Shinichi Saito

Activation of p53-mediated transcription is a critical cellular response to DNA damage. p53 stability and site-specific DNA-binding activity and, therefore, transcriptional activity, are modulated by post-translational modifications including phosphorylation and acetylation. Here we show that p53 is acetylated in vitro at separate sites by two different histone acetyltransferases (HATs), the coactivators p300 and PCAF. p300 acetylates Lys-382 in the carboxy-terminal region of p53, whereas PCAF acetylates Lys-320 in the nuclear localization signal. Acetylations at either site enhance sequence-specific DNA binding. Using a polyclonal antisera specific for p53 that is phosphorylated or acetylated at specific residues, we show that Lys-382 of human p53 becomes acetylated and Ser-33 and Ser-37 become phosphorylated in vivo after exposing cells to UV light or ionizing radiation. In vitro, amino-terminal p53 peptides phosphorylated at Ser-33 and/or at Ser-37 differentially inhibited p53 acetylation by each HAT. These results suggest that DNA damage enhances p53 activity as a transcription factor in part through carboxy-terminal acetylation that, in turn, is directed by amino-terminal phosphorylation.

SIRT1 is a mammalian homolog of the Saccharomyces cerevisiae chromatin silencing factor Sir2. Dominant-negative and overexpression studies have implicated a role for SIRT1 in deacetylating the p53 tumor suppressor protein to dampen apoptotic and cellular senescence pathways. To elucidate SIRT1 function in normal cells, we used gene-targeted mutation to generate mice that express either a mutant SIRT1 protein that lacks part of the catalytic domain or has no detectable SIRT1 protein at all. Both types of SIRT1 mutant mice and cells had essentially the same phenotypes. SIRT1 mutant mice were small, and exhibited notable developmental defects of the retina and heart, and only infrequently survived postnatally. Moreover, SIRT1-deficient cells exhibited p53 hyperacetylation after DNA damage and increased ionizing radiation-induced thymocyte apoptosis. In SIRT1-deficient embryonic fibroblasts, however, p53 hyperacetylation after DNA damage was not accompanied by increased p21 protein induction or DNA damage sensitivity. Together, our observations provide direct evidence that endogenous SIRT1 protein regulates p53 acetylation and p53-dependent apoptosis, and show that the function of this enzyme is required for specific developmental processes.