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NF-Y Mediates the Transcriptional Inhibition of thecyclin B1, cyclin B2, and cdc25CPromoters upon Induced G2 Arrest

2001; Elsevier BV; Volume: 276; Issue: 8 Linguagem: Inglês

10.1074/jbc.m006052200

ISSN

1083-351X

Autores

Isabella Manni, Giuseppina Mazzaro, Aymone Gurtner, Roberto Mantovani, Ulrike Haugwitz, Karen Krause, Kurt Engeland, Ada Sacchi, Silvia Soddu, Giulia Piaggio,

Tópico(s)

Ubiquitin and proteasome pathways

Resumo

During normal cell cycles, the function of mitotic cyclin-cdk1 complexes, as well as of cdc25C phosphatase, is required for G2 phase progression. Accordingly, the G2 arrest induced by DNA damage is associated with a down-regulation of mitotic cyclins, cdk1, and cdc25C phosphatase expression. We found that the promoter activity of these genes is repressed in the G2 arrest induced by DNA damage. We asked whether the CCAAT-binding NF-Y modulates mitoticcyclins, cdk1, and cdc25C gene transcription during this type of G2 arrest. In our experimental conditions, the integrity of the CCAAT boxes ofcyclin B1, cyclin B2, and cdc25Cpromoters, as well as the presence of a functional NF-Y complex, is strictly required for the transcriptional inhibition of these promoters. Furthermore, a dominant-negative p53 protein, impairing doxorubicin-induced G2 arrest, prevents transcriptional down-regulation of the mitotic cyclins, cdk1, and cdc25C genes. We conclude that, as already demonstrated for cdk1, NF-Y mediates the transcriptional inhibition of the mitotic cyclins and the cdc25C genes during p53-dependent G2 arrest induced by DNA damage. These data suggest a transcriptional regulatory role of NF-Y in the G2 checkpoint after DNA damage. During normal cell cycles, the function of mitotic cyclin-cdk1 complexes, as well as of cdc25C phosphatase, is required for G2 phase progression. Accordingly, the G2 arrest induced by DNA damage is associated with a down-regulation of mitotic cyclins, cdk1, and cdc25C phosphatase expression. We found that the promoter activity of these genes is repressed in the G2 arrest induced by DNA damage. We asked whether the CCAAT-binding NF-Y modulates mitoticcyclins, cdk1, and cdc25C gene transcription during this type of G2 arrest. In our experimental conditions, the integrity of the CCAAT boxes ofcyclin B1, cyclin B2, and cdc25Cpromoters, as well as the presence of a functional NF-Y complex, is strictly required for the transcriptional inhibition of these promoters. Furthermore, a dominant-negative p53 protein, impairing doxorubicin-induced G2 arrest, prevents transcriptional down-regulation of the mitotic cyclins, cdk1, and cdc25C genes. We conclude that, as already demonstrated for cdk1, NF-Y mediates the transcriptional inhibition of the mitotic cyclins and the cdc25C genes during p53-dependent G2 arrest induced by DNA damage. These data suggest a transcriptional regulatory role of NF-Y in the G2 checkpoint after DNA damage. nuclear factor Y Adriamycin phosphate-buffered saline chloramphenicol acetyltransferase luciferase cytomegalovirus In mammalian cells, progression through the G2 phase of the cell cycle is mediated by the activity of a specific set of proteins, which includes mitotic cyclins A, B1, and B2, mitotic kinase cdk1 (alias p34cdc2), and mitotic phosphatase cdc25C. The kinase activity of cdk1 during the G2 phase is dependent on its dephosphorylated status of specific residues triggered by the phosphatase activity of cdc25C protein (1Gould K.L. Nurse P. Nature. 1989; 342: 39-45Crossref PubMed Scopus (851) Google Scholar, 2Pines J. Hunter T. Cell. 1989; 58: 833-846Abstract Full Text PDF PubMed Scopus (693) Google Scholar, 3Pines J. Hunter T. Nature. 1990; 346: 760-763Crossref PubMed Scopus (528) Google Scholar, 4Parker L.L. Piwnica-Worms H. Science. 1992; 257: 1955-1957Crossref PubMed Scopus (536) Google Scholar, 5McGowan C.H. Russell P. EMBO J. 1993; 12: 75-85Crossref PubMed Scopus (384) Google Scholar, 6Liu F. Stanton J.J. Wu Z. Piwnica-Worms H. Mol. Cell. Biol. 1997; 17: 571-583Crossref PubMed Scopus (263) Google Scholar), as well as on the levels of cyclins A and Bs. In proliferating cells, oscillations of mitotic cyclin amounts are tightly regulated at the transcriptional level (7Henglein B. Chenivesse X. Wang J. Eick D. Brechot C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5490-5494Crossref PubMed Scopus (244) Google Scholar, 8Hwang A. Maity A. McKenna W.G. Muschel R.J. J. Biol. Chem. 1995; 270: 28419-28424Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 9Lucibello F.C. Truss M. Zwicker J. Ehlert F. Beato M. Muller R. EMBO J. 1995; 14: 132-142Crossref PubMed Scopus (87) Google Scholar, 10Piaggio G. Farina A. Perrotti D. Manni I. Fuschi P. Sacchi A. Gaetano C. Exp. Cell Res. 1995; 216: 396-402Crossref PubMed Scopus (54) Google Scholar, 11Zwicker J. Gross C. Lucibello F.C. Truss M. Ehlert F Engeland K. Muller R. Nucleic Acids Res. 1995; 23: 3822-3830Crossref PubMed Scopus (91) Google Scholar, 12Katula K.S. Wright K.L. Paul H. Surman D.R. Nuckolls F.J. Smith J.W. Ting J.P. Yates J. Cogswell J.P. Cell Growth Differ. 1997; 8: 11-20PubMed Google Scholar). In particular, activation of cdk1 does not occur until sufficient cyclin B1 protein is synthesized (13Solomon M.J. Glotzer M. Lee H. Philipe M. Kirshner S. Cell. 1990; 63: 1013-1034Abstract Full Text PDF PubMed Scopus (503) Google Scholar). The accumulation of mitotic cyclins and cdk1 correlates with nascent gene expression, and their mRNAs can only be detected in particular phases of the cell cycle (2Pines J. Hunter T. Cell. 1989; 58: 833-846Abstract Full Text PDF PubMed Scopus (693) Google Scholar, 7Henglein B. Chenivesse X. Wang J. Eick D. Brechot C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5490-5494Crossref PubMed Scopus (244) Google Scholar, 10Piaggio G. Farina A. Perrotti D. Manni I. Fuschi P. Sacchi A. Gaetano C. Exp. Cell Res. 1995; 216: 396-402Crossref PubMed Scopus (54) Google Scholar, 14Dalton S. EMBO J. 1992; 11: 797-1804Crossref Scopus (335) Google Scholar).Cell cycle progression through the G2 phase is controlled by the G2 checkpoint. This checkpoint ensures correct DNA synthesis during cell proliferation and genome integrity after DNA damage. In the latter condition, cells can arrest at the G1and/or the G2 checkpoint, depending on cell type, cell cycle phase, and checkpoint integrity (15Kastan M.B. Onyekwere O. Sidransky D. Vogelstein B. Craig R.W. Cancer Res. 1991; 51: 6304-6311PubMed Google Scholar, 16Maity A. McKenna W.G. Muschel R.J. Radiother. Oncol. 