p53 binds to a constitutively nucleosome free region of the mdm2 gene

The mdm2 oncogene is a p53 responsive gene which contains both a p53 independent and a p53 dependent promoter (P1 and P2 respectively), We have utilized ligation mediated PCR genomic footprinting in order to investigate the intra-nuclear binding of p53 to the mdm2 P2 promoter. The DNase I protection pattern in nuclei from murine cells lacking p53 has been compared to the protection pattern in cells containing a temperature sensitive p53-Val135. At 32 degrees C p53-Val135 assumes a wild-type conformation while at 37 degrees C this p53 is conformationally mutant. We observed clear wild-type p53 dependent protection of the putative p53 response elements (REs) as well as protection of the adjacent TATA box. Interestingly the protection pattern observed with purified wild-type p53 on naked DNA showed less nucleotide sequence protection than the protection observed to be p53 dependent in nuclei. Constitutive DNase I hypersensitivity at both the mdm2 P1 and P2 promoters was detected by indirect Southern blot analysis. DNase I hypersensitivity reflects altered chromatin conformations resulting, most likely, from the absence of nucleosomes. Taken together our findings suggest that the mdm2 P2 promoter is maintained in a nucleosome free state which is pre-primed for transcriptional activation by p53.