Kinetics of p53 Binding to Promoter Sites In Vivo

Abstract

Downstream target genes of p53 are thought to mediate its tumor-suppressive activity, but it is unknown whether differential transactivation of these genes is regulated at the level of p53 binding to their promoters. To address this issue, p53 binding in vivo to consensus sites in the p21 Waf1 , MDM2, and PIG3 promoters was investigated in cells exposed to adriamycin (ADR) or ionizing radiation as well as in an inducible p53 cell line. p53-DNA complexes were cross-linked in vivo by treating the cells with formaldehyde and processed by chromatin immunoprecipitation-PCR. This methodology allowed for the analysis of relevant p53-DNA complexes by preventing redistribution of cellular components upon collection of cell extracts. Increased p53 binding to the p21 Waf1 , MDM2, and PIG3 promoters occurred within 2 h after p53 activation; however, significant increases in PIG3 transcription did not occur until 15 h after p53 binding. Gel shift analyses indicated that p53 had lower affinity for the consensus binding site in the PIG3 promoters compared to its consensus sites in the p21 and MDM2 genes, which suggests that additional factors may be required to stabilize the interaction of p53 with the PIG3 promoter. Further, acetylated p53 (Lys382) was found in chemically cross-linked complexes at all promoter sites examined after treatment of cells with ADR. In summary, the kinetics of p53 binding in vivo to target gene regulatory regions does not uniformly correlate with target gene mRNA expression for the p53 target genes examined. Our results suggest that target genes with low-affinity p53 binding sites may require additional events and will have delayed kinetics of induction compared to those with high-affinity binding sites.

Mechanisms of regulatory diversity within the p53 transcriptional network

“…It is well demonstrated that p53 has different affinities for distinct p53REs, which would allow the p53 transcriptional program to be modulated by the nuclear concentration of p53 (Resnick-Silverman et al, 1998;Szak et al, 2001). At low concentrations of p53, only target genes carrying high affinity p53REs would be regulated.…”

Differential regulation of survivin by p53 contributes to cell cycle dependent apoptosis

“…Hundreds of p53-responsive genes have been identified [10][11][12]. It was reported that differential transregulation by p53, which means transactivation or transrepression of different subsets of p53 target genes, was important to determine what downstream event would be elicited [13]. Nonetheless, more details about the differential transregulation of p53 remains to be elucidated.…”

Decision making by p53: life, death and cancer

“…The need for additional p53 partners may be of particular importance in the case of genes harboring low-affinity p53BS, which may fail to be engaged effectively by the limited concentrations of p53 attained under physiological conditions. 37 It is noteworthy that many, albeit not all, proapoptotic p53 target genes harbor p53BS of rather low binding affinity. Consequently, this subclass of genes may rely more heavily on the availability of cooperating proteins of the type described above, whereas cell cycle inhibitory genes may be turned on by p53 as a default option.…”