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CDCA4 Is an E2F Transcription Factor Family-induced Nuclear Factor That Regulates E2F-dependent Transcriptional Activation and Cell Proliferation

2006; Elsevier BV; Volume: 281; Issue: 47 Linguagem: Inglês

10.1074/jbc.m603800200

ISSN

1083-351X

Autores

Reiko Hayashi, Yuya Goto, Ryuji Ikeda, Kazunari K. Yokoyama, Kenichi Yoshida,

Tópico(s)

Epigenetics and DNA Methylation

Resumo

The TRIP-Br1/p34SEI-1 family proteins participate in cell cycle progression by coactivating E2F1- or p53-dependent transcriptional activation. Here, we report the identification of human CDCA4 (also know as SEI-3/Hepp) as a novel target gene of transcription factor E2F and as a repressor of E2F-dependent transcriptional activation. Analysis of CDCA4 promoter constructs showed that an E2F-responsive sequence in the vicinity of the transcription initiation site is necessary for the E2F1–4-induced activation of CDCA4 gene transcription. Chromatin immunoprecipitation analysis demonstrated that E2F1 and E2F4 bound to an E2F-responsive sequence of the human CDCA4 gene. Like TRIP-Br1/p34SEI-1 and TRIP-Br2 (SEI-2), the transactivation domain of CDCA4 was mapped within C-terminal acidic region 175–241. The transactivation function of the CDCA4 protein was inhibited by E2F1–4 and DP2, but not by E2F5–8. Inhibition of CDCA4 transactivation activity by E2F1 partially interfered with retinoblastoma protein overexpression. Conversely, CDCA4 suppressed E2F1–3-induced reporter activity. CDCA4 (but not acidic region-deleted CDCA4) suppressed E2F1-regulated gene promoter activity. These findings suggest that the CDCA4 protein functions as a suppressor at the E2F-responsive promoter. Small interfering RNA-mediated knockdown of CDCA4 expression in cancer cells resulted in up-regulation of cell growth rates and DNA synthesis. The CDCA4 protein was detected in several human cells and was induced as cells entered the G1/S phase of the cell cycle. Taken together, our results suggest that CDCA4 participates in the regulation of cell proliferation, mainly through the E2F/retinoblastoma protein pathway. The TRIP-Br1/p34SEI-1 family proteins participate in cell cycle progression by coactivating E2F1- or p53-dependent transcriptional activation. Here, we report the identification of human CDCA4 (also know as SEI-3/Hepp) as a novel target gene of transcription factor E2F and as a repressor of E2F-dependent transcriptional activation. Analysis of CDCA4 promoter constructs showed that an E2F-responsive sequence in the vicinity of the transcription initiation site is necessary for the E2F1–4-induced activation of CDCA4 gene transcription. Chromatin immunoprecipitation analysis demonstrated that E2F1 and E2F4 bound to an E2F-responsive sequence of the human CDCA4 gene. Like TRIP-Br1/p34SEI-1 and TRIP-Br2 (SEI-2), the transactivation domain of CDCA4 was mapped within C-terminal acidic region 175–241. The transactivation function of the CDCA4 protein was inhibited by E2F1–4 and DP2, but not by E2F5–8. Inhibition of CDCA4 transactivation activity by E2F1 partially interfered with retinoblastoma protein overexpression. Conversely, CDCA4 suppressed E2F1–3-induced reporter activity. CDCA4 (but not acidic region-deleted CDCA4) suppressed E2F1-regulated gene promoter activity. These findings suggest that the CDCA4 protein functions as a suppressor at the E2F-responsive promoter. Small interfering RNA-mediated knockdown of CDCA4 expression in cancer cells resulted in up-regulation of cell growth rates and DNA synthesis. The CDCA4 protein was detected in several human cells and was induced as cells entered the G1/S phase of the cell cycle. Taken together, our results suggest that CDCA4 participates in the regulation of cell proliferation, mainly through the E2F/retinoblastoma protein pathway. The E2F family of transcription factors integrates cellular signals and coordinates cell proliferation (1Nevins J.R. Cell Growth & Differ. 