Transcriptional repression in response to p53

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DESCRIPTION (provided by applicant) DNA damage induces arrest in the G2 phase of the cell cycle, a response conserved from yeast to humans. In animal cells, p53 ensures that the arrest is stably maintained and cells do not enter mitosis with damaged DMA, which could compromise genomic integrity and contribute to tumorigenesis. One way that p53 contributes to the stable arrest is by triggering the down regulation of proteins normally needed for mitosis. This effect requires Rb family proteins which form a complex with E2F proteins and repress the transcription of cell cycle-regulated genes. Our analysis of the cdc2 and plkl promoters shows that repression by p53 requires a previously identified DMA element called the CDE/CHR. The CDE partially resembles an E2F site, while the CHR has no similarity. In our first Specific Aim we propose to use chromatin immunoprecipitation (ChlP) to determine if Rb and E2F proteins are associated with the CDE/CHR regions of the cdc2 and plkl promoters in vivo when p53 expression is induced. The CDE/CHR elements of the cdc2 and plkl promoters are found very close to the start sites of transcription raising the possibility that binding of Rb/E2F to these elements may physcially block the association of components of the preinitiation complex. This hypothesis will be tested in our second Aim using ChlP and promoter modification. Other groups have suggested that CCAAT elements in several promoters, including cdc2 and plkl are important for repression by p53. In our third Aim, we propose to use ChlP and gel shift analysis to analyze the cdc2 and plkl promoters in cells overexpressing p53 to examine this model. These studies will provide detailed information about the mechanisms used by p53 to repress two genes required for mitosis, and may uncover new mechanisms used by Rb/E2F to regulate gene expression. Since p53 is frequently mutated in cancer, our studies should provide new insight into how gene expression is deranged during tumorigenesis.