The human p73 promoter:: Characterization and identification of functional E217 binding sites

p73, a member of the p53 family, is overexpressed in many cancers. To understand the mechanism(s) underlying this overexpression, we have undertaken a detailed characterization of the human p73 promoter. The promoter is strongly activated in cells expressing exogenous E2F1 and suppressed by exogenous Rb. At least three functional E2F binding sites, located immediately upstream of exon 1 (at -284, -155 and -132) mediate this induction. 5' serially deleted promoter constructs and constructs harboring mutated E2F sites were analyzed for their response to exogenously expressed E2F1 or Rb to establish functionality of these sites. Authenticity of E2F sites was further confirmed by electrophoretic mobility shift assay (EMSA) using E2F1/DP1 heterodimers synthesized in vitro, followed by competition assays with unlabeled wild-type or mutant oligonucleotides and supershift analysis using anti-E2F1 antibodies. In vivo binding of E2F1 to the p73 promoter was demonstrated using nuclear extracts prepared from E2F1-inducible Saos2 cells. The region conferring the highest promoter activity was found to reside between -113 to -217 of the p73 gene. Two of the three functional E2F sites(at -155 and -132) reside within this region. Our results suggest that regulation of p73 expression is primarily mediated through binding of E2F1 to target sites at -155 and -132.