cellular transformation, cell growth, and proliferation. Plasmids, Antibodies, and Reagents—pGL2-p21-Luc, pGL2-p21-Luc WT –131 bp, and pGL2-p21-Luc WT –101 bp were kindly provided by Dr. Yoshihiro Sowa (Kyoto Perpetual University of Medicine, Japan). Constructions of pcDNA3-FBI-1 and pcDNA3-FBI-1ΔPOZ are reported elsewhere (21Lee 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). Human p53 cDNA was cloned into pcDNA3.1 (Invitrogen) to prepare pcDNA3.1-p53. Sp1ZFDBD (amino acids 622–778) and FBI-1ZFDBD (amino acids 382–490) were prepared by cloning PCR-amplified cDNA fragments into pGEX4T1. To prepare GST-POZFBI-1, a cDNA fragment encoding the POZ domain of FBI-1 was cloned into pGEX4T3. The lentiviruses M1.4-FBI-1-FLAG, -GFP, or -LacZ were obtained from Vectorcorea (Korea). The sequences of all the constructs used in this study were verified. Various pGL2 promoter-Luc and expression vectors for p53 and FBI-1 were prepared by standard cloning procedures. Various purified recombinant proteins were prepared from Escherichia coli BL21(DE3) transformed with pGEX4T1 protein expression vectors (Amersham Biosciences). Primary and secondary antibodies were obtained from Upstate (Charlottesville, VA), Chemicon (Temecula, CA), Calbiochem, Santa Cruz Biotechnology (Santa Cruz, CA), Dako, Vector Laboratories, and Abcam (Cambridge, UK). Lipofectamine reagents (Invitrogen) were used for transfection. Buffer recipes, PCR amplification procedures, siRNA sequences, and primer sequences are available upon request. Unless otherwise noted, all other chemical reagents were purchased from Sigma. Cell Culture—Human osteosarcoma Saos-2 cell lines, lacking endogenous p53, were purchased from ATCC (Manassas, VA). Saos-2 cells were cultured in McCoy's 5A medium containing 15% fetal bovine serum. All other cell lines were cultured in Dulbecco's modified Eagles medium containing 10% fetal bovine serum (Invitrogen). HeLa cells cultured with Polybrene (8 μg/ml) were incubated (37 °C, 5% CO2) for 6–8 h with 350 μl of 4 × 107 transducing units/ml LentiM1.4-FLAG-FBI-1 (experimental) or LentiM1.4eGFP and LentiM1.4LacZ (control). NIH/3T3 cells were infected with His- and Myc-tagged LentiM1.4-FBI-1 (experimental) or LentiM1.4 (control). Puromycin was used to select stable cells after 2–3 days. Doxycycline (1 μg/ml)-inducible FBI-1-overexpressing cells were prepared by transfecting mammalian Flp-In T-REx host HEK293T cells (Invitrogen) with pOG44:pcDNA5/FRT/TO-FBI-1 plasmid DNA (9:1). Stable FBI-1-overexpressing cells were selected with hygromycin (300 μg/ml) and blasticidin (15 μg/ml). Transcriptional Analysis—pGL2-Hdm2-Luc, pGL2-ARF-Luc, and pGL2-p53-Luc were prepared by cloning the promoter fragments into pGL2-Luc Basic (Promega, WI). WT or mutant p21-Luc reporter plasmids, pcDNA3-FBI-1, pcDNA3.1-p53, and pCMV-LacZ were transiently transfected into HeLa cells. Luciferase activity, measured at 36 h, was normalized by cotransfected β-galactosidase activity or protein concentration. Chromatin Immunoprecipitation (ChIP)—A ChIP assay (Upstate) was used to investigate whether FBI-1 and FRE interact in vivo. Subconfluent HeLa cells and Saos-2 cells were transfected for 48 h with 1 μg of pGL2-p21-Luc and 3 μg of either pcDNA3 or pcDNA3-FBI-1-FLAG. HeLa cells and Drosophila SL2 cells were fixed with 1% formaldehyde, washed, lysed with SDS lysis buffer, and sonicated into DNA fragments of 500–1000 bp. Rest of ChIP assays was performed as described elsewhere (19Jeon B.N. Yoo J.Y. Choi W.I. Lee C.E. Yoon H.G
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