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Characterization of the Condensin Component Cnap1 and Protein Kinase Melk as Novel E2F Target Genes Down-regulated by 1,25-Dihydroxyvitamin D3

2005; Elsevier BV; Volume: 280; Issue: 45 Linguagem: Inglês

10.1074/jbc.m503587200

ISSN

1083-351X

Autores

Lieve Verlinden, Guy Eelen, Ine Beullens, Mark Van Camp, Paul Van Hummelen, Kristof Engelen, Ruth Van Hellemont, Kathleen Marchal, Bart De Moor, Floris Foijer, Hein te Riele, Monique Beullens, Mathieu Bollen, Chantal Mathieu, Roger Bouillon, Annemieke Verstuyf,

Tópico(s)

Vitamin D Research Studies

Resumo

1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) has potent antiproliferative effects characterized by a hampered G1/S transition. cDNA microarrays were used to monitor expression of 21,492 genes in MC3T3-E1 mouse osteoblasts at 1, 6, 12, 24, and 36 h after treatment with 1,25(OH)2D3. Statistical analysis revealed a cluster of genes that were strongly down-regulated by 1,25(OH)2D3 and which not only function in cell cycle regulation and DNA replication but also mediate checkpoint control, DNA repair, chromosome modifications, and mitosis. Because many of these genes were shown earlier to be regulated by the transcriptional repressor E2F4, the intergenic regions of these 1,25(OH)2D3-down-regulated genes were searched for the presence of E2F binding sites. This led to the characterization of two novel E2F target genes, chromosome condensation-related SMC-associated protein 1 (Cnap1) and maternal embryonic leucine zipper kinase (Melk). Transfection studies and site-directed mutagenesis confirmed Cnap1 and Melk to be bona fide E2F targets. Repression of Cnap1 and Melk by 1,25(OH)2D3 was confirmed not only in MC3T3-E1 cells but also in several other bone-unrelated cell types. This down-regulation as well as the antiproliferative effect of 1,25(OH)2D3 depended on the pocket proteins p107 and p130 because 1,25(OH)2D3 failed to repress these E2F target genes and lost its antiproliferative action in p107–/–;p130–/– cells but not in pRb–/– cells. 1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) has potent antiproliferative effects characterized by a hampered G1/S transition. cDNA microarrays were used to monitor expression of 21,492 genes in MC3T3-E1 mouse osteoblasts at 1, 6, 12, 24, and 36 h after treatment with 1,25(OH)2D3. Statistical analysis revealed a cluster of genes that were strongly down-regulated by 1,25(OH)2D3 and which not only function in cell cycle regulation and DNA replication but also mediate checkpoint control, DNA repair, chromosome modifications, and mitosis. Because many of these genes were shown earlier to be regulated by the transcriptional repressor E2F4, the intergenic regions of these 1,25(OH)2D3-down-regulated genes were searched for the presence of E2F binding sites. This led to the characterization of two novel E2F target genes, chromosome condensation-related SMC-associated protein 1 (Cnap1) and maternal embryonic leucine zipper kinase (Melk). Transfection studies and site-directed mutagenesis confirmed Cnap1 and Melk to be bona fide E2F targets. Repression of Cnap1 and Melk by 1,25(OH)2D3 was confirmed not only in MC3T3-E1 cells but also in several other bone-unrelated cell types. This down-regulation as well as the antiproliferative effect of 1,25(OH)2D3 depended on the pocket proteins p107 and p130 because 1,25(OH)2D3 failed to repress these E2F target genes and lost its antiproliferative action in p107–/–;p130–/– cells but not in pRb–/– cells. Active complexes between cyclin D and cyclin-dependent kinases 4/6 regulate the transition through the G1/S restriction point by phosphorylation of the retinoblastoma protein (pRb) 3The abbreviations used are: pRbretinoblastoma