1998; 9: 585-593PubMed Google Scholar, 2Dyson N. Genes Dev. 1998; 12: 2245-2262Crossref PubMed Scopus (1988) Google Scholar). Studies in recent years have identified E2Fs as important transcriptional regulators of the expression of many genes involved not only in DNA replication and cell cycle progression, but also in DNA damage repair, apoptosis, and cell differentiation and development (3Dimova D.K. Dyson N.J. Oncogene. 2005; 24: 2810-2826Crossref PubMed Scopus (612) Google Scholar, 4Bracken A.P. Ciro M. Cocito A. Helin K. Trends Biochem. Sci. 2004; 29: 409-417Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar). Among the E2F family members, E2F1–5 possess a transcriptional activation domain at the C terminus and can induce transcription from target promoters together with dimerization partner DP1 or DP2 (5La Thangue N.B. Curr. Opin. Cell Biol. 1994; 6: 443-450Crossref PubMed Scopus (142) Google Scholar, 6Slansky J.E. Farnham P.J. Curr. Top. Microbiol. Immunol. 1996; 208: 1-30Crossref PubMed Scopus (239) Google Scholar). In contrast, E2F6 lacks a transcriptional activation domain and has been shown to compete for E2F-binding sites on promoters and to repress their activity (7Trimarchi J.M. Lees J.A. Nat. Rev. Mol. Cell Biol. 2002; 3: 11-20Crossref PubMed Scopus (973) Google Scholar). The E2F family is often subdivided into activator E2Fs (E2F1–3) and repressor E2Fs (E2F4–6) based on the pattern of their interactions with retinoblastoma tumor suppressor pocket-binding protein (pRb) 2The abbreviations used are: pRb, retinoblastoma tumor suppressor pocket-binding protein; HDAC, histone deacetylase; CBP, cAMP-responsive element-binding protein-binding protein; PHD, plant homeodomain; siRNA, small interfering RNA; DBD, DNA-binding domain; RT, reverse transcription; TAD, transactivation domain; GST, glutathione S-transferase; GFP, green fluorescent protein; HA, hemagglutinin; RNAi, RNA interference; FBS, fetal bovine serum; BrdUrd, 5-bromo-2′-deoxyuridine; PBS, phosphate-buffered saline; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; ChIP, chromatin immunoprecipitation; BisTris, 2-[bis(2-hydroxyethyl)-amino]-2-(hydroxymethyl)propane-1,3-diol; MES, 4-morpholineethanesulfonic acid. family members (8Chellappan S.P. Hiebert S. Mudryj M. Horowitz J.M. Nevins J.R. Cell. 1991; 65: 1053-1061Abstract Full Text PDF PubMed Scopus (1107) Google Scholar, 9DeGregori J. Biochim. Biophys. Acta. 2002; 1602: 131-150PubMed Google Scholar). The pRb proteins contact directly with the E2F C-terminal activation domain and silence it (10Helin K. Wu C.L. Fattaey A.R. Lees J.A. Dynlacht B.D. Ngwu C. Harlow E. Genes Dev. 1993; 7: 1850-1861Crossref PubMed Scopus (421) Google Scholar, 11Flemington E.K. Speck S.H. Kaelin W.G.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6914-6918Crossref PubMed Scopus (290) Google Scholar). E2F1–3 can all bind exclusively to pRb, but the pRb relatives p107 and p130 interact specifically with repressor E2Fs. Among repressor E2Fs, E2F4 uniquely interacts with the pRb, p107, and p130 proteins (9DeGregori J. Biochim. Biophys. Acta. 2002; 1602: 131-150PubMed Google Scholar). E2F7 and E2F8, very recently identified as members of the E2F family, share unique structural features, including the absence of dimerization, pRb binding, and transcriptional activation domains (12de Bruin A. Maiti B. Jakoi L. Timmers C. Buerki R. Leone G. J. Biol. Chem. 2003; 278: 42041-42049Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar, 13Di Stefano L. Jensen M.R. Helin K. EMBO J. 2003; 22: 6289-6298Crossref PubMed Scopus (216) Google Scholar, 14Logan N. Delavaine L. Graham A. Reilly C. Wilson J. Brummelkamp T.R. Hijmans E.M. Bernards R. La Thangue N.B. Oncogene. 