1994; 31: 1-13Abstract Full Text PDF PubMed Scopus (270) Google Scholar, 17Cox L.S. Lane D.P. Bioessays. 1995; 17: 501-508Crossref PubMed Scopus (310) Google Scholar). The G1/G2 arrest after DNA damage is regulated, at least in part, by the activities of the tumor suppressor genep53. Indeed, γ-irradiated cells, knockout for thep53 gene, progress from the G2 to the M phase and maintain DNA content of 4n because of cytokinesis failure (18Bunz F. Dutriaux A. Lengauer C. Waldman T. Zhou S. Brown J.P. Sedlvy J.M. Kinzler K.W. Vogelstein B. Science. 1998; 282: 1497-1500Crossref PubMed Scopus (2517) Google Scholar). Overexpression of an exogenous wild type p53 induces G2arrest associated with down-regulation of cyclin B1, cyclin A, and cdk1 expression (19Innocente S.A. Abrahamson J.L. Cogswell J.P. Lee J.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2147-2152Crossref PubMed Scopus (380) Google Scholar, 20Sugrue M.M. Shin D.Y. Lee S.W. Aaronson S.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9648-9653Crossref PubMed Scopus (259) Google Scholar). In agreement with these observations, it has been reported that wild type p53 overexpression in p53-null cells can suppress the transcriptional activity of cyclin A,cyclin B1, and cdk1 promoters (19Innocente S.A. Abrahamson J.L. Cogswell J.P. Lee J.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2147-2152Crossref PubMed Scopus (380) Google Scholar, 21Yamamoto M. Yoshida M. Ono K. Fujita T. Ohtani-Fujita N. Sakai T. Nikaido T. Exp. Cell Res. 1994; 210: 94-101Crossref PubMed Scopus (97) Google Scholar, 22Passalaris T.M. Benati J.A. Gewin L. Kiyono T. Galloway D.A. Mol. Cell. Biol. 1999; 19: 5872-5881Crossref PubMed Scopus (101) Google Scholar, 23Taylor W.R. DePrimo S.E. Agarwal A. Agarwal M.L. Schonthal A.H. Katula K.S. Stark G.R. Mol. Biol. Cell. 1999; 10: 3607-3622Crossref PubMed Scopus (164) Google Scholar, 24Yun J. Chae H.D. Choy H.E. Chung J. Yoo H.S. Han M.H. Shin D.Y. J. Biol. Chem. 1999; 274: 29677-29682Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). However, the specific molecular mechanisms responsible for the down-regulation of the transcription of these genes in the G2 arrest are still unknown.The CCAAT motif, G/AG/ACCAATC/GA/GC/G, is present in 30% of the eukaryotic promoters of tissue-specific, housekeeping, and cell cycle-regulatory classes of genes (25Bucher P. J. Mol. Biol. 1990; 212: 563-578Crossref PubMed Scopus (970) Google Scholar). NF-Y1 has been shown to bind to more than 120 CCAAT-containing promoters (26Mantovani R. Nucleic Acids Res. 1998; 26: 1135-1143Crossref PubMed Scopus (443) Google Scholar). It is composed of three subunits, NF-YA, -B, and -C, whose highly conserved genes have been cloned in mammals, yeast, plants, and parasites (27Hooft van Huijsduijnen R. Li X.Y. Black D. Matthes H. Benoist C. Mathis D. EMBO J. 1990; 9: 119-127Crossref Scopus (210) Google Scholar, 28Li X.Y. Hooft van Huijsduijnen R. Mantovani R. Benoist C. Mathis D.J. Biol. 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The promoters of cyclin A, cyclin B1, cyclin B2,cdk1, and cdc25C genes all contain CCAAT boxes, and it has been demonstrated that NF-Y modulates, at least in part, their activity during the cell cycle (11Zwicker J. Gross C. Lucibello F.C. Truss M. Ehlert F Engeland K. Muller R. Nucleic Acids Res. 1995; 23: 3822-3830Crossref PubMed Scopus (91) Google Scholar, 33Farina A. Manni I. Fontemaggi G. Tiainen M. Cenciarelli C. Bellorini M. Mantovani R. Sacchi A. Piaggio G. Oncogene. 1999; 18: 2818-2827Crossref PubMed Scopus (96) Google Scholar, 34Bolognese F. Wasner M. Lange-zu Dohna C. Gurtner A. Ronchi A. Muller H. Manni I. Mossner J. Piaggio G. Mantovani R. Engeland K. Oncogene. 1999; 18: 1845-1853Crossref PubMed Scopus (107) Google Scholar). Thus, we asked whether NF-Y is involved in the modulation of these promoter activities during the G2 arrest induced by DNA damage.For this purpose, we induced a G2 arrest by doxorubicin (Adriamycin; ADR)-mediated DNA damage in C2C12 nontransformed skeletal muscle cells, which possess a wild type p53 (35Soddu S. Blandino G. Scardigli R. Coen S. Marchetti A. Rizzo M.G. Bossi G. Imino L. Crescenzi M. Sacchi A. J. Cell Biol. 1996; 134: 193-204Crossref PubMed Scopus (121) Google Scholar). We found that ADR treatment down-regulates the promoter activities of the mitoticcyclins, cdk1, and cdc25C genes. This down-regulation involves a molecular mechanism requiring, at least in part, the CCAAT boxes and the transcription factor NF-Y. By using a dominant-negative p53 protein (DD-p53), we also show that the NF-Y-dependent down-regulation of mitoticcyclins, cdk1, and cdc25C occurs only in the presence of a functional p53 protein.DISCUSSIONThe progression through the G2 phase of the cell cycle is regulated in part by the cyclin A-cdk1 and cyclin B-cdk1 mitotic complexes (49Pines J. Hunter T. New Biol. 1990; 2: 389-401PubMed Google Scholar, 50Pagano M. Pepperkok R. Verde F. Ansorge W. Draetta G. EMBO J. 1992; 11: 961-971Crossref PubMed Scopus (1114) Google Scholar). Although it has been described that after DNA damage the mitotic entry is inhibited (18Bunz F. Dutriaux A. Lengauer C. Waldman T. Zhou S. Brown J.P. Sedlvy J.M. Kinzler K.W. Vogelstein B. Science. 1998; 282: 1497-1500Crossref PubMed Scopus (2517) Google Scholar, 43Flatt P.M. Tang L.J. Scatena C.D. Szak S.T. Pietenpol J.A. Mol. Cell. Biol. 2000; 20: 4210-4223Crossref PubMed Scopus (144) Google Scholar, 51de Toledo S.M. Azzam E.I. Keng P. Laffrenier S. Little J.B. Cell Growth Differ. 