Identification of Two p53 Target Genes by Modified Chromosomal Immunoprecipitation and Inverse PCR

Direct target loci for the transcription factor p53 were identified using a modified version of chromosomal immunoprecipitation combined with inverse PCR. HeLa cells were irradiated to drive a DNA damage response, and subjected to sequential chromosomal immunoprecipitation with antibodies against the large subunit of RNA polymerase II and against p53. Inverse PCR with degenerate oligonucleotides specific for the p53 binding site was then performed on immunoprecipitated DNA, and fragments containing putative p53 target genes were subcloned and sequenced. Two sequences were identified which contain near-consensus p53 binding sites as well as recognition sites for the core transcriptional machinery including RNA polymerase II and Sp1. Cotransfection of vectors containing these sequences linked to a reporter with p53 expression vectors resulted in stimulation of transcription. Application of the technology described herein may expedite identification of target loci for a wide variety of transcription factors.