Repression of new p53 targets revealed by ChIP on chip experiments.
Following DNA-damage, the tumor suppressor p53 activates G1/S blocking and apoptotic genes, and represses other genes, including those involved in G2/M transition. In this latter system, it acts through the CCAAT-binding histone-like NF-Y. Several groups have reported that p53 is associated to promoters in unstressed conditions. We developed an oligo-based array containing 179 human promoters, enriched in genes involved in the DNA-damage and ER-stress response. We performed ChIP on chip experiments with p53 and NF-Y in cells under normal growing conditions. We identified 46 new p53 targets and noted (i) a significant enrichment in genes of the ER-stress response, including crucial regulators such as XBP1 and C/EBPbeta (ii) genes whose products are involved in the regulation of p53 function. Several genes were validated by conventional ChIP. DNA-damage dependent PCAF-mediated acetylation was observed on most, but not all promoters. The effect of p53 activation was checked by RT-PCR and transfections in HCT116 wt, E6 and p53-/- cells: most promoters were actively repressed upon Adriamycin treatment or following p53 transfection in p53-/- cells. In particular, the behaviour of some of the genes (BRCA1, RAD23 and RAD17) is consistent with a feedback loop regulation on p53 levels. Finally, there is a large overlap (66%) between p53 and NF-Y targets. Our data reinstate the physiological importance of p53 promoter recognition and direct transcriptional repression as a mechanism to cope with DNA-damage.
Ceribelli
Michele
M
Dipartimento di Scienze Biomolecolari e Biotecnologie, Università degli Studi di Milano, Milano, Italy.
Alcalay
Myriam
M
Viganò
Maria Alessandra
MA
Mantovani
Roberto
R
D000477:Alkylating Agents; D019313:BRCA1 Protein / Q000235:genetics / Q000378:metabolism; D023081:CCAAT-Binding Factor / Q000235:genetics / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D004249:DNA Damage / Q000235:genetics; D045643:DNA Repair Enzymes / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D015536:Down-Regulation; D004317:Doxorubicin; D020869:Gene Expression Profiling / Q000379:methods*; D045325:HCT116 Cells; D051548:Histone Acetyltransferases; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D015203:Reproducibility of Results; D014157:Transcription Factors; D014158:Transcription, Genetic; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*; D050880:p300-CBP Transcription Factors; D000099224:p300-CBP-Associated Factor
D000477:Alkylating Agents; D019313:BRCA1 Protein; D023081:CCAAT-Binding Factor; D018797:Cell Cycle Proteins; D045643:DNA Repair Enzymes; D004268:DNA-Binding Proteins; D004317:Doxorubicin; D051548:Histone Acetyltransferases; D012333:RNA, Messenger; D014157:Transcription Factors; D016159:Tumor Suppressor Protein p53; D050880:p300-CBP Transcription Factors; D000099224:p300-CBP-Associated Factor; C113575:Rad17 protein, human; C086985:RAD23A protein, human