1998; 9: 87-96Google Scholar), the molecular mechanism sustaining the G2 arrest is not completely elucidated. In this report, we show that during the ADR-mediated G2 arrest, a decrease of protein expression levels of cyclin A, cyclin B1, cdk1, and cdc25C caused, at least in part, by a transcriptional level of regulation, is observed. Indeed, we demonstrate that after ADR treatment the promoter activities of thecyclin A, cyclin B1, cyclin B2,cdk1, and cdc25C genes are down-regulated. These results indicate that key molecules that control G2/M transition in the normal cell cycle are transcriptionally modulated during the G2 checkpoint induced by DNA damage.The repression of cyclin B1, cyclin B2, andcdc25C promoters, occurring in the G2 arrest, requires the integrity of the CCAAT boxes present in the 5′ region of these genes, suggesting a role for the transcription factor NF-Y (Figs.3 and 4). It has been demonstrated previously that NF-Y binds the CCAAT boxes of cyclin B1, cyclin B2, andcdc25C promoters (11Zwicker J. Gross C. Lucibello F.C. Truss M. Ehlert F Engeland K. Muller R. Nucleic Acids Res. 1995; 23: 3822-3830Crossref PubMed Scopus (91) Google Scholar, 33Farina A. Manni I. Fontemaggi G. Tiainen M. Cenciarelli C. Bellorini M. Mantovani R. Sacchi A. Piaggio G. Oncogene. 1999; 18: 2818-2827Crossref PubMed Scopus (96) Google Scholar, 34Bolognese F. Wasner M. Lange-zu Dohna C. Gurtner A. Ronchi A. Muller H. Manni I. Mossner J. Piaggio G. Mantovani R. Engeland K. Oncogene. 1999; 18: 1845-1853Crossref PubMed Scopus (107) Google Scholar). Here we show that a functional NF-Y complex is required for the inhibition of cyclin B1, cyclin B2, and cdc25C promoters during G2 arrest. Indeed, the expression of a dominant-negative NF-YA abolishes the ADR-mediated repression of these promoters. Moreover, as already demonstrated, this is true also for thecdk1 promoter (24Yun J. Chae H.D. Choy H.E. Chung J. Yoo H.S. Han M.H. Shin D.Y. J. Biol. Chem. 1999; 274: 29677-29682Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar).The down-regulation of expression and transcription of mitotic cyclin-cdk1 complexes, as well as of cdc25C phosphatase, after DNA damage requires the presence of a functional p53 protein (Fig. 8). In agreement with this result it has been reported that overexpression of p53 in p53-null cells suppresses the transcriptional activity of cyclin A, cyclin B1, cyclin B2, and cdk1 promoters (19Innocente S.A. Abrahamson J.L. Cogswell J.P. Lee J.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2147-2152Crossref PubMed Scopus (380) Google Scholar, 21Yamamoto M. Yoshida M. Ono K. Fujita T. Ohtani-Fujita N. Sakai T. Nikaido T. Exp. Cell Res. 1994; 210: 94-101Crossref PubMed Scopus (97) Google Scholar, 22Passalaris T.M. Benati J.A. Gewin L. Kiyono T. Galloway D.A. Mol. Cell. Biol. 1999; 19: 5872-5881Crossref PubMed Scopus (101) Google Scholar, 23Taylor W.R. DePrimo S.E. Agarwal A. Agarwal M.L. Schonthal A.H. Katula K.S. Stark G.R. Mol. Biol. Cell. 1999; 10: 3607-3622Crossref PubMed Scopus (164) Google Scholar, 24Yun J. Chae H.D. Choy H.E. Chung J. Yoo H.S. Han M.H. Shin D.Y. J. Biol. Chem. 1999; 274: 29677-29682Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 52Krause K. Wasner M. Reinhard W. Haugwitz U. Lange-zu Dohna C. Mossner J. Engeland K. Nucleic Acids Res. 2000; 28: 4410-4418Crossref PubMed Scopus (147) Google Scholar). Nevertheless, this is the first evidence that the cdc25C promoter activity is down-regulated also.None of the tested promoters contains a canonical DNA binding site for p53, thus our results lead to the speculation that p53 could interfere with the function of NF-Y. Indeed, it has been described that p53 protein can repress transcription by binding to and preventing the function of specific transcription factors (53Farmer G. Friedlander P. Colgan J. Manley J.L. Prives C. Nucleic Acids Res. 1996; 24: 4281-4288Crossref PubMed Scopus (61) Google Scholar, 54Gopalkrishnan R.V. Lam E.W.F. Kedinger C. J. Biol. Chem. 1998; 273: 10972-10978Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). In shift experiments performed with anti NF-Y we observed that NF-Y is present on the CCAAT boxes of cyclin B1 and cyclin B2promoters before and after ADR-treatment. 2I. Manni, unpublished observations Thus, p53 does not interfere with the DNA binding of NF-Y. Another possibility is that p53 interferes with the NF-Y recruitment of coactivators and/or general transcription factors. Indeed, it has been shown that p53 and NF-Y bind to overlapping domains of the p300 coactivator (55Avantaggiati M.L. Ogryzko V. Gardner K. Levine A.S. Kelly K. Cell. 1997; 89: 1175-1184Abstract Full Text Full Text PDF PubMed Scopus (588) Google Scholar, 56Li Q. Herrler M. Landsberger N. Kaludov N. Ogryzko V.V. Nakatani Y. Wolffe A.P. EMBO J. 1998; 17: 6300-6315Crossref PubMed Scopus (178) Google Scholar), and both p53 and NF-Y bind to TATA-binding protein (57Farmer G. Colgan J. Nakatani Y. Manley J.L. Prives C. Mol. Cell. Biol. 1996; 16: 4295-4304Crossref PubMed Google Scholar, 58Bellorini M. Lee D.K. Dantonel J.C. Zemzoumi K. Roeder R.G. Tora L. Mantovani R. Nucleic Acids Res. 1997; 25: 2174-2181Crossref PubMed Scopus (103) Google Scholar).Interestingly, NF-Y was shown also by other groups to be required for the p53-mediated inhibition of cdk1 transcription (23Taylor W.R. DePrimo S.E. Agarwal A. Agarwal M.L. Schonthal A.H. Katula K.S. Stark G.R. Mol. Biol. Cell. 1999; 10: 3607-3622Crossref PubMed Scopus (164) Google Scholar, 24Yun J. Chae H.D. Choy H.E. Chung J. Yoo H.S. Han M.H. Shin D.Y. J. Biol. Chem. 1999; 274: 29677-29682Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Furthermore, NF-Y binds the CCAAT box contained in the cyclin A promoter (11Zwicker J. Gross C. Lucibello F.C. Truss M. Ehlert F Engeland K. Muller R. Nucleic Acids Res. 1995; 23: 3822-3830Crossref PubMed Scopus (91) Google Scholar). These findings lead to the speculation that NF-Y modulates cdk1 and cyclin A transcription after ADR-mediated G2 arrest by the same mechanism described here for the cyclin B1, cyclin B2, andcdc25C genes.It has been shown recently that an endogenous p53 