protein;1,25(OH)2D31,25-dihydroxyvitamin D3MEFmurine embryonic fibroblastQRT-PCRquantitative real-time PCRwtwild type and other members of the pocket protein family, p107 and p130. The phosphorylation status of these pocket proteins determines their association with members of the E2F family of transcriptional regulators, which play a pivotal role in mediating gene expression during cell proliferation. These E2F proteins can be allocated to four subclasses. Upon release by their pocket protein pRb, E2Fs 1–3 function as transcriptional activators in late G1 and in S phase. E2F4 and E2F5 act as transcriptional repressors in quiescent and early G1 cells by associating with p107 or p130 (1Classon M. Dyson N. Exp. Cell Res. 2001; 264: 135-147Crossref PubMed Scopus (205) Google Scholar, 2Cam H. Dynlacht B.D. Cancer Cell. 2003; 3: 311-316Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar). In quiescent cells repression of the promoter activity of E2F target genes is associated with the recruitment of E2F4 and p130 and low levels of histone acetylation. By late G1, these proteins are largely replaced by activator E2Fs in concert with histone acetylation and gene activation. It is, therefore, likely that two pathways, one controlled by pRb and the other by p130/p107, regulate distinct downstream events required for G1 progression and G1/S transition (2Cam H. Dynlacht B.D. Cancer Cell. 2003; 3: 311-316Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar, 3Stevens C. La Thangue N.B. Arch. Biochem. Biophys. 2003; 412: 157-169Crossref PubMed Scopus (181) Google Scholar). Recently, the transcriptional repressor E2F6 was proposed to make up the third subclass of E2F proteins (4Attwooll C. Oddi S. Cartwright P. Prosperini E. Agger K. Steensgaard P. Wagener C. Sardet C. Moroni M.C. Helin K. J. Biol. Chem. 2005; 280: 1199-1208Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar, 5Giangrande P.H. Zhu W. Schlisio S. Sun X. Mori S. Gaubatz S. Nevins J.R. Genes Dev. 2004; 18: 2941-2951Crossref PubMed Scopus (84) Google Scholar), whereas E2F7 and E2F8 form the last subclass and are thought to regulate a subset of E2F target genes during the cell cycle (6Maiti 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 (152) Google Scholar, 7Logan 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 (82) Google Scholar). retinoblastoma protein; 1,25-dihydroxyvitamin D3 murine embryonic fibroblast quantitative real-time PCR wild type 1,25-Dihydroxyvitamin D3 (1,25(OH)2D3), the active metabolite of vitamin D3, acts on bone and mineral homeostasis and also inhibits proliferation and induces differentiation of various normal and malignant cells (8Haussler M.R. Whitfield G.K. Haussler C.A. Hsieh J.C. Thompson P.D. Selznick S.H. Dominguez C.E. Jurutka P.W. J. Bone Miner. Res. 1998; 13: 325-349Crossref PubMed Scopus (1234) Google Scholar). However, the exact molecular mechanism behind this growth-inhibitory effect is unknown. 1,25(OH)2D3 has a cell cycle-specific effect leading to an accumulation of cells in the G1 phase of the cell cycle (9Wang Q.M. Luo X. Studzinski G.P. Cancer Res. 1997; 57: 2851-2855PubMed Google Scholar). It has been shown previously that 1,25(OH)2D3 reduces the activity of the cyclin D1-cyclin-dependent kinase 4/6 complex, which may contribute to its antiproliferative effect (10Jensen S.S. Madsen M.W. Lukas J. Binderup L. Bartek J. Mol. Endocrinol. 