In various human diseases, altered gene expression patterns are often the result of deregulated gene-specific transcription factor activity. To further understand disease on a molecular basis, the comprehensive analysis of transcription factor signaling networks is required. We developed an experimental approach, combining chromatin immunoprecipitation (ChIP) with a yeast-based assay, to screen the genome for transcription factor binding sites that link to transcriptionally regulated target genes. We used the tumor suppressor p53 to demonstrate the effectiveness of the method. Using primary and immortalized, nontransformed cultures of human mammary epithelial cells, we isolated over 100 genomic DNA fragments that contain novel p53 binding sites. This approach led to the identification and validation of novel p53 target genes involved in diverse signaling pathways, including growth factor signaling, protein kinase/phosphatase signaling, and RNA binding. Our results yield a more complete understanding of p53-regulated signaling pathways, and this approach could be applied to any number of transcription factors to further elucidate complex transcriptional networks.

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The mechanism by which the p53 family of proteins coordinately regulates select target genes after various types of cell stress is not well understood. To further define factors that dictate regulation of target genes, we examined the binding of p53, DNp63a and RNA polymerase II (pol II) to the regulatory regions of select target genes in primary human epidermal keratinocytes (HEKs) using chromatin immunoprecipitation. In rapidly proliferating cells, we observed constitutive binding of DNp63a and varying levels of p53 binding, to consensus sites in target genes involved in cell cycle arrest, DNA repair and apoptosis. Following genotoxic stress, p53 occupancy increased whereas DNp63a occupancy decreased at the majority of binding sites examined. Microarray analysis of transcripts isolated from HEKs ectopically expressing p53 and DNp63a revealed an inverse regulation of select target genes by the two family members. Collectively, our results suggest that DNp63a can function as a repressor of select p53 target genes involved in growth arrest, DNA repair and apoptosis, and that the location of the p53 consensus binding site(s) in a target gene may dictate whether pol II is constitutively bound in proliferating cells.