protein sustains a G2 arrest induced by ADR through a pRb-dependent decrease of cyclin B1 and cdk1 expression (43Flatt P.M. Tang L.J. Scatena C.D. Szak S.T. Pietenpol J.A. Mol. Cell. Biol. 2000; 20: 4210-4223Crossref PubMed Scopus (144) Google Scholar). However, the pRb-dependent inhibition of cyclin B1 expression does not seem to be caused by direct binding of pRB/E2F family proteins to the cyclin B1 promoter. Indeed, as assessed by chromatin cross-linked immunoprecipitation, an anti-E2F1 antibody does not immunoprecipitate, from cycling cells, chromatin containing the cyclin B1 promoter, whereas an anti-NF-Y antibody does. 3S. Sciortino, unpublished observations. The transcriptional regulation of the expression of mitotic kinase complexes is not the only mechanism that sustains a G2block. Indeed, it has been demonstrated recently that in a human colorectal cancer cell line, the p53-dependent G2 arrest after γ-irradiation is the result of induction of 14–3-3ς expression (59Hermeking H. Lengauer C. Polyak K. He T.C. Zhang L. Thiagalingam S. Kinzler K.W. Vogelstein B. Mol. Cell. 1997; 1: 3-11Abstract Full Text Full Text PDF PubMed Scopus (1100) Google Scholar). Once overexpressed, 14–3-3ς blocks the cell cycle contributing to the nuclear exclusion of cdk1-cyclin B1 complexes and cdc25C phosphatase (60Chan T.A. Hermeking H. Lengauer C. Kinzler K.W. Vogelstein B. Nature. 1999; 401: 616-620Crossref PubMed Scopus (809) Google Scholar). Thus, after DNA damage, transcriptional down-regulation of mitotic kinase complexes (this paper) and their cytoplasmic segregation (56Li Q. Herrler M. Landsberger N. Kaludov N. Ogryzko V.V. Nakatani Y. Wolffe A.P. EMBO J. 1998; 17: 6300-6315Crossref PubMed Scopus (178) Google Scholar) might concomitantly sustain the G2 checkpoint.In summary, this work provides evidences that in muscle cells, the molecular mechanism responsible for the G2 checkpoint induced by ADR-mediated DNA damage involves the ability of the NF-Y transcription factor to prevent the transcription of key regulatory molecules essential for the progression through the G2phase of the cell cycle. This finding opens the question of whether other genes, controlled by NF-Y during the cell cycle (26Mantovani R. Nucleic Acids Res. 1998; 26: 1135-1143Crossref PubMed Scopus (443) Google Scholar), are targets of its activity in the cell cycle checkpoints. In mammalian cells, progression through the G2 phase of the cell cycle is mediated by the activity of a specific set of proteins, which includes mitotic cyclins A, B1, and B2, mitotic kinase cdk1 (alias p34cdc2), and mitotic phosphatase cdc25C. The kinase activity of cdk1 during the G2 phase is dependent on its dephosphorylated status of specific residues triggered by the phosphatase activity of cdc25C protein (1Gould K.L. Nurse P. Nature. 1989; 342: 39-45Crossref PubMed Scopus (851) Google Scholar, 2Pines J. Hunter T. Cell. 1989; 58: 833-846Abstract Full Text PDF PubMed Scopus (693) Google Scholar, 3Pines J. Hunter T. Nature. 1990; 346: 760-763Crossref PubMed Scopus (528) Google Scholar, 4Parker L.L. Piwnica-Worms H. Science. 1992; 257: 1955-1957Crossref PubMed Scopus (536) Google Scholar, 5McGowan C.H. Russell P. EMBO J. 1993; 12: 75-85Crossref PubMed Scopus (384) Google Scholar, 6Liu F. Stanton J.J. Wu Z. Piwnica-Worms H. Mol. Cell. Biol. 1997; 17: 571-583Crossref PubMed Scopus (263) Google Scholar), as well as on the levels of cyclins A and Bs. In proliferating cells, oscillations of mitotic cyclin amounts are tightly regulated at the transcriptional level (7Henglein B. Chenivesse X. Wang J. Eick D. Brechot C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5490-5494Crossref PubMed Scopus (244) Google Scholar, 8Hwang A. Maity A. McKenna W.G. Muschel R.J. J. Biol. Chem. 1995; 270: 28419-28424Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 9Lucibello F.C. Truss M. Zwicker J. Ehlert F. Beato M. Muller R. EMBO J. 1995; 14: 132-142Crossref PubMed Scopus (87) Google Scholar, 10Piaggio G. Farina A. Perrotti D. Manni I. Fuschi P. Sacchi A. Gaetano C. Exp. Cell Res. 1995; 216: 396-402Crossref PubMed Scopus (54) Google Scholar, 11Zwicker J. Gross C. Lucibello F.C. Truss M. Ehlert F Engeland K. Muller R. Nucleic Acids Res. 1995; 23: 3822-3830Crossref PubMed Scopus (91) Google Scholar, 12Katula K.S. Wright K.L. Paul H. Surman D.R. Nuckolls F.J. Smith J.W. Ting J.P. Yates J. Cogswell J.P. Cell Growth Differ. 1997; 8: 11-20PubMed Google Scholar). In particular, activation of cdk1 does not occur until sufficient cyclin B1 protein is synthesized (13Solomon M.J. Glotzer M. Lee H. Philipe M. Kirshner S. Cell. 1990; 63: 1013-1034Abstract Full Text PDF PubMed Scopus (503) Google Scholar). The accumulation of mitotic cyclins and cdk1 correlates with nascent gene expression, and their mRNAs can only be detected in particular phases of the cell cycle (2Pines J. Hunter T. Cell. 1989; 58: 833-846Abstract Full Text PDF PubMed Scopus (693) Google Scholar, 7Henglein B. Chenivesse X. Wang J. Eick D. Brechot C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5490-5494Crossref PubMed Scopus (244) Google Scholar, 10Piaggio G. Farina A. Perrotti D. Manni I. Fuschi P. Sacchi A. Gaetano C. Exp. Cell Res. 1995; 216: 396-402Crossref PubMed Scopus (54) Google Scholar, 14Dalton S. EMBO J. 1992; 11: 797-1804Crossref Scopus (335) Google Scholar). Cell cycle progression through the G2 phase is controlled by the G2 checkpoint. This checkpoint ensures correct DNA synthesis during cell proliferation and genome integrity after DNA damage. In the latter condition, cells can arrest at the G1and/or the G2 checkpoint, depending on cell type, cell cycle phase, and checkpoint integrity (15Kastan M.B. Onyekwere O. Sidransky D. Vogelstein B. Craig R.W. Cancer Res. 1991; 51: 6304-6311PubMed Google Scholar, 16Maity A. McKenna W.G. Muschel R.J. Radiother. Oncol. 1994; 31: 1-13Abstract Full Text PDF PubMed Scopus (270) Google Scholar, 17Cox L.S. Lane D.P. Bioessays. 