The p53 tumor suppressor gene is the most frequent target for genetic alterations in cancer, with mutations occurring in approximately 50% of all human tumors. P53 functions as a sequence-specific DNA binding protein that has been shown to regulate transcription of a number of target genes involved in the critical processes of cell progression, apoptosis, and oxidative stress. This protein is known to function either as an activator of repressor of gene expression by binding to a canonical DNA recognition sequence as a homotetramer within target promoters. P53 responds to diverse cellular signaling pathways that modulate its activity post-translationally mainly by phosphorylation and acetylation. Recently, several protein co-factors have been found associated with p53 and are implicated in p53 dependent transcriptional activity. Among these are histone acetylases (HATs) and deacetylases (HDACs) which raises the intriguing question of how p53 regulates target gene expression through chromatin. Although much is known about p53, little information is available regarding the mechanism by which it activates of represses specific genes. For example, the regulation of cell cycle arrest and apoptosis by p53 are mechanistically distinct processes yet the underlying basis for this is poorly understood.
The specific aims of this grant are to develop an in vitro system in which to analyze the mechanism of p53- dependent transcriptional regulation of natural target promoters when assembled into chromatin. This system will be used to define the roles of specific co-factors, chromatin remodeling or modifying complexes, and post-translational modifications in p53-mediated gene regulation. First, an in vitro system will be devised in which to analyze p53-dependent transcription of a natural target promoter, p21, when assembled into chromatin, in the presence of the histone acetylase, p300. Second, other chromatin remodeling or modifying complexes will be examined for their ability to facilitate transcriptional activation of the chromatin-assembled p21 promoter by p53. Third, the effects of distinct modifications of p53, such as phosphorylation by various kinases, on DNA binding, chromatin accessibility, and transcriptional activation of chromatin-assembled target genes will be ascertained. Fourth, recently identified proteins that stimulate p53-dependent p53-dependent activation of p21 in vivo will be tested for their ability to directly regulate p53 function at the level of transcription of chromatin accessibility.