2004; 23: 5138-5150Crossref PubMed Scopus (85) Google Scholar, 15Maiti B. Li J. de Bruin A. Gordon F. Timmers C. Opavsky R. Patil K. Tuttle J. Cleghorn W. Leone G. J. Biol. Chem. 2005; 280: 18211-18220Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 16Logan N. Graham A. Zhao X. Fisher R. Maiti B. Leone G. La Thangue N.B. Oncogene. 2005; 24: 5000-5004Crossref PubMed Scopus (98) Google Scholar, 17Christensen J. Cloos P. Toftegaard U. Klinkenberg D. Bracken A.P. Trinh E. Heeran M. Di Stefano L. Helin K. Nucleic Acids Res. 2005; 33: 5458-5470Crossref PubMed Scopus (131) Google Scholar). E2F1 binds DNA as a heterodimer with the DP1 or DP2 protein and achieves activation through a C-terminal activation domain (10Helin K. Wu C.L. Fattaey A.R. Lees J.A. Dynlacht B.D. Ngwu C. Harlow E. Genes Dev. 1993; 7: 1850-1861Crossref PubMed Scopus (421) Google Scholar, 18Qin X.Q. Chittenden T. Livingston D.M. Kaelin W.G.J. Genes Dev. 1992; 6: 953-964Crossref PubMed Scopus (358) Google Scholar), which has been shown to interact directly with coactivators such as TATA-binding protein and MDM2 (19Hagemeier C. Cook A. Kouzarides T. Nucleic Acids Res. 1993; 21: 4998-5004Crossref PubMed Scopus (114) Google Scholar, 20Martin K. Trouche D. Hagemeier C. Sorensen T.S. La Thangue N.B. Kouzarides T. Nature. 1995; 375: 691-694Crossref PubMed Scopus (452) Google Scholar). Many transcription factors recruit histone acetyltransferase and histone deacetylase (HDAC) activities. The appearance of histone acetyltransferases, including cAMP-responsive element-binding protein-binding protein (CBP), p300, p300/CBP-associated factor (PCAF), and Tip60, correlates with the timing of induction of E2F-dependent transcription (21Ferreira R. Naguibneva I. Mathieu M. Ait-Si-Ali S. Robin P. Pritchard L.L. Harel-Bellan A. EMBO Rep. 2001; 2: 794-799Crossref PubMed Scopus (87) Google Scholar, 22Rayman J.B. Takahashi Y. Indjeian V.B. Dannenberg J.H. Catchpole S. Watson R.J. te Riele H. Dynlacht B.D. Genes Dev. 2002; 16: 933-947Crossref PubMed Scopus (248) Google Scholar, 23Taubert S. Gorrini C. Frank S.R. Parisi T. Fuchs M. Chan H.M. Livingston D.M. Amati B. Mol. Cell. Biol. 2004; 24: 4546-4556Crossref PubMed Scopus (171) Google Scholar). Indeed, p300 and CBP interact with the activation domain of E2F1 and stimulate E2F1-mediated activation as coactivators (24Trouche D. Cook A. Kouzarides T. Nucleic Acids Res. 1996; 24: 4139-4145Crossref PubMed Scopus (104) Google Scholar, 25Morris L. Allen K.E. La Thangue N.B. Nat. Cell Biol. 2000; 2: 232-239Crossref PubMed Scopus (124) Google Scholar). On the other hand, HDAC1–3 activity is implicated in the pRb-mediated repression of E2F-regulated promoters in the G1 phase (26Harbour J.W. Dean D.C. Genes Dev. 2000; 14: 2393-2409Crossref PubMed Scopus (966) Google Scholar, 27Brehm A. Miska E.A. McCance D.J. Reid J.L. Bannister A.J. Kouzarides T. Nature. 1998; 391: 597-601Crossref PubMed Scopus (1084) Google Scholar, 28Brehm A. Kouzarides T. Trends Biochem. Sci. 1999; 24: 142-145Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). To achieve cell cycle arrest into the G1 phase, pRb also requires the activity of Brg1/Brm, the two known human homologs of the yeast nucleosome-remodeling complex SWI2/SNF2 (29Dunaief J.L. Strober B.E. Guha S. Khavari P.A. Alin K. Luban J. Begemann M. Crabtree G.R. Goff S.P. Cell. 1994; 79: 119-130Abstract Full Text PDF PubMed Scopus (558) Google Scholar). Prohibitin and TopBP1 are known as Brg1/Brm-dependent E2F1-mediated repressors (30Wang S. Zhang B. Faller D.V. EMBO J. 2002; 21: 3019-3028Crossref PubMed Scopus (115) Google Scholar). TopBP1, a DNA replication initiator/DNA repair protein, is induced by E2F1 and recruits Brg1/Brm to repress E2F1-induced apoptosis (31Liu K. Luo Y. Lin F.