1995; 17: 501-508Crossref PubMed Scopus (310) Google Scholar). The G1/G2 arrest after DNA damage is regulated, at least in part, by the activities of the tumor suppressor genep53. Indeed, γ-irradiated cells, knockout for thep53 gene, progress from the G2 to the M phase and maintain DNA content of 4n because of cytokinesis failure (18Bunz F. Dutriaux A. Lengauer C. Waldman T. Zhou S. Brown J.P. Sedlvy J.M. Kinzler K.W. Vogelstein B. Science. 1998; 282: 1497-1500Crossref PubMed Scopus (2517) Google Scholar). Overexpression of an exogenous wild type p53 induces G2arrest associated with down-regulation of cyclin B1, cyclin A, and cdk1 expression (19Innocente S.A. Abrahamson J.L. Cogswell J.P. Lee J.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2147-2152Crossref PubMed Scopus (380) Google Scholar, 20Sugrue M.M. Shin D.Y. Lee S.W. Aaronson S.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9648-9653Crossref PubMed Scopus (259) Google Scholar). In agreement with these observations, it has been reported that wild type p53 overexpression in p53-null cells can suppress the transcriptional activity of cyclin A,cyclin B1, and cdk1 promoters (19Innocente S.A. Abrahamson J.L. Cogswell J.P. Lee J.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2147-2152Crossref PubMed Scopus (380) Google Scholar, 21Yamamoto M. Yoshida M. Ono K. Fujita T. Ohtani-Fujita N. Sakai T. Nikaido T. Exp. Cell Res. 1994; 210: 94-101Crossref PubMed Scopus (97) Google Scholar, 22Passalaris T.M. Benati J.A. Gewin L. Kiyono T. Galloway D.A. Mol. Cell. Biol. 1999; 19: 5872-5881Crossref PubMed Scopus (101) Google Scholar, 23Taylor W.R. DePrimo S.E. Agarwal A. Agarwal M.L. Schonthal A.H. Katula K.S. Stark G.R. Mol. Biol. Cell. 1999; 10: 3607-3622Crossref PubMed Scopus (164) Google Scholar, 24Yun J. Chae H.D. Choy H.E. Chung J. Yoo H.S. Han M.H. Shin D.Y. J. Biol. Chem. 1999; 274: 29677-29682Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). However, the specific molecular mechanisms responsible for the down-regulation of the transcription of these genes in the G2 arrest are still unknown. The CCAAT motif, G/AG/ACCAATC/GA/GC/G, is present in 30% of the eukaryotic promoters of tissue-specific, housekeeping, and cell cycle-regulatory classes of genes (25Bucher P. J. Mol. Biol. 1990; 212: 563-578Crossref PubMed Scopus (970) Google Scholar). NF-Y1 has been shown to bind to more than 120 CCAAT-containing promoters (26Mantovani R. Nucleic Acids Res. 1998; 26: 1135-1143Crossref PubMed Scopus (443) Google Scholar). It is composed of three subunits, NF-YA, -B, and -C, whose highly conserved genes have been cloned in mammals, yeast, plants, and parasites (27Hooft van Huijsduijnen R. Li X.Y. Black D. Matthes H. Benoist C. Mathis D. EMBO J. 1990; 9: 119-127Crossref Scopus (210) Google Scholar, 28Li X.Y. Hooft van Huijsduijnen R. Mantovani R. Benoist C. Mathis D.J. Biol. Chem. 1992; 267: 8984-8990Abstract Full Text PDF Google Scholar, 29McNabb D.S. Xing Y. Guarente L. Genes Dev. 1995; 9: 47-58Crossref PubMed Scopus (232) Google Scholar, 30Sinha S. Maity S.N. Seldin M.F. de Crombrugghe B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1624-1628Crossref PubMed Scopus (250) Google Scholar, 31Bellorini M. Zemzoumi K. Farina A. Berthelsen J. Piaggio G. Mantovani R. Gene (Amst.). 1997; 93: 119-125Crossref Google Scholar). All three subunits are required for CCAAT binding (32Caretti G. Motta M.C. Mantovani R. Mol. Cell. Biol. 1999; 19: 8591-8603Crossref PubMed Scopus (59) Google Scholar, 33Farina A. Manni I. Fontemaggi G. Tiainen M. Cenciarelli C. Bellorini M. Mantovani R. Sacchi A. Piaggio G. Oncogene. 1999; 18: 2818-2827Crossref PubMed Scopus (96) Google Scholar). The promoters of cyclin A, cyclin B1, cyclin B2,cdk1, and cdc25C genes all contain CCAAT boxes, and it has been demonstrated that NF-Y modulates, at least in part, their activity during the cell cycle (11Zwicker J. Gross C. Lucibello F.C. Truss M. Ehlert F Engeland K. Muller R. Nucleic Acids Res. 1995; 23: 3822-3830Crossref PubMed Scopus (91) Google Scholar, 33Farina A. Manni I. Fontemaggi G. Tiainen M. Cenciarelli C. Bellorini M. Mantovani R. Sacchi A. Piaggio G. Oncogene. 1999; 18: 2818-2827Crossref PubMed Scopus (96) Google Scholar, 34Bolognese F. Wasner M. Lange-zu Dohna C. Gurtner A. Ronchi A. Muller H. Manni I. Mossner J. Piaggio G. Mantovani R. Engeland K. Oncogene. 1999; 18: 1845-1853Crossref PubMed Scopus (107) Google Scholar). Thus, we asked whether NF-Y is involved in the modulation of these promoter activities during the G2 arrest induced by DNA damage. For this purpose, we induced a G2 arrest by doxorubicin (Adriamycin; ADR)-mediated DNA damage in C2C12 nontransformed skeletal muscle cells, which possess a wild type p53 (35Soddu S. Blandino G. Scardigli R. Coen S. Marchetti A. Rizzo M.G. Bossi G. Imino L. Crescenzi M. Sacchi A. J. Cell Biol. 1996; 134: 193-204Crossref PubMed Scopus (121) Google Scholar). We found that ADR treatment down-regulates the promoter activities of the mitoticcyclins, cdk1, and cdc25C genes. This down-regulation involves a molecular mechanism requiring, at least in part, the CCAAT boxes and the transcription factor NF-Y. By using a dominant-negative p53 protein (DD-p53), we also show that the NF-Y-dependent down-regulation of mitoticcyclins, cdk1, and cdc25C occurs only in the presence of a functional p53 protein. DISCUSSIONThe progression through the G2 phase of the cell cycle is regulated in part by the cyclin A-cdk1 and cyclin B-cdk1 mitotic complexes (49Pines J. Hunter T. New Biol. 1990; 2: 389-401PubMed Google Scholar, 50Pagano M. Pepperkok R. Verde F. Ansorge W. Draetta G. EMBO J. 1992; 11: 961-971Crossref PubMed Scopus (1114) Google Scholar). Although it has been described that after DNA damage the mitotic entry is inhibited (18Bunz F. Dutriaux A. Lengauer C. Waldman T. Zhou S. Brown J.P. Sedlvy J.M. Kinzler K.W. Vogelstein B. Science. 1998; 282: 1497-1500Crossref PubMed Scopus (2517) Google Scholar, 43Flatt P.M. Tang L.J. Scatena C.D. Szak S.T. Pietenpol J.A. Mol. Cell. Biol. 2000; 20: 4210-4223Crossref PubMed Scopus (144) Google Scholar, 51de Toledo S.M. Azzam E.I. Keng P. Laffrenier S. Little J.B. Cell Growth Differ. 