Direct p53 Transcriptional Repression: In Vivo Analysis of CCAAT-Containing G₂/M Promoters

Abstract

In response to DNA damage, p53 activates G₁/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 G₂/M transition. Like most G₂/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 αC 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 G₂/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 G₂/M transition, and (iii) delineate a new role for PCAF in cell cycle control.

ACKNOWLEDGMENTS

We thank Y. Nakatani for the gift of PCAF antibodies and T. Halazonetis, S. Berger, M. L. Avvantaggiati, and S. McMahon for the p53 reagents. We thank B. Amati for many helpful discussions and S. Soddu for helpful comments on the article. R.M. thanks V. Rossi for inspiration.

C.I. and S.D.A. are the recipients of a FIRC fellowship; A.G. is the recipient of a MIUR-FIRB contract; R.M., G.P., and G.D.S. are supported by grants from AIRC; R.M. and G.D.S. are supported by MIUR-COFIN; R.M. and G.P. are supported by FIRB and Ministero della Sanitá (R.F. 02/184); and R.M. is supported by Fondazione Cariplo. M.D. acknowledges support from Wilhelm Sander Stiftung.