Direct p53 transcriptional repression: in vivo analysis of CCAAT-containing G2/M promoters.
In response to DNA damage, p53 activates G(1)/S blocking and apoptotic genes through sequence-specific binding. p53 also represses genes with no target site, such as those for Cdc2 and cyclin B, key regulators of the G(2)/M transition. Like most G(2)/M promoters, they rely on multiple CCAAT boxes activated by NF-Y, whose binding to DNA is temporally regulated during the cell cycle. NF-Y associates with p53 in vitro and in vivo through the alphaC helix of NF-YC (a subunit of NF-Y) and a region close to the tetramerization domain of p53. Chromatin immunoprecipitation experiments indicated that p53 is associated with cyclin B2, CDC25C, and Cdc2 promoters in vivo before and after DNA damage, requiring DNA-bound NF-Y. Following DNA damage, p53 is rapidly acetylated at K320 and K373 to K382, histones are deacetylated, and the release of PCAF and p300 correlates with the recruitment of histone deacetylases (HDACs)-HDAC1 before HDAC4 and HDAC5-and promoter repression. HDAC recruitment requires intact NF-Y binding sites. In transfection assays, PCAF represses cyclin B2, and a nonacetylated p53 mutant shows a complete loss of repression potential, despite its abilities to bind NF-Y and to be recruited on G(2)/M promoters. These data (i) detail a strategy of direct p53 repression through associations with multiple NF-Y trimers that is independent of sequence-specific binding of p53 and that requires C-terminal acetylation, (ii) suggest that p53 is a DNA damage sentinel of the G(2)/M transition, and (iii) delineate a new role for PCAF in cell cycle control.
Imbriano
Carol
C
Dipartimento di Scienze Biomolecolari e Biotecnologie, Università di Milano, Via Celoria 26, 20133 Milan, Italy.
Gurtner
Aymone
A
Cocchiarella
Fabienne
F
Di Agostino
Silvia
S
Basile
Valentina
V
Gostissa
Monica
M
Dobbelstein
Matthias
M
Del Sal
Giannino
G
Piaggio
Giulia
G
Mantovani
Roberto
R
D000107:Acetylation; D000123:Acetyltransferases / Q000378:metabolism; D000818:Animals; D023081:CCAAT-Binding Factor / Q000378:metabolism*; D002453:Cell Cycle / Q000235:genetics / Q000502:physiology; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism; D002455:Cell Division; D047369:Chromatin Immunoprecipitation; D004249:DNA Damage / Q000235:genetics / Q000502:physiology*; D015536:Down-Regulation; D016195:G2 Phase; D051548:Histone Acetyltransferases; D006655:Histone Deacetylases / Q000378:metabolism / Q000502:physiology; D006801:Humans; D051379:Mice; D009154:Mutation / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics / Q000502:physiology*; D025941:Protein Interaction Mapping; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D050880:p300-CBP Transcription Factors; D000099224:p300-CBP-Associated Factor
D000123:Acetyltransferases; D023081:CCAAT-Binding Factor; D018797:Cell Cycle Proteins; D051548:Histone Acetyltransferases; D006655:Histone Deacetylases; D012097:Repressor Proteins; D014157:Transcription Factors; D016159:Tumor Suppressor Protein p53; D050880:p300-CBP Transcription Factors; D000099224:p300-CBP-Associated Factor