2001; 15: 1370-1380Crossref PubMed Scopus (234) Google Scholar). In the present study a cDNA microarray was performed to examine the expression profile of 21,492 genes in MC3T3-E1 cells treated with 1,25(OH)2D3 for different times up to 36 h. Statistical analysis revealed a cluster of down-regulated genes involved in cell cycle regulation and in DNA replication but also in checkpoint control, DNA repair, chromosome transactions, and mitosis. Approximately 30% of the genes in this cluster are known E2F targets, and in silico promoter analysis demonstrated an additional 20% of the genes to contain E2F binding sites in their promoter. Four of these genes were selected for further analysis, namely Cnap1, Melk, retroviral integration site 2 (Ris2), and enhancer of Zeste homolog 2 (Ezh2). Expression of these genes was growth-regulated as were the promoter activities of Cnap1 and Melk. Mutational analysis revealed that the identified E2F binding sites were required for transactivation by E2F family members. Rather than being key genes responsible for the antiproliferative effect of 1,25(OH)2D3, these genes are suggested to be part of the general mechanism by which the pocket proteins translate the effect of 1,25(OH)2D3 and regulate a large number of E2F target genes. Because p107–/–;p130–/–-cells no longer responded to the antiproliferative activity of 1,25(OH)2D3, we suggest that 1,25(OH)2D3 exerts this growth-inhibitory effect by means of the repressive activity of p107/p130·E2F complexes rather than by affecting pRb-related E2F activity, as previously suggested. Cell Culture—MC3T3-E1 cells (Riken Cell Bank, Tsukuba, Japan) and GR cells were cultured as previously described (11Eelen G. Verlinden L. Van Camp M. Van Hummelen P. Marchal K. De Moor B. Mathieu C. Carmeliet G. Bouillon R. Verstuyf A. J. Bone Miner. Res. 2004; 19: 133-146Crossref PubMed Google Scholar). Wild type, pRb, p107, and p130 nullizygous as well as p107 p130 double nullizygous murine embryonic fibroblasts (wt, pRb–/–, p107–/–, p130–/–, and p107–/–;p130–/– murine embryonic fibroblasts (MEFs)) were cultured in Dulbecco's modified Eagle's medium with 4.5 mg/ml glucose with 10% fetal bovine serum, 2 mm glutaMAX-I, 100 units/ml penicillin, and 100 μg/ml streptomycin (Invitrogen). Proliferation Assays—Antiproliferative effects of 1,25(OH)2D3 were measured by [3H]thymidine incorporation or by analysis of cell cycle distribution as previously described (11Eelen G. Verlinden L. Van Camp M. Van Hummelen P. Marchal K. De Moor B. Mathieu C. Carmeliet G. Bouillon R. Verstuyf A. J. Bone Miner. Res. 2004; 19: 133-146Crossref PubMed Google Scholar, 12Verlinden L. Verstuyf A. Van Camp M. Marcelis S. Sabbe K. Zhao X.Y. De Clercq P. Vandewalle M. Bouillon R. Cancer Res. 2000; 60: 2673-2679PubMed Google Scholar). Total RNA Extraction—Total RNA for microarray analysis was extracted with TRizol LS reagent (Invitrogen). Total RNA for quantitative RT-PCR analysis was isolated with the RNeasy kit (Qiagen, Hilden, Germany). Construction of Microarrays—The mouse gene set consisted of 5 separate microarrays containing in total 21,492 cDNA fragments. The clone set was composed from the 8000 collection of Incyte (Mouse Gem I, Incyte, Wilmington, DE) and from the 15,000 collection of National Institute of Aging (HGMP Resource Centre, Cambridge, UK). A complete description of the array content and the printing procedures can be downloaded from ArrayExpress (www.ebi.ac.uk/arrayexpress) with accession number A-MECP-146. RNA Labeling and Hybridization—Antisense RNA amplification, RNA labeling, and hybridization were performed as previously described (11Eelen G. Verlinden L. Van Camp M. Van Hummelen P. Marchal K. De Moor B. Mathieu C. Carmeliet G. Bouillon R. Verstuyf A. J. Bone Miner. Res. 2004; 19: 133-146Crossref PubMed Google Scholar). All protocols can be downloaded from www.microarrays.be or via ArrayExpress (www.ebi.ac.uk/arrayexpress) with accession number P-MEXP578-582. Scanning and Microarray Data Analysis—Array slides were scanned using a Generation III scanner (Amersham Biosciences) with wavelength settings at 532 nm (Cy3 signal) and 635 nm (Cy5 signal). Image analysis was performed with ArrayVision (Imaging Research Inc., St. Catharines, Ontario, Canada). Spot intensities were measured as artifact-removed total intensities (ARVol). Spot intensities were normalized using a Loess-fit (13Yang Y.H. Dudoit S. Luu P. Lin D.M. Peng V. Ngai J. Speed T.P. Nucleic Acids Res. 2002; 30: 15-24Crossref PubMed Scopus (2831) Google Scholar) for removing nonlinear dye related variation followed by a global analysis of variance normalization (14Engelen K. Coessens B. Marchal K. De Moor B. Bioinformatics. 