-T. Lin W.-C. Genes Dev. 2004; 18: 673-686Crossref PubMed Scopus (125) Google Scholar). Among transcriptional regulator proteins, TRIP-Br1/p34SEI-1 (hereafter referred to as TRIP-Br1; transcriptional regulator interacting with PHD bromodomain 1) belongs to a novel family of proteins that share the N-terminal SERTA (for SEI-1, RBT1, and TARA) motif (32Calgaro S. Boube M. Cribbs D.L. Bourbon H.M. Genetics. 2001; 160: 547-560Crossref Google Scholar). TRIP-Br1 was originally reported to activate cyclin D1-Cdk4 by antagonizing the inhibitory effect of p16INK4a during the late G1 phase of the cell cycle (33Sugimoto M. Nakamura T. Ohtani N. Hampson L. Hampson I.N. Shimamoto A. Furuichi Y. Okumura K. Niwa S. Taya Y. Hara E. Genes Dev. 1999; 13: 3027-3033Crossref PubMed Scopus (94) Google Scholar). The SERTA motif has been shown to be responsible for the interaction between TRIP-Br1 and Cdk4. Interestingly, TRIP-Br1 and TRIP-Br2 (SEI-2) make a direct functional contact with E2F1/DP1, stimulating E2F1 transcriptional activity (34Hsu S.I. Yang C.M. Sim K.G. Hentschel D.M. O'Leary E. Bonventre J.V. EMBO J. 2001; 20: 2273-2285Crossref PubMed Scopus (94) Google Scholar). RBT1 (replication protein A-binding transactivator 1), a SERTA motif-containing protein, has also been characterized as a transcriptional coactivator, but uniquely binds the second subunit of replication protein A (35Cho J.M. Song D.J. Bergeron J. Benlimame N. Wold M.S. Alaoui-Jamali M.A. Nucleic Acids Res. 2000; 28: 3478-3485Crossref PubMed Scopus (24) Google Scholar). Recently, in addition to TRIP-Br1 and TRIP-Br2, SEI-3 (hereafter referred to as CDCA4, a HUGO Gene Nomenclature Committee-approved gene symbol; characterized previously as murine Hepp (36Abdullah J.M. Jing X. Spassov D.S. Nachtman R.G. Jurecic R. Blood Cells Mol. Dis. 2001; 27: 667-676Crossref PubMed Scopus (21) Google Scholar)) was characterized as a coactivator of p53-dependent transcriptional activation possibly through interacting with CBP or the ING (inhibitor of growth) family of chromatin-associated proteins, although the growth inhibition induced by overexpression of those proteins was p53-independent (37Watanabe-Fukunaga R. Iida S. Shimizu Y. Nagata S. Fukunaga R. Genes Cells. 2005; 10: 851-860Crossref PubMed Scopus (41) Google Scholar). As an approach toward a better understanding of the full extent of gene expression under the control of the E2F/pRb pathway in cell proliferation, we characterized a series of DNA replication initiators (38Yoshida K. Inoue I. Oncogene. 2004; 23: 3802-3812Crossref PubMed Scopus (84) Google Scholar, 39Yoshida K. Inoue I. Oncogene. 2004; 23: 6250-6260Crossref PubMed Scopus (43) Google Scholar), including members of the novel CDCA (cell division cycle-associated) family of genes as candidate genes, using DNA microarray experiments. 3K. Yoshida, unpublished data. The genes of the CDCA family are characterized by the association of their expression patterns with those of known cell cycle genes such as CDC2, CDC7, and cyclins (40Walker M.G. Curr. Cancer Drug Targets. 2001; 1: 73-83Crossref PubMed Scopus (57) Google Scholar). We focused initially on human CDCA4 to determine whether the CDCA4 protein is functionally related to TRIP-Br1 and TRIP-Br2. Interestingly, CDCA4 repressed E2F1-induced transcriptional activation, although TRIP-Br1 and TRIP-Br2 coactivated it. Small interfering RNA (siRNA)-mediated gene knockdown of CDCA4 resulted in an increase in cell proliferation independent of the p53 status. We suggest a model in which CDCA4 functions as a critical modulator of cell proliferation and serves as a negative feedback regulator of activator E2Fs by inhibiting E2F-dependent transcriptional activation. Bioinformatics—Bioinformatics analysis was performed as described previously (41Yoshida K. Biochem. Biophys. Res. Commun. 