1998; 9: 87-96Google Scholar), the molecular mechanism sustaining the G2 arrest is not completely elucidated. In this report, we show that during the ADR-mediated G2 arrest, a decrease of protein expression levels of cyclin A, cyclin B1, cdk1, and cdc25C caused, at least in part, by a transcriptional level of regulation, is observed. Indeed, we demonstrate that after ADR treatment the promoter activities of thecyclin A, cyclin B1, cyclin B2,cdk1, and cdc25C genes are down-regulated. These results indicate that key molecules that control G2/M transition in the normal cell cycle are transcriptionally modulated during the G2 checkpoint induced by DNA damage.The repression of cyclin B1, cyclin B2, andcdc25C promoters, occurring in the G2 arrest, requires the integrity of the CCAAT boxes present in the 5′ region of these genes, suggesting a role for the transcription factor NF-Y (Figs.3 and 4). It has been demonstrated previously that NF-Y binds the CCAAT boxes of cyclin B1, cyclin B2, andcdc25C promoters (11Zwicker J. Gross C. Lucibello F.C. Truss M. Ehlert F Engeland K. Muller R. Nucleic Acids Res. 1995; 23: 3822-3830Crossref PubMed Scopus (91) Google Scholar, 33Farina A. Manni I. Fontemaggi G. Tiainen M. Cenciarelli C. Bellorini M. Mantovani R. Sacchi A. Piaggio G. Oncogene. 1999; 18: 2818-2827Crossref PubMed Scopus (96) Google Scholar, 34Bolognese F. Wasner M. Lange-zu Dohna C. Gurtner A. Ronchi A. Muller H. Manni I. Mossner J. Piaggio G. Mantovani R. Engeland K. Oncogene. 1999; 18: 1845-1853Crossref PubMed Scopus (107) Google Scholar). Here we show that a functional NF-Y complex is required for the inhibition of cyclin B1, cyclin B2, and cdc25C promoters during G2 arrest. Indeed, the expression of a dominant-negative NF-YA abolishes the ADR-mediated repression of these promoters. Moreover, as already demonstrated, this is true also for thecdk1 promoter (24Yun J. Chae H.D. Choy H.E. Chung J. Yoo H.S. Han M.H. Shin D.Y. J. Biol. Chem. 1999; 274: 29677-29682Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar).The down-regulation of expression and transcription of mitotic cyclin-cdk1 complexes, as well as of cdc25C phosphatase, after DNA damage requires the presence of a functional p53 protein (Fig. 8). In agreement with this result it has been reported that overexpression of p53 in p53-null cells suppresses the transcriptional activity of cyclin A, cyclin B1, cyclin B2, and cdk1 promoters (19Innocente S.A. Abrahamson J.L. Cogswell J.P. Lee J.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2147-2152Crossref PubMed Scopus (380) Google Scholar, 21Yamamoto M. Yoshida M. Ono K. Fujita T. Ohtani-Fujita N. Sakai T. Nikaido T. Exp. Cell Res. 1994; 210: 94-101Crossref PubMed Scopus (97) Google Scholar, 22Passalaris T.M. Benati J.A. Gewin L. Kiyono T. Galloway D.A. Mol. Cell. Biol. 1999; 19: 5872-5881Crossref PubMed Scopus (101) Google Scholar, 23Taylor W.R. DePrimo S.E. Agarwal A. Agarwal M.L. Schonthal A.H. Katula K.S. Stark G.R. Mol. Biol. Cell. 1999; 10: 3607-3622Crossref PubMed Scopus (164) Google Scholar, 24Yun J. Chae H.D. Choy H.E. Chung J. Yoo H.S. Han M.H. Shin D.Y. J. Biol. Chem. 1999; 274: 29677-29682Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 52Krause K. Wasner M. Reinhard W. Haugwitz U. Lange-zu Dohna C. Mossner J. Engeland K. Nucleic Acids Res. 2000; 28: 4410-4418Crossref PubMed Scopus (147) Google Scholar). Nevertheless, this is the first evidence that the cdc25C promoter activity is down-regulated also.None of the tested promoters contains a canonical DNA binding site for p53, thus our results lead to the speculation that p53 could interfere with the function of NF-Y. Indeed, it has been described that p53 protein can repress transcription by binding to and preventing the function of specific transcription factors (53Farmer G. Friedlander P. Colgan J. Manley J.L. Prives C. Nucleic Acids Res. 1996; 24: 4281-4288Crossref PubMed Scopus (61) Google Scholar, 54Gopalkrishnan R.V. Lam E.W.F. Kedinger C. J. Biol. Chem. 1998; 273: 10972-10978Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). In shift experiments performed with anti NF-Y we observed that NF-Y is present on the CCAAT boxes of cyclin B1 and cyclin B2promoters before and after ADR-treatment. 2I. Manni, unpublished observations Thus, p53 does not interfere with the DNA binding of NF-Y. Another possibility is that p53 interferes with the NF-Y recruitment of coactivators and/or general transcription factors. Indeed, it has been shown that p53 and NF-Y bind to overlapping domains of the p300 coactivator (55Avantaggiati M.L. Ogryzko V. Gardner K. Levine A.S. Kelly K. Cell. 1997; 89: 1175-1184Abstract Full Text Full Text PDF PubMed Scopus (588) Google Scholar, 56Li Q. Herrler M. Landsberger N. Kaludov N. Ogryzko V.V. Nakatani Y. Wolffe A.P. EMBO J. 1998; 17: 6300-6315Crossref PubMed Scopus (178) Google Scholar), and both p53 and NF-Y bind to TATA-binding protein (57Farmer G. Colgan J. Nakatani Y. Manley J.L. Prives C. Mol. Cell. Biol. 1996; 16: 4295-4304Crossref PubMed Google Scholar, 58Bellorini M. Lee D.K. Dantonel J.C. Zemzoumi K. Roeder R.G. Tora L. Mantovani R. Nucleic Acids Res. 1997; 25: 2174-2181Crossref PubMed Scopus (103) Google Scholar).Interestingly, NF-Y was shown also by other groups to be required for the p53-mediated inhibition of cdk1 transcription (23Taylor W.R. DePrimo S.E. Agarwal A. Agarwal M.L. Schonthal A.H. Katula K.S. Stark G.R. Mol. Biol. Cell. 1999; 10: 3607-3622Crossref PubMed Scopus (164) Google Scholar, 24Yun J. Chae H.D. Choy H.E. Chung J. Yoo H.S. Han M.H. Shin D.Y. J. Biol. Chem. 1999; 274: 29677-29682Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Furthermore, NF-Y binds the CCAAT box contained in the cyclin A promoter (11Zwicker J. Gross C. Lucibello F.C. Truss M. Ehlert F Engeland K. Muller R. Nucleic Acids Res. 1995; 23: 3822-3830Crossref PubMed Scopus (91) Google Scholar). These findings lead to the speculation that NF-Y modulates cdk1 and cyclin A transcription after ADR-mediated G2 arrest by the same mechanism described here for the cyclin B1, cyclin B2, andcdc25C genes.It