2003; 19: 893-894Crossref PubMed Scopus (15) Google Scholar). The obtained expression data were clustered with the AQBC algorithm (15De Smet F. Mathys J. Marchal K. Thijs G. De Moor B. Moreau Y. Bioinformatics. 2002; 18: 735-746Crossref PubMed Scopus (149) Google Scholar). Subsequently, the intergenic regions of all the genes in the resulting clusters were selected using the Ensembl mart data base release 18.1 (16Kasprzyk A. Keefe D. Smedley D. London D. Spooner W. Melsopp C. Hammond M. Rocca-Serra P. Cox T. Birney E. Genome Res. 2004; 14: 160-169Crossref PubMed Scopus (324) Google Scholar). The intergenic region is defined as the region upstream of the transcription start, limited to 2 kilobases, and the 5′-untranslated region, limited to the first intron. These intergenic regions (both direct and indirect strands) were then screened with a position-specific weight matrix of E2F, downloaded from the jaspar data base (jaspar.cgb.ki.se) (17Lenhard B. Wasserman W.W. Bioinformatics. 2002; 18: 1135-1136Crossref PubMed Scopus (142) Google Scholar, 18Sandelin A. Alkema W. Engstrom P. Wasserman W.W. Lenhard B. Nucleic Acids Res. 2004; 32: D91-D94Crossref PubMed Google Scholar). The screening was performed using the MotifScanner algorithm with prior set to 0.2 and a mouse-specific zero-order background model (19Thijs G. Moreau Y. De Smet F. Mathys J. Lescot M. Rombauts S. Rouze P. De Moor B. Marchal K. Bioinformatics. 2002; 18: 331-332Crossref PubMed Scopus (62) Google Scholar). Plasmids—A TK-TATA luciferase reporter vector served as a control vector for the same reporter construct in which six artificial E2F binding sites were cloned (20Hofman K. Swinnen J.V. Verhoeven G. Heyns W. Biochem. Biophys. Res. Commun. 2001; 283: 97-101Crossref PubMed Scopus (45) Google Scholar). The pGL3-Basic reporter vector (Promega, Madison, WI) was used as control for pGL3-Basic vectors in which both short and long fragments of the intergenic regions of mouse Melk and Cnap1 were cloned. Site-directed mutagenesis of the E2F binding sites in these promoter regions was performed by use of the QuikChange II site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the instructions of the manufacturer. The sequences of the primers used are available upon request. Expression plasmids pcDNA-HA-E2F1, -E2F2, -E2F3, and -E2F4 were kind gifts of Dr. J. Nevins (Duke University Medical Center, Durham, NC). The cytomegalovirus-hemagglutinin-E2F5 expression plasmid was a kind gift of Dr. J Magae (Institute of Research and Innovation, Chiba, Japan). The β-galactosidase expression vector pcDNA3.1(-)/Myc-His/lacZ and the pcDNA3.1/Myc-His vector were obtained from Invitrogen. Transfection Assays—Exponentially growing MC3T3-E1 cells were transfected with FuGENE 6 (Roche Diagnostics) in 24-well dishes (2 × 105 cells/well) with 100 ng of luciferase reporter vector (or representative control vector), 50 ng of the different E2F constructs (or the empty pcDNA3.1/Myc-His), and 10 ng of pcDNA3.1(–)/Myc-His/lacZ. Cells were lysed 48 h after transfection (with reporter lysis buffer, Roche Diagnostics), and luciferase activity was measured with the luciferase assay system (Promega) and normalized to β-galactosidase activity, measured with the Galacto-Light Plus System (Applied