2005; 331: 669-674Crossref PubMed Scopus (40) Google Scholar). The TRANSFAC program (motif. genome.jp/) was used to determine the transcription factor-binding elements (42Heinemeyer T. Chen X. Karas H. Kel A.E. Kel O.V. Liebich I. Meinhardt T. Reuter I. Schacherer F. Wingender E. Nucleic Acids Res. 1999; 27: 318-322Crossref PubMed Scopus (271) Google Scholar). The amino acid sequences of human TRIP-Br1 family proteins were aligned using the ClustalW Version 1.83 program (clustalw.genome.jp/) (43Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (56228) Google Scholar). The domain structure of the CDCA4 protein was searched using the Pfam program (pfam.wustl.edu/) and BLAST algorithms (www.ncbi.nlm.nih.gov/) (44Altschul S.F. Gish W. Miller W. Myers E.W. Lipman D.J. J. Mol. Biol. 1990; 215: 403-410Crossref PubMed Scopus (72097) Google Scholar). Construction of Plasmids—Human CDCA4 promoter fragments were generated by PCR from genomic DNA and ligated into the KpnI-digested pGL3-Basic vector (Promega Corp.). PCR primers were designed to amplify 1125-bp (–1097/+28; pGL3-ABCD), 875-bp (–847/+28; pGL3-BCD), 775-bp (–747/+28; pGL3-CD), 709-bp (–681/+28; pGL3-D), and 594-bp (–681/–88; pGL3-delD) fragments of the CDCA4 promoter sequences, which are numbered relative to the transcription initiation site at position +1 described in the NCBI Uni-Gene Database (Genome View). The GenBank™ accession number of the genomic clone used for the design of the PCR primers was AL512356. The forward (F) and reverse (R) PCR primers used were as follows: –1097F, 5′-CACGCCACTGTTGTCCAGCC-3′; –847F, 5′-TTTGCCCCAGCCATTCCTCT-3′; –747F, 5′-TCACTCCCAGGTTTCCCGAC-3′; –681F, 5′-CTGCGGATTGATCTGAGGAT-3′; –88R, 5′-CTTCCCGCGCTGCGGCGACG-3′; and +28R, 5′-TCCACAAAGGGTCGACGCTG-3′. KpnI sites were added to the forward and reverse primers to facilitate subcloning. Motif D (5′-TGTGGCGC-3′) was mutated (5′-TGTGAAGC-3′) for pGL3-mutD. To construct the Gal4 DNA-binding domain (DBD) and the CDCA4 fusion protein expression vector, reverse transcription (RT)-PCR was performed as described below. The PCR products were digested with BamHI and EcoRI and ligated into their respective sites in the pCMV-BD vector (Stratagene). The Gen-Bank™ accession number of the cDNA clone used for the design of the PCR primers was NM_145701. The forward (F) and reverse (R) PCR primers used were as follows (with the BamHI sites in the forward primers and the EcoRI sites in the reverse primers underlined): DBD/1F, 5′-CGGGATCCTTTGCACGAGGACTGAAGAGG-3′; DBD/37F, 5′-CGGGATCCCTCCTGGACATGTCTCTG-3′; DBD/75F, 5′-CGGGATCCCAGGATGGGACGTGGCGCACA-3′; DBD/175F, 5′-CGGGATCCAGCTGCATGGAAGAGCTG-3′; DBD/174R, 5′-CGGAATTCTCAGGGGTTTTTAGTCTCCAG-3′; and DBD/241R, 5′-CGGAATTCTCAGGTCTCCACCAGGATCTC-3′. The full-length transactivation domain (TAD) of E2F1 was also cloned into the pCMV-BD vector. The GenBank™ accession number of the cDNA clone used for the design of the PCR primers was NM_005225. The PCR primers used were 5′-CGGGATCCGCCTTGGCCGGGGCCCCT-3′, 5′-CGGGATCCCTTAAGAGCAAACAAGGCCCG-3′, and 5′-CGGAATTCTCAGAAATCCAGGGGGGTGAG-3′ (with the BamHI sites in the forward primers and the EcoRI sites in the reverse primers underlined). The N-terminally FLAG-tagged CDCA4 and glutathione S-transferase (GST)-tagged CDCA4 expression vectors were constructed as follows. Briefly, PCR products were digested with BamHI and EcoRI and ligated into their respective sites in the pCMV-TAG2B (Stratagene) and pGEX-6P-1 (Amersham Biosciences) vectors, respectively. The forward and reverse PCR primers used were DBD/1F and DBD/241R, respectively. The enhanced green fluorescent protein (GFP)-CDCA4 fusion protein