has been shown recently that an endogenous p53 protein sustains a G2 arrest induced by ADR through a pRb-dependent decrease of cyclin B1 and cdk1 expression (43Flatt P.M. Tang L.J. Scatena C.D. Szak S.T. Pietenpol J.A. Mol. Cell. Biol. 2000; 20: 4210-4223Crossref PubMed Scopus (144) Google Scholar). However, the pRb-dependent inhibition of cyclin B1 expression does not seem to be caused by direct binding of pRB/E2F family proteins to the cyclin B1 promoter. Indeed, as assessed by chromatin cross-linked immunoprecipitation, an anti-E2F1 antibody does not immunoprecipitate, from cycling cells, chromatin containing the cyclin B1 promoter, whereas an anti-NF-Y antibody does. 3S. Sciortino, unpublished observations. The transcriptional regulation of the expression of mitotic kinase complexes is not the only mechanism that sustains a G2block. Indeed, it has been demonstrated recently that in a human colorectal cancer cell line, the p53-dependent G2 arrest after γ-irradiation is the result of induction of 14–3-3ς expression (59Hermeking H. Lengauer C. Polyak K. He T.C. Zhang L. Thiagalingam S. Kinzler K.W. Vogelstein B. Mol. Cell. 1997; 1: 3-11Abstract Full Text Full Text PDF PubMed Scopus (1100) Google Scholar). Once overexpressed, 14–3-3ς blocks the cell cycle contributing to the nuclear exclusion of cdk1-cyclin B1 complexes and cdc25C phosphatase (60Chan T.A. Hermeking H. Lengauer C. Kinzler K.W. Vogelstein B. Nature. 1999; 401: 616-620Crossref PubMed Scopus (809) Google Scholar). Thus, after DNA damage, transcriptional down-regulation of mitotic kinase complexes (this paper) and their cytoplasmic segregation (56Li Q. Herrler M. Landsberger N. Kaludov N. Ogryzko V.V. Nakatani Y. Wolffe A.P. EMBO J. 1998; 17: 6300-6315Crossref PubMed Scopus (178) Google Scholar) might concomitantly sustain the G2 checkpoint.In summary, this work provides evidences that in muscle cells, the molecular mechanism responsible for the G2 checkpoint induced by ADR-mediated DNA damage involves the ability of the NF-Y transcription factor to prevent the transcription of key regulatory molecules essential for the progression through the G2phase of the cell cycle. This finding opens the question of whether other genes, controlled by NF-Y during the cell cycle (26Mantovani R. Nucleic Acids Res. 1998; 26: 1135-1143Crossref PubMed Scopus (443) Google Scholar), are targets of its activity in the cell cycle checkpoints. The progression through the G2 phase of the cell cycle is regulated in part by the cyclin A-cdk1 and cyclin B-cdk1 mitotic complexes (49Pines J. Hunter T. New Biol. 1990; 2: 389-401PubMed Google Scholar, 50Pagano M. Pepperkok R. Verde F. Ansorge W. Draetta G. EMBO J. 1992; 11: 961-971Crossref PubMed Scopus (1114) Google Scholar). Although it has been described that after DNA damage the mitotic entry is inhibited (18Bunz F. Dutriaux A. Lengauer C. Waldman T. Zhou S. Brown J.P. Sedlvy J.M. Kinzler K.W. Vogelstein B. Science. 1998; 282: 1497-1500Crossref PubMed Scopus (2517) Google Scholar, 43Flatt P.M. Tang L.J. Scatena C.D. Szak S.T. Pietenpol J.A. Mol. Cell. Biol. 2000; 20: 4210-4223Crossref PubMed Scopus (144) Google Scholar, 51de Toledo S.M. Azzam E.I. Keng P. Laffrenier S. Little J.B. Cell Growth Differ. 1998; 9: 87-96Google Scholar), the molecular mechanism sustaining the G2 arrest is not completely elucidated. In this report, we show that during the ADR-mediated G2 arrest, a decrease of protein expression levels of cyclin A, cyclin B1, cdk1, and cdc25C caused, at least in part, by a transcriptional level of regulation, is observed. Indeed, we demonstrate that after ADR treatment the promoter activities of thecyclin A, cyclin B1, cyclin B2,cdk1, and cdc25C genes are down-regulated. These results indicate that key molecules that control G2/M transition in the normal cell cycle are transcriptionally modulated during the G2 checkpoint induced by DNA damage. The repression of cyclin B1, cyclin B2, andcdc25C promoters, occurring in the G2 arrest, requires the integrity of the CCAAT boxes present in the 5′ region of these genes, suggesting a role for the transcription factor NF-Y (Figs.3 and 4). It has been demonstrated previously that NF-Y binds the CCAAT boxes of cyclin B1, cyclin B2, andcdc25C promoters (11Zwicker J. Gross C. Lucibello F.C. Truss M. Ehlert F Engeland K. Muller R. Nucleic Acids Res. 1995; 23: 3822-3830Crossref PubMed Scopus (91) Google Scholar, 33Farina A. Manni I. Fontemaggi G. Tiainen M. Cenciarelli C. Bellorini M. Mantovani R. Sacchi A. Piaggio G. Oncogene. 1999; 18: 2818-2827Crossref PubMed Scopus (96) Google Scholar, 34Bolognese F. Wasner M. Lange-zu Dohna C. Gurtner A. Ronchi A. Muller H. Manni I. Mossner J. Piaggio G. Mantovani R. Engeland K. Oncogene. 1999; 18: 1845-1853Crossref PubMed Scopus (107) Google Scholar). Here we show that a functional NF-Y complex is required for the inhibition of cyclin B1, cyclin B2, and cdc25C promoters during G2 arrest. Indeed, the expression of a dominant-negative NF-YA abolishes the ADR-mediated repression of these promoters. Moreover, as already demonstrated, this is true also for thecdk1 promoter (24Yun J. Chae H.D. Choy H.E. Chung J. Yoo H.S. Han M.H. Shin D.Y. J. Biol. Chem. 1999; 274: 29677-29682Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). The down-regulation of expression and transcription of mitotic cyclin-cdk1 complexes, as well as of cdc25C phosphatase, after DNA damage requires the presence of a functional p53 protein (Fig. 8). In agreement with this result it has been reported that overexpression of p53 in p53-null cells suppresses the transcriptional activity of cyclin A, cyclin B1, cyclin B2, and cdk1 promoters (19Innocente S.A. Abrahamson J.L. Cogswell J.P. Lee J.