Biosystems, Foster City, CA). To measure growth-dependent induction of the Cnap1 and Melk promoter activities, growth-arrested MC3T3-E1 cells (48 h in α-minimal essential medium with 0.1% fetal bovine serum) were transfected with 100 ng of luciferase reporter vector (or control vector) and 10 ng of pcDNA3.1(-)/Myc-His/lacZ. The next day cells were released into the cell cycle by the addition of α-minimal essential medium with 15% fetal bovine serum. The effect of 1,25(OH)2D3 treatment on the promoter activities of Cnap1 and Melk was determined by transfection of MC3T3-E1 cells and wt or p107–/–;p130–/– MEFs with 100 ng of luciferase reporter vector (or control vector) and 10 ng of pcDNA3.1(–)/Myc-His/lacZ. The day after transfection MC3T3-E1 cells were stimulated with 10–8 m 1,25(OH)2D3, and luciferase and β-galactosidase activities were assessed after a 24-h incubation period with 1,25(OH)2D3. wt and p107–/–;p130–/– MEFs were stimulated for 48 h with 10–7 m 1,25(OH)2D3. Quantitative Real-time PCR—cDNA production, PCR reactions, and subsequent quantification was performed as described previously (21Overbergh L. Valckx D. Waer M. Mathieu C. Cytokine. 1999; 11: 305-312Crossref PubMed Scopus (522) Google Scholar). PCR primers and fluorogenic probes (6-carboxyfluorescein as reporter and 6-carboxytetramethylrhodamine as quencher dye) for mouse Ezh2, Ris2, Cnap1, Melk, VDR, CYP24, and β-actin were purchased from Eurogentec (Seraing, Belgium). Sequences of primers and probes are available upon request. Chromatin Immunoprecipitation Reactions—Chromatin immunoprecipitation assays were based on a previously described protocol (22Vaisanen S. Dunlop T.W. Frank C. Carlberg C. J. Steroid Biochem. Mol. Biol. 2004; 89–90: 277-279Crossref PubMed Scopus (18) Google Scholar) with minor modifications. In brief, 106 MC3T3-E1 cells were cross-linked with formaldehyde (1%) for 10 min. After lysis of the cells, samples were sonicated with a Branson Sonifer 250 to generate DNA fragments with an average length of 500 bp. Subsequently, samples were incubated overnight with 10 μg of anti-E2F1-antibody (sc-193x, Santa Cruz Biotechnology, Santa Cruz, CA) or irrelevant antibody (“mock”, rabbit anti-mouse immunoglobulins, Dako, Denmark) at 4 °C with rotation. After collection and elution of immunocomplexes, cross-links were reversed, and DNA was recovered with a QIAquick spin kit (Qiagen) and eluted in 30 μl. 4 μl of recovered DNA was used for PCR analysis. PCR products were analyzed by standard gel electrophoresis. The sequences of the primers used are available upon request. Statistics—Statistical analysis was performed with the software program NCSS (NCSS, Kaysville, UT). All results are expressed as the means and S.E. of at least three independent experiments. Analysis of variance analyses were followed by a Bonferroni multiple comparison test or a Student's t test. p < 0.05 was accepted as significant. Genes Down-regulated after Treatment with 1,25(OH)2D3 Cluster into Distinct Functional Groups—Analysis of microarray data led to the identification of a cluster of 94 different genes, which were similarly down-regulated by 1,25(OH)2D3 (TABLE ONE). Down-regulation started at 12–24 h after treatment, and the degree of down-regulation at 36 h ranged from 1.5- to 4.3-fold. 1,25(OH)2D3 not only decreased the expression of genes that are involved in cell cycle regulation and DNA replication but also that of genes that mediate checkpoint control, DNA repair, chromosome transactions, and mitosis.TABLE ONEIdentification of genes downregulated by 1,25(OH)2D3 treatment and their expression profileNameAccession no.Gene expression in MC3T3-E1 cells treated with 10-8 m 1,25(OH)2D3 relative to gene expression in vehicle-treated cellsFunction1 h6 h12 h24 h36 hCell Cycle Regulation