expression vector was constructed by PCR. The PCR products were digested with EcoRI and BamHI and ligated into their respective sites in the pEGFP-N2 vector (Clontech). The forward (F) and reverse (R) PCR primers used were as follows: 1F/GFP, 5′-CGGAATTCGCCGCCATGTTTGCACGAGGACTGAAG-3′; 73F/GFP, 5′-CGGAATTCGCCGCCATGACGCAGGATGGGACGTG-3′; 174R/GFP, 5′-CGGGATCCGGGTTTTTAGTCTCCAGCGT-3′; and 241R/GFP, 5′-CGGGATCCCGGTCTCCACCAGGATCTCCAC-3′. The pcDNA3-HA-E2F1, pcDNA3-HA-E2F2, pcDNA3-HA-E2F3, pcDNA3-HA-E2F4, and pcDNA3-HA-E2F6 expression plasmids (where HA denotes hemagglutinin) were a gift from Dr. Joseph R. Nevins (Duke University Medical Center). The pCMV-HA-E2F5, pCMV-HA-DP2, pCMV-HA-pRb, pCMV-HA-p107, and pCMV-HA-p130 expression plasmids were supplied by Dr. Junji Magae (Institute of Research and Innovation, Kashiwa, Chiba, Japan). The pCMV-Myc5-E2F7 and pCMV-Myc5-E2F8 expression vectors were provided by Dr. Gustavo Leone (Ohio State University). The pRcCMV-pRb expression vector was a gift from Dr. Robert A. Weinberg (Whitehead Institute for Biomedical research, Massachusetts Institute of Technology). The pMT3-HA-mTRIP-Br1 and pMT3-HA-hTRIP-Br2 expression plasmids were a gift from Dr. Stephen I-Hong Hsu (National University of Singapore). The FLAG-tagged p300 and HA-tagged CBP expression plasmids were obtained from the BioResource Center (Tsukuba Institute, RIKEN). pF-Bmi1 was kindly provided by Dr. Jay L. Hess (University of Pennsylvania Medical Center), pBJ5-Brg1 by Dr. Weidong Wang (National Institutes of Health, Baltimore), and pcDNA3-TopBP1-Myc by Dr. Junjie Chen (Mayo Clinic, Rochester, MN) and Dr. Kazuhiko Yamane (Case Western Reserve University, Cleveland, OH). The human Brm expression vector (4102) was a gift from Dr. Christian Muchardt (Institut Pasteur, Paris, France). The pTAG4A-p53 expression vector was described previously (45Goto Y. Hayashi R. Ogawa H. Eguchi I. Muramatsu T. Oshida Y. Ohtani K. Yoshida K. Biochim. Biophys. Acta. 2006; 1759: 60-68Crossref PubMed Scopus (35) Google Scholar). As a mock transfection, pcDNA3 (Invitrogen) or pCMV-based vectors were used. The reporter plasmids pGL3-MCM7 (pGL3-0.46 kb) and pGL3-geminin (pGL3-4.98 kb) were described previously (38Yoshida K. Inoue I. Oncogene. 2004; 23: 3802-3812Crossref PubMed Scopus (84) Google Scholar). The pE2F-TA-Luc and pTA-Luc vectors were purchased from Clontech. Cell Culture and RNA Interference (RNAi)—HeLa (RCB0007, BioResource Center) and A549 cells (TKG0184, Cell Resource Center for Biomedical Research, Institute of Development, Aging, and Cancer, Tohoku University) were cultured in Earle's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum (FBS; Bioserum, Fukuyama, Hiroshima, Japan), 1% nonessential amino acids (Invitrogen), and antibiotics/antimycotics (Invitrogen). Saos-2 cells (TKG0469, Cell Resource Center for Biomedical Research) were cultured in McCoy's 5A medium (Invitrogen) supplemented with 10% FBS and antibiotics/antimycotics. WI-38 cells (IFO50075, Health Science Research Resources Bank, Osaka, Japan) were cultured in Earle's modified Eagle's medium supplemented with 10% FBS and antibiotics/antimycotics. To measure the growth-dependent induction of human CDCA4 gene expression, Saos-2 cell growth was arrested in the G0 phase by incubation in the absence of FBS for 3 days, and the cells were reintroduced into the cell cycle by culture with 20% FBS. Cell lysates were recovered at 0, 14, and 24 h after serum stimulation. To determine the number of viable cells in culture, the WST-1 assay (Roche Applied Science) and the CellTiter-Glo luminescent cell viability assay (Promega Corp.), which are based on quantification of the ATP present, were performed in accordance with the manufacturers' protocols. For RNAi, 2 × 105 cells were transfected