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2147-2152Crossref PubMed Scopus (380) Google Scholar, 21Yamamoto M. Yoshida M. Ono K. Fujita T. Ohtani-Fujita N. Sakai T. Nikaido T. Exp. Cell Res. 1994; 210: 94-101Crossref PubMed Scopus (97) Google Scholar, 22Passalaris T.M. Benati J.A. Gewin L. Kiyono T. Galloway D.A. Mol. Cell. Biol. 1999; 19: 5872-5881Crossref PubMed Scopus (101) Google Scholar, 23Taylor W.R. DePrimo S.E. Agarwal A. Agarwal M.L. Schonthal A.H. Katula K.S. Stark G.R. Mol. Biol. Cell. 1999; 10: 3607-3622Crossref PubMed Scopus (164) Google Scholar, 24Yun J. Chae H.D. Choy H.E. Chung J. Yoo H.S. Han M.H. Shin D.Y. J. Biol. Chem. 1999; 274: 29677-29682Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 52Krause K. Wasner M. Reinhard W. Haugwitz U. Lange-zu Dohna C. Mossner J. Engeland K. Nucleic Acids Res. 2000; 28: 4410-4418Crossref PubMed Scopus (147) Google Scholar). Nevertheless, this is the first evidence that the cdc25C promoter activity is down-regulated also. None of the tested promoters contains a canonical DNA binding site for p53, thus our results lead to the speculation that p53 could interfere with the function of NF-Y. Indeed, it has been described that p53 protein can repress transcription by binding to and preventing the function of specific transcription factors (53Farmer G. Friedlander P. Colgan J. Manley J.L. Prives C. Nucleic Acids Res. 1996; 24: 4281-4288Crossref PubMed Scopus (61) Google Scholar, 54Gopalkrishnan R.V. Lam E.W.F. Kedinger C. J. Biol. Chem. 1998; 273: 10972-10978Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). In shift experiments performed with anti NF-Y we observed that NF-Y is present on the CCAAT boxes of cyclin B1 and cyclin B2promoters before and after ADR-treatment. 2I. Manni, unpublished observations Thus, p53 does not interfere with the DNA binding of NF-Y. Another possibility is that p53 interferes with the NF-Y recruitment of coactivators and/or general transcription factors. Indeed, it has been shown that p53 and NF-Y bind to overlapping domains of the p300 coactivator (55Avantaggiati M.L. Ogryzko V. Gardner K. Levine A.S. Kelly K. Cell. 1997; 89: 1175-1184Abstract Full Text Full Text PDF PubMed Scopus (588) Google Scholar, 56Li Q. Herrler M. Landsberger N. Kaludov N. Ogryzko V.V. Nakatani Y. Wolffe A.P. EMBO J. 1998; 17: 6300-6315Crossref PubMed Scopus (178) Google Scholar), and both p53 and NF-Y bind to TATA-binding protein (57Farmer G. Colgan J. Nakatani Y. Manley J.L. Prives C. Mol. Cell. Biol. 1996; 16: 4295-4304Crossref PubMed Google Scholar, 58Bellorini M. Lee D.K. Dantonel J.C. Zemzoumi K. Roeder R.G. Tora L. Mantovani R. Nucleic Acids Res. 1997; 25: 2174-2181Crossref PubMed Scopus (103) Google Scholar). Interestingly, NF-Y was shown also by other groups to be required for the p53-mediated inhibition of cdk1 transcription (23Taylor W.R. DePrimo S.E. Agarwal A. Agarwal M.L. Schonthal A.H. Katula K.S. Stark G.R. Mol. Biol. Cell. 1999; 10: 3607-3622Crossref PubMed Scopus (164) Google Scholar, 24Yun J. Chae H.D. Choy H.E. Chung J. Yoo H.S. Han M.H. Shin D.Y. J. Biol. Chem. 1999; 274: 29677-29682Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Furthermore, NF-Y binds the CCAAT box contained in the cyclin A promoter (11Zwicker J. Gross C. Lucibello F.C. Truss M. Ehlert F Engeland K. Muller R. Nucleic Acids Res. 1995; 23: 3822-3830Crossref PubMed Scopus (91) Google Scholar). These findings lead to the speculation that NF-Y modulates cdk1 and cyclin A transcription after ADR-mediated G2 arrest by the same mechanism described here for the cyclin B1, cyclin B2, andcdc25C genes. It has been shown recently that an endogenous p53 protein sustains a G2 arrest induced by ADR through a pRb-dependent decrease of cyclin B1 and cdk1 expression (43Flatt P.M. Tang L.J. Scatena C.D. Szak S.T. Pietenpol J.A. Mol. Cell. Biol. 2000; 20: 4210-4223Crossref PubMed Scopus (144) Google Scholar). However, the pRb-dependent inhibition of cyclin B1 expression does not seem to be caused by direct binding of pRB/E2F family proteins to the cyclin B1 promoter. Indeed, as assessed by chromatin cross-linked immunoprecipitation, an anti-E2F1 antibody does not immunoprecipitate, from cycling cells, chromatin containing the cyclin B1 promoter, whereas an anti-NF-Y antibody does. 3S. Sciortino, unpublished observations. The transcriptional regulation of the expression of mitotic kinase complexes is not the only mechanism that sustains a G2block. Indeed, it has been demonstrated recently that in a human colorectal cancer cell line, the p53-dependent G2 arrest after γ-irradiation is the result of induction of 14–3-3ς expression (59Hermeking H. Lengauer C. Polyak K. He T.C. Zhang L. Thiagalingam S. Kinzler K.W. Vogelstein B. Mol. Cell. 1997; 1: 3-11Abstract Full Text Full Text PDF PubMed Scopus (1100) Google Scholar). Once overexpressed, 14–3-3ς blocks the cell cycle contributing to the nuclear exclusion of cdk1-cyclin B1 complexes and cdc25C phosphatase (60Chan T.A. Hermeking H. Lengauer C. Kinzler K.W. Vogelstein B. Nature. 1999; 401: 616-620Crossref PubMed Scopus (809) Google Scholar). Thus, after DNA damage, transcriptional down-regulation of mitotic kinase complexes (this paper) and their cytoplasmic segregation (56Li Q. Herrler M. Landsberger N. Kaludov N. Ogryzko V.V. Nakatani Y. Wolffe A.P. EMBO J. 1998; 17: 6300-6315Crossref PubMed Scopus (178) Google Scholar) might concomitantly sustain the G2 checkpoint. In summary, this work provides evidences that in muscle cells, the molecular mechanism responsible for the G2 checkpoint induced by ADR-mediated DNA damage involves the ability of the NF-Y transcription factor to prevent the transcription of key regulatory molecules essential for the progression through the G2phase of the cell cycle. This finding opens the question of whether other genes, controlled by NF-Y during the cell cycle (26Mantovani R. Nucleic Acids Res. 1998; 26: 1135-1143Crossref PubMed Scopus (443) Google Scholar), are targets of its activity in the cell cycle checkpoints. We thank Stephen Dalton for humancdk1 promoter, Mark Wasner for cyclin B2promoter, and Pidder Yansen-Durr for cyclin A promoter; Moshe Oren for LDDSN packaging cells; Marco Crescenzi for helpful discussion; Giulio Tibursi for technical advice; Antonio Giordano and Antonio De Luca for anti-cdk9 antibody; and Daniela Bona for computing assistance.

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