Ccna2BG0696881.061.091.080.520.43Cyclin-dependent protein kinase regulator activity Ccna2BG0735181.000.860.780.490.43 Ccnb1AA3963241.071.150.990.520.39Cyclin-dependent protein kinase regulator activity (G2/M) Ccnb1BG0784261.011.071.130.540.40 Cdc20BG0786381.011.180.890.540.38Cell division cycle protein; key regulator of the cell cycle Cdc2aM38724/X164611.020.920.890.490.50Cell division cycle protein; kinase activity (G2/M) Cdc2aBG0648460.941.050.910.450.34DNA replication Ask pendingBG0820351.051.140.670.670.51Regulation of S phase of mitotic cell cycle; kinase activity Cdc451BG0631391.001.040.590.560.41Initiation of DNA replication Cdc6AA0484261.020.840.760.400.29Cell division cycle 6 homolog ∼DNA replication; recruitment of Mcm proteins Cdc6AA1898361.120.830.820.720.43 Cdc6BG0770121.010.800.640.350.23 Chaf1aBG0704520.930.950.740.360.27Chromatin assembly factor 1 subunit A and B involved in DNA replication and repair Chaf1bBG0728350.671.121.020.520.36 CTF18BG0634231.031.100.690.620.39Chromosome transmission fidelity factor Fen1BG0635900.950.950.830.430.34Flap structure-specific endonuclease 1 ∼DNA replication Fignl1BG0782120.970.900.960.490.42Fidgetin-like 1; ATP and nucleotide binding FoxM1AA0667411.011.050.720.470.36Transcription factor; essential for DNA replication and mitosis FoxM1BG0874680.921.150.900.520.32 Lig1U19604/M360670.940.890.790.480.37Ligase 1, DNA- and ATP-dependent; involved in DNA replication Lig1U19604/M360670.960.960.830.550.38 Lig1W666261.190.901.100.580.36 Lig1BG0791730.930.980.890.420.36 Mcm2AA0118390.991.010.810.570.44Minichromosome maintenance deficient mitotins; proteins involved in initiation of DNA replication Mcm2BG0746680.890.720.730.550.44 Mcm3BG0650550.980.930.860.430.28 Mcm4AA2597881.030.920.850.380.31 Mcm5BG0648650.920.960.860.410.27 Mcm7BG0747210.901.020.940.730.28 PCNABG0645980.960.920.880.440.40Proliferating cell nuclear antigen; regulator of DNA replication PCNAAA1169470.910.950.850.510.43 Pol ϵBG0697320.880.880.920.460.47Polymerase (DNA directed) ϵ; involved in DNA replication and repair Pol ϵ2BG0714800.951.030.850.450.55 Prim1AA2599000.890.880.650.510.40Part of DNA-polα-primase complex; ∼DNA replication Rfc3BG0683090.850.900.680.540.50Replication factor C (activator 1); ∼DNA replication Ris2BG0646841.000.920.770.590.50Retroviral integration site 2; DNA replication factor Rpa2BG0753720.890.880.730.530.38Replication protein A2 Rrm2BG0766130.950.840.530.340.28Ribonucleotide reductase M2; catalyzes formation of deoxyribonucleotides from ribonucleotides Rrm2BG0781380.950.750.560.350.30 Tk1AA0418340.951.150.620.310.23Thymidine kinase 1; involved in DNA metabolism Tk1BG0777450.830.960.690.390.31 UmpsBG0632910.981.020.840.590.55Uridine monophosphate synthetaseCheckpoints Bub1bBG0694211.001.080.980.530.42Essential for spindle checkpoint activation Mad2l1AA0028950.981.010.830.550.40Mitotic arrest deficient-like 1 (yeast); component of mitotic spindle assembly checkpoint Mad2l1BG0678601.020.970.880.520.36 Tlk1AA4662881.010.790.690.390.33Tousled-like kinase 1; ∼chromatin modificationDNA repair Chaf1aBG0704520.930.950.740.360.27Chromatin assembly factor 1 subunit A and B involved in DNA replication and repair Chaf1bBG0728350.871.121.020.520.36 Exo1NM_0120121.010.910.800.690.45Exonuclease 1; 5′-3′ exonuclease activity Fen1BG0635900.950.950.830.430.34Flap-structure specific endonuclease 1; ∼DNA replication PCNABG0645980.960.920.880.440.40Proliferating cell nuclear antigen PCNAAA1169470.910.950.850.510.43Proliferating cell nuclear antigen Pol ϵBG0697320.880.880.920.460.47Polymerase (DNA directed) ϵ; involved in DNA replication and repair Pol ϵ2BG0714800.951.030.850.450.55 Rad51D134730.920.870.910.540.36RAD51 homolog (Saccharomyces cerevisiae); involved in homologous recombination and repair of DNA; interacts