with Lipofectamine 2000 (Invitrogen) following the manufacturer's instructions. Cells were plated onto a 6-well plate prior to transfections. Briefly, 125 pmol of annealed siRNA duplex (Ambion, Inc.) and 2.5 μl of transfection reagent were incubated in 0.5 ml of Opti-MEM I reduced serum medium (Invitrogen) for 15 min to facilitate complex formation. The resulting mixture was added to cells cultured in 2 ml of modified Eagle's medium. Twenty-four hours after the first siRNA transfection (day 1), cells were split 1:1 to maintain the cells in the log phase. Three hours after splitting, the second siRNA transfection was performed. The target sequences of the oligonucleotides used were as follows: CDCA4-1, 5′-GGUGUGUUUUCUUUUGUGCtt-3′ (sense) and 5′-GCACAAAAGAAAACACACCtt-3′ (antisense); and CDCA4-2, 5′-GCUUUCACAAGUCACUUGAtt-3′ (sense) and 5′-UCAAGUGACUUGUGAAAGCtt-3′ (antisense). Silencer negative control #1 siRNA (Ambion, Inc.) was used as a negative control. The cells were lysed after 24 h (day 2) and 72 or 96 h (day 3 or 4, respectively) following the second RNAi transfection to isolate total RNA and protein, respectively. 5-Bromo-2′-deoxyuridine (BrdUrd) Incorporation Assay—To determine DNA synthesis based on BrdUrd, labeling was performed (BrdUrd labeling and detection kit III, Roche Applied Science) in accordance with the manufacturer's protocol. Control or CDCA4 siRNA was transfected into HeLa cells as described above. Twenty-four hours after the second siRNA transfection (day 2), 3 × 103 cells were plated onto a 96-well plate. The next day, cells were prelabeled with 10 μm BrdUrd for 14 h before treatment. Cells were washed twice with cold phosphate-buffered saline (PBS) and fixed in 70% ethanol and 0.5 m HCl for 30 min at –20 °C. Cells were then washed three times with PBS and treated with nuclease. After incubation for 30 min at 37 °C, the cells were washed three times with PBS, and 1% bovine serum albumin-containing PBS including peroxidase-conjugated anti-BrdUrd monoclonal antibody was added to each well. After incubation for 30 min at 37 °C, cells were washed three times with PBS. BrdUrd labeling was visualized using peroxidase substrate and was measured at 405 nm with a reference wavelength at 490 nm. Luciferase Reporter Assay—For the reporter assay, 2 × 104 cells were transfected with FuGENE 6 (Roche Applied Science) following the manufacturer's instructions. Briefly, 200 ng of expression plasmid, 200 ng of firefly luciferase reporter plasmid (pGL3-Basic or pE2F-TA-Luc), and 0.6 ng of Renilla luciferase reporter plasmid (pRL-TK, Promega Corp.) on a 24-well dish were used for each transfection. For TAD mapping, 200 ng of pCMV-BD construct, 200 ng of firefly luciferase reporter plasmid (pFR-Luc, Stratagene), and 0.6 ng of Renilla luciferase reporter plasmid (pRL-TK) on 24-well dish were used for each transfection. The cells were harvested 24 or 48 h after transfection, and a luciferase assay was performed using the Dual-Luciferase reporter assay system (Promega Corp.) in accordance with the manufacturer's protocol. The results were read with a luminescence microplate reader (Wallac 1420 ARVOsx multilabel counter, PerkinElmer Life Sciences). Experiments were performed at least in triplicate. As a control for transfection efficiency, the firefly luciferase activity values were normalized to Renilla luciferase activity values. Data are presented as the means ± S.D. Statistical differences were analyzed using Student's paired t test. A p value of <0.05 was considered to indicate a statistically significant difference. RT-PCR—Total cellular RNA was extracted from the cultured cells using the RNeasy mini kit (Qiagen Inc.) following the manufacturer's instructions. Control and E2F1 adenoviral vectors and virus were kindly provided