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One of the most frequent alterations in breast cancer is deregulation of the p53 tumor suppression signaling pathways. The tumor suppressor p53 is a sequence-specific transcription factor that is activated in response to various cellular stresses. It has been predicted that there are over 1500 consensus p53 binding sites present in the human genome. However, only a subset of p53 binding sites and correlated target genes have been identified to date, and characterization of p53 signaling pathways in their entirety is not yet complete. The goal of this study is to further define and characterize such pathways through the identification of novel genes that are directly regulated by p53. Using chromatin immunoprecipitation followed by a yeast selection system we have isolated over 100 genomic DNA fragments that contain novel p53 binding sites. The new DNA fragments obtained have been mapped to various regions of the human genome, and four novel p53 target genes have been identified and validated. This research will lead to a more complete understanding of p53-regulated signaling pathways in mammary epithelial cells.