also with BRCA1 and BRCA2 Rad51D134730.940.930.840.610.37 Rad51BG0729041.031.120.890.460.37 Rad51ap1AA3867690.910.830.760.550.53RAD51-associated protein 1 Rfc3BG0683090.850.900.680.540.50Replication factor C (activator 1); ∼DNA replication Rpa2BG0753720.890.880.730.530.38Replication protein A2Chromatin assembly, modification, condensation, segregation CenpaBG0720560.981.200.900.550.43Centromere autoantigen A; involved in chromosome organisation and biogenesis CenpaBG0828811.101.150.950.610.51 CenphAA1985240.900.880.870.520.42Centromere autoantigen H; kinetochore protein involved in chromosome segregation CenphBG0716830.980.860.770.380.41 Chaf1aBG0704520.930.950.740.360.27Chromatin assembly factor 1 subunit A and B involved in DNA replication and repair Chaf1bBG0728350.871.121.020.520.36 Cnap1BG0825660.961.110.870.470.40Chromosome condensation-related SMC-associated protein 1 Espl1BG0718610.911.100.930.380.48Extra spindle poles like-1 (S. cerevisiae) Ezh2BG0749311.020.910.960.430.42Enhancer of Zeste homolog H2afzBG0651100.980.981.010.580.44H2A histone family, member Z; involved in chromosome organization and biogenesis H2afzBG0651110.980.941.000.570.45 Hmgb3BG0787000.970.960.960.490.54High mobility group box 3 Hmgn2BG0788060.950.951.050.500.45High mobility group nucleosomal binding domain 2 NaspBG0768051.000.890.820.580.44Nuclear autoantigenic sperm protein (histone binding) Nusap1AA2657890.830.940.770.470.43Nucleolar- and spindle-associated protein 1 Pcnt2BG0718450.921.030.930.490.56Pericentrin2 ∼spindle assembly, microtubule organizing center Smc2like1BG0778441.051.010.980.440.41SMC2 structural maintenance of chromosomes 2-like 1 Suv39h1AA0509070.930.971.030.630.54Suppressor of variegation 3-9 homolog 1 (Drosophila); involved in chromatin modification Suv39h1BG0876790.891.040.860.420.39 Tlk1AA4662881.010.790.690.390.33Tousled-like kinase 1; ∼chromatin modificationMitosis AnillinBG0639790.950.900.880.490.41Actin binding protein involved in cytokinesis Cdca5BG0687990.990.860.880.550.34Cell division cycle associated 5; ∼cytokinesis Cdca8BG0782991.001.040.890.560.49Cell division cycle associated 8; ∼cytokinesis Ect2AA2670001.030.941.150.540.37Oncogene involved in regulation of cytokinesis FoxM1AA0667411.011.050.720.470.38Transcription factor; essential for DNA replication and mitosis FoxM1BG0874680.921.150.900.520.32 IncenpBG0769091.011.040.660.440.39Inner centromere protein; involved in cytokinesis Kif20aAA1771971.151.200.990.570.41Kinesin family member 20A; microtubule associated complex Kif22AA0081891.021.130.800.500.33Kinesin family member 22; microtubule associated complex Kif23BG0683241.041.050.990.410.42Kinesin family member 23; microtubule associated complex Kif23BG0686661.141.031.060.400.44 MelkBG0768920.930.930.720.400.28Maternal embryonic leucine zipper kinase Nek2AA2683491.031.110.920.510.38Nima (never in mitosis gene a)-related expressed kinase 2 involved in centrosome separation and cytokinesis Nek2BG0658260.991.051.070.600.53 Prc1AA2545521.031.000.890.470.40Protein regulator of cytokinesis Racgap1AA1405231.121.011.050.580.39Rac GTPase-activating protein 1; regulates cytokinesis Spag5AA0867961.001.100.850.580.42Sperm assoc. antigen 5; localizes to mitotic spindles Suv39h1AA0509070.930.971.030.630.54Suppressor of variegation 3-9 homolog 1 (Drosophila); involved in chromatin modification Suv39h1BG0876790.891.040.860.420.39Miscellaneous AaasBG0833430.860.970.690.430.38Alias Aladin; involved in nucleocytoplasmic transport Exosc8BG0885410.940.970.870.440.44Exosome component 8; involved in (r)RNA processing Kpna2BG0664420.950.991.080.430.37Karyopherin (importin) a2 involved in nuclear transport Lsm3AA2706520.910.960.950.720.56U6 small RNA processing; involved in mRNA processing Nup37BG0816081.011.010.850.580.58Nucleoporin 