Mechanisms of transcriptional repression of cell-cycle G2/M promoters by p63.
p63 is a developmentally regulated transcription factor related to p53, which activates and represses specific genes. The human AEC (Ankyloblepharon-Ectodermal dysplasia-Clefting) and EEC (Ectrodactyly-Ectodermal dysplasia-Cleft lip/palate) syndromes are caused by missense mutations of p63, within the DNA-binding domain (EEC) or in the C-terminal sterile alpha motif domain (AEC). We show here that p63 represses transcription of cell-cycle G(2)/M genes by binding to multiple CCAAT core promoters in immortalized and primary keratinocytes. The CCAAT-activator NF-Y and DeltaNp63alpha are associated in vivo and a conserved alpha-helix of the NF-YC histone fold is required. p63 AEC mutants, but not an EEC mutant, are incapable to bind NF-Y. DeltaNp63alpha, but not the AEC mutants repress CCAAT-dependent transcription of G(2)/M genes. Chromatin immunoprecipitation recruitment assays establish that the AEC mutants are not recruited to G(2)/M promoters, while normally present on 14-3-3sigma, which contains a sequence-specific binding site. Surprisingly, the EEC C306R mutant activates transcription. Upon keratinocytes differentiation, NF-Y and p63 remain bound to G(2)/M promoters, while HDACs are recruited, histones deacetylated, Pol II displaced and transcription repressed. Our data indicate that NF-Y is a molecular target of p63 and that inhibition of growth activating genes upon differentiation is compromised by AEC missense mutations.
Testoni
Barbara
B
Dipartimento di Scienze Biomolecolari e Biotecnologie, Università di Milano Via Celoria 26, 20133 Milano, Italy.
Mantovani
Roberto
R
D023081:CCAAT-Binding Factor / Q000737:chemistry / Q000378:metabolism*; D002454:Cell Differentiation; D002455:Cell Division; D002460:Cell Line; D002971:Cleft Lip / Q000235:genetics; D002972:Cleft Palate / Q000235:genetics; D004268:DNA-Binding Proteins; D004476:Ectodermal Dysplasia / Q000235:genetics*; D016195:G2 Phase; D020868:Gene Silencing; D016147:Genes, Tumor Suppressor; D018816:Genes, cdc; D006801:Humans; D015603:Keratinocytes / Q000378:metabolism; D009056:Mouth Abnormalities / Q000235:genetics*; D020125:Mutation, Missense; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D013577:Syndrome; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D014157:Transcription Factors; D014158:Transcription, Genetic; D025521:Tumor Suppressor Proteins
D023081:CCAAT-Binding Factor; D004268:DNA-Binding Proteins; D010750:Phosphoproteins; D012097:Repressor Proteins; C115051:TP63 protein, human; D015534:Trans-Activators; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins