The p53 tumor suppressor protein is likely the most well studied mammalian transcription factor. Yet important questions remain about the mechanisms by which p53 regulates expression of its many target genes that control cell outcomes such as cell cycle arrest, apoptosis, senescence and others. Most intriguing is the issue as to how its target genes are selected, since such targets are not uniformly induced by p53, but instead each requires specific p53 modifications, co-factors or cellular milieus. Ever since its discovery as a sequence-specific transcriptional activator there have been numerous attempts to elucidate how p53 finds and binds specific sequences in the promoters of genes that it regulates and how this leads to induction of transcription. One particular issue that has not yet been well addressed is how and when p53 locates its site in nucleosomes within chromatin. Further, the role of acetylation of key p53 lysine residues and how they regulate p53 function is still mysterious. Finally, there have been scant few studies linking p53 to nuclear architecture and function including the nuclear transport machinery. Going forward we plan to ask the following questions: How does p53 identify its sites within nucleosomes? In Aim 1 we will build on our discovery that p53 protein can only bind efficiently to nucleosomes when it has been acetylated at certain lysine residues within its C-terminus or core DNA binding domain. We have correspondingly demonstrated marked variation in nucleosome density and remodeling at p53 binding sites within different target genes in vivo and we will examine how acetylation of p53 regulates its interaction with nucleosomes within such genes in cells. How does p53 select target genes that regulate the cell cycle? In Aim 2 we have discovered that mutation of the 2 lysines (K351 and K357) within the p53 tetramerization domain (one or both of which are likely acetylated), generating p53(2KQ), causes a dramatic alteration in the promoters to which p53 binds and activates, resulting in cell death but impaired cell cycle arrest. We will seek to clarify how p53(2KQ) interacts differentially with target genes such as miR-34a, determine when K351/357 are acetylated and clarify why p53(2KQ) cannot arrest cells. What is the relationship between nuclear transport and tumor suppression? Aim3 builds on our discovery that a component of the nuclear pore complex, Nup98, that is found in chromosomal translocations with numerous partners, is required for p21 mRNA accumulation by a post-transcriptional step that we postulate links nuclear transport and mRNA stability. It will also elucidate a possible role for Nup98 as a new tumor suppressor.
The work proposed in this grant will focus on two general areas that have considerable relevance for cancer biology. First, building on our novel observations, we will delve into the mechanism by which acetylation of p53 regulates its ability to activate its target genes that mediate cellular outcomes such as arrest and apoptosis. Several inhibitors of deacetylases (HDACs) have now been tested in pre-clinical models and are in clinical trials. HDAC inhibitors in some cases require p53 and our studies may therefore reveal how they work in that context. Second, our discovery that the nuclear pore component Nup98 plays an unanticipated role in post-transcriptional regulation of p53 target gene mRNA accumulation in liver cancer cell lines may be linked to our preliminary evidence that a significant number of hepatocellular carcinomas have reduced levels of Nup98. Further, Nup98 has been found in chromosomal translocations with a large number of partners in some leukemias and its deregulation in that regard may inform how it functions as a tumor suppressor.
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