Functional analysis of p53 response elements in the human Polycystic Kidney Disease Gene 1 (PKD‐1) promoter

Abstract

We have previously shown that p53 represses human PKD1 expression in vivo and in vitro, and that this effect is dependent on histone deacetylase activity (JBC, 281, p.31234, 2006). The 5′‐regulatory region of PKD1 contains 4 putative p53 response elements (p53RE4‐1) at positions: −2.7kb, −1.2kb, −0.8kb and −0.2kb respectively. In this study, we delineate the differential roles of the p53RE's in transcriptional repression of the PKD1 gene. We performed site‐directed mutagenesis of distal p53RE4, proximal p53RE1. The wild‐type and mutant PKD1 promoter‐reporter constructs were co‐transfected with a p53 expression plasmid in IMCD3, HCT116(p53+/+) and HCT116(p53−/−) cells. p53RE4 deletion decreased PKD1 baseline promoter activity (2.4, 1.8 and 1.4‐fold) in IMCD3, HCT116(p53+/+) and HCT116(p53−/−) cells, respectively, and significantly enhanced p53‐mediated repression in all cell lines, indicating that p53RE4 is a positive cis‐response element. In contrast, deletion of the p53RE1 induced a 1.6‐fold increase in baseline PKD1 promoter activity; this was observed in p53+/+ but not p53−/ − cells. p53RE1 failed to mediate repression of a known p53‐target gene ‐the bradykinin B2 receptor‐ indicating that p53RE1 functions in a context‐specific manner. Chromatin Immunoprecipitation assays in IMCD3 cells revealed the DNA regions surrounding p53RE1/2 are occupied by HDAC 1 and 2. We conclude that the balance between p53RE2 and p53RE1 occupancy by p53 and HDACs determines the final response of PKD1 to p53 within a given cell.Supported by NIH grant DK‐062250