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.
Xiao
G
G
The Institute for Biomolecular Structure and Function, and Department of Biological Sciences, Hunter College, New York, NY 10021, USA.
White
D
D
Bargonetti
J
J
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism* / Q000648:ultrastructure; D018983:DNA Footprinting; D003850:Deoxyribonuclease I; D051379:Mice; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000235:genetics; D009687:Nuclear Proteins; D009707:Nucleosomes / Q000648:ultrastructure; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D051736:Proto-Oncogene Proteins c-mdm2; D015183:Restriction Mapping; D016385:TATA Box; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism*
D009363:Neoplasm Proteins; D009687:Nuclear Proteins; D009707:Nucleosomes; D011518:Proto-Oncogene Proteins; D016159:Tumor Suppressor Protein p53; C497207:Mdm2 protein, mouse; D051736:Proto-Oncogene Proteins c-mdm2; D003850:Deoxyribonuclease I