by Dr. Kiyoshi Ohtani (Tokyo Medical and Dental University). The virus preparation and the virus infection procedure were as described (45Goto Y. Hayashi R. Ogawa H. Eguchi I. Muramatsu T. Oshida Y. Ohtani K. Yoshida K. Biochim. Biophys. Acta. 2006; 1759: 60-68Crossref PubMed Scopus (35) Google Scholar, 46Schwarz J.K. Bassing C.H. Kovesdi I. Datto M.B. Blazing M. George S. Wang X.F. Nevins J.R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 483-487Crossref PubMed Scopus (154) Google Scholar). The RT step was performed as recommended by Invitrogen. Briefly, 500 ng of extracted RNA, oligo(dT) primer, and 1× annealing buffer were diluted in 8 μl of RNase/DNase-free water, heated to 65 °C for 5 min, and then chilled on ice. For first-strand cDNA synthesis, a heat-denatured RNA solution, together with 2× first-strand reaction mixture and SuperScript III/RNaseOUT enzyme mixture, was added to make up 20 μlof the reaction mixture, followed by incubation at 50 °C for 50 min, heating to 85 °C for 5 min, and cooling on ice. To amplify CDCA4 cDNA, primers 1F/GFP and 241R/GFP were used. To check the mRNA abundances in 16 different human tissues, human MTC multiple tissue cDNA Panels I and II (Clontech) were used in accordance with the manufacturer's instructions. The primer set for amplification of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was included in the kit. PCR was then performed as follows: denaturation at 94 °C for 3 min, followed by 25–30 cycles at 94 °C for 15 s, 60 °C for 30 s, and 68 °C for 1 min. To amplify ORC1 (NM_004153) and CDC25A (NM_001789) cDNAs, the following primers were used: ORC1, 5′-CTGAGAGCCATCCTCGCAGA-3′ and 5′-GCTGGGCATGGTGGCATGTG-3′; and CDC25A, 5′-TAGGTGGGCTCCACAGGATG-3′ and 5′-AAGTCTGCCCCAGCTCCTTG-3′. PCR was then performed as follows: denaturation at 94 °C for 3 min, followed by 27 cycles at 94 °C for 15 s, 60 °C for 30 s, and 68 °C for 1 min. As a control, a GAPDH primer set (R&D Systems) was used. The amplified products were separated on 1% agarose gels and visualized by ultraviolet transillumination. Chromatin Immunoprecipitation (ChIP)—ChIP assays were performed using the EZ-Chip kit (Upstate) following the manufacturer's instructions with some modification (38Yoshida K. Inoue I. Oncogene. 2004; 23: 3802-3812Crossref PubMed Scopus (84) Google Scholar, 39Yoshida K. Inoue I. Oncogene. 2004; 23: 6250-6260Crossref PubMed Scopus (43) Google Scholar). Briefly, 2 × 106 A549 cells were used for each ChIP; asynchronously growing cells were treated with formaldehyde at a final concentration of 1% to create protein-DNA cross-links; and the cross-linked chromatins were then extracted, diluted with SDS lysis buffer containing protease inhibitor mixture, and sheared by sonication (Branson Sonifier II) on ice to fragments with an average length of ∼1000 bp. After being precleared with protein G-agarose at 4 °C for 1 h, the chromatin was divided into equally sized samples for immunoprecipitation with 2–5 μgof anti-E2F1 (catalog no. sc-193), anti-E2F4 (catalog no. sc-866), or anti-E2F6 (catalog no. sc-22823) antibody (Santa Cruz Biotechnology, Inc.) or anti-FLAG polyclonal antibody (Sigma). After overnight incubation at 4 °C, preblocked protein G-agarose was added, and the resultant immunocomplexes were pelleted by centrifugation after 1 h of incubation at 4 °C and washed with the buffer supplied with the kit. The immunoprecipitates were resuspended in TE buffer (10 mm Tris-HCl (pH 7.6) and 1 mm EDTA), and the cross-links were reversed by incubation with proteinase K and RNase A. After phenol/chloroform extraction and ethanol precipitation, the pellets were resuspended in 50 μl of distilled water and analyzed by PCR. As an input control, 0.5% volume of ch

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