37; involved in protein transport Nup43BG0825710.980.950.760.470.45Nucleoporin 43; involved in protein transport Nup93BG0637930.971.000.960.650.58Nucleoporin 93; involved in protein transport NurimAA2703640.900.920.650.600.50Nuclear envelope membrane protein NurimBG0715340.881.000.610.320.32Nuclear envelope membrane protein OdcBG0696470.760.940.760.480.45Ornithine decarboxylase; ∼polyamine biosynthesis PbkAA0363220.980.961.000.420.35PDZ binding kinase; protein kinase activity PbkAA4155790.930.930.950.560.46 PbkBG0636241.051.051.050.380.30 StathminAA2653960.901.000.790.390.29Regulation of microtubule filament system Tacc3W851661.070.971.050.550.46Transforming, acidic coiled-coll-containing protein 3 Tacc3BG0687590.981.051.150.740.40 Tacc3AA1901231.011.121.140.510.41 Tacc3BG0837650.991.091.080.490.43 Tagnl2BG0775500.870.990.720.610.51Transgelin 2; actin-associated protein, function unknown Tcf19BG0692940.920.970.680.580.38Transcription factor 19; ∼DNA dependent transcription Timm50BG0831000.861.090.790.550.65Translocase of inner mitochondrial membrane 50 homolog; determines sensitivity to apoptotic signals Timm50BG0710690.801.180.710.530.56 Xpo1AA1055460.961.000.940.450.43Exportin 1; involved in protein nucleus exportRiken clones 1700021F05RikAA2454920.900.970.710.450.28 2410015N17RikBG0637580.941.040.630.500.45 2610005B21RikBG0717040.991.050.990.440.47 2610019103RikW899660.991.030.890.610.42Proliferation associated nuclear element 1 2610040C18RikBG0715550.930.920.820.420.45∼Chromatin structure and dynamics 2610528A17RikAA5112421.000.940.830.510.51 2610528M18RikBG0747100.970.980.980.440.42 2810417H13RikBG0765691.090.920.980.480.44 2810417H13RikBG0767241.150.860.810.340.29 2810417H13RikBG0732300.980.930.730.360.38 2810475A17RikBG0780650.930.981.010.520.50Membrane protein F730047E07RikAA2882480.970.980.960.590.50ESTs ESTAU0186870.900.970.670.620.35 ESTAW5382201.080.980.870.600.46 ESTBG0647040.860.860.740.580.48 ESTBG0688851.101.050.850.600.45 ESTBG0746580.940.970.940.720.41 Open table in a new tab Approximately 30% of the genes in this cluster were known E2F targets. Therefore, the remaining genes in this cluster were screened for E2F binding sites in their promoter. An additional 20% of the genes was found to contain E2F binding sites. Four of these genes were selected for further study based on the highly conserved E2F binding sites in their promoter (Cnap1, Ezh2, and Ris2), on the one hand, and on their overexpression in undifferentiated cancers, on the other hand (Cnap1, Ezh2, and Melk) (23Rhodes D.R. Yu J. Shanker K. Deshpande N. Varambally R. Ghosh D. Barrette T. Pandey A. Chinnaiyan A.M. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 9309-9314Crossref PubMed Scopus (810) Google Scholar). Expression Analysis of Cnap1, Melk, Ris2, and Ezh2 in 1,25(OH)2D3-treated Cells—Quantitative real-time PCR (QRT-PCR) experiments were performed in MC3T3-E1 cells to monitor the expression profile of these genes at different time points up to 72 h after treatment with a single dose of 1,25(OH)2D3 (10–10 m) (Fig. 1). The expression of all 4 genes decreased as soon as 6 h after treatment. A maximal 5-fold reduction was observed at 48–72 h after treatment. Growth-dependent Expression of Cnap1, Melk, Ris2, and Ezh2—To determine whether the expression of Cnap1, Melk, Ris2, and Ezh2 was growth-regulated, MC3T3-E1 cells were serum-starved for 48 h and subsequently stimulated to re-enter the cell cycle by the addition of serum (Fig. 2). As shown in Fig. 2B, mRNA transcripts of all four genes strongly increased after the addition of serum and peaked at the G1/S transition (16–20 h after re-feeding, Fig. 2A), which suggested that the regulation of these genes was growth-dependent. Growth-dep

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