The p53 tumor suppressor gene is frequently inactivated by mutations in human cancers, p53 is a sequence-specific transcription factor, whose activity is regulated by DNA damage, p53 induces expression of genes that cause cell cycle arrest or apoptosis, and represses genes that oppose these processes, hence promoting repair of damage or programmed cell death. Replacement of normal p53 with a transcriptionallyinactive point mutant in the mouse model leads to tumors with the same frequency as the absence of p53, suggesting that transcriptional regulation by p53 is its key function. Regulation of p53's activity is complex; p53 is subject to numerous DNA damage-induced posttranslational modifications. In the previous grant period we demonstrated that acetylation in the C-terminus of p53, at lysines 320, 373, 381 and 382, is important for activating p53 in response to DNA damage, leading to transcriptional activation and cell cycle arrest. Based on these observations we hypothesize that an acetylation cascade exists whereby the same enzymes acetylate p53 (as factor acetyltransferases, or FATs) and then histones (as histone acetyltransferases, or HATs). Other preliminary data obtained during the previous grant period identified new regulatory pathways for p53 function. First, we as well as others previously identified in yeast and mammalian cells conserved proteins, Gcn5/PCAF and Ada2 that interact and function as partners in an acetylation module within a larger complex. We have now discovered a novel human Ada2 (Ada2b), which is in a distinct protein complex but may recruit the Gcn5 complex to target genes. In addition, we have examined substrates of the Peutz-Jeager kinase, LKB1, previously shown to function with p53. We have discovered that LKB1 phosphorylates both p53 and histone H2B, and thus may, like the acetyltransferases, function in a cascade, signaling from p53 to chromatin. Based on these observations, we propose a model of gene regulation by p53 involving interrelated covalent modifications of both p53 and histones. Aspects of this hypothesis will be tested during the proposed studies.
Specific Aim 1 will explore mechanisms of p53 acetylation and possible interplay with p53 phosphorylation and methylation. We will determine whether p53 is dually phosphorylated at Ser-378 and acetylated at Lys-382.
Specific Aim 2 will investigate patterns of histone modifications at p53 promoters and will test the factor/histone code cascade hypothesis for p53 regulation.
Specific Aim 3 will determine whether p53 requires the novel Ada2b coactivator for activation, and specifically whether Ada2b functions with Gcn5 (acetylation) and Brgl (nucleosome remodeling) complexes for regulation of p53-dependent genes.
Specific Aim 4 will determine the role of p53 and histone H2B phosphorylation by the kinase LKB1 in cell cycle arrest and apoptosis. The proposed studies will advance our understanding of the mechanisms by which p53 activates gene expression raising the likelihood for pharmacologic regulation of p53 function in cancer patients in the future. ? ?
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