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p53 binds to a constitutively nucleosome free region of the mdm2 gene
G Xiao1, D White, J Bargonetti
1The Institute for Biomolecular Structure and Function, and Department of Biological Sciences, Hunter College, New York, NY 10021, USA.
Abstract:
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.
Insights
The mdm2 oncogene
Area of Science:
- Molecular Biology
- Cancer Genetics
Background:
- The MDM2 oncogene is a p53-responsive gene with two promoters: P1 (p53-independent) and P2 (p53-dependent).
- Understanding p53 binding to the MDM2 P2 promoter is crucial for comprehending gene regulation in cancer.
Purpose of the Study:
- To investigate the intra-nuclear binding of the tumor suppressor protein p53 to the MDM2 P2 promoter using genomic footprinting techniques.
- To compare p53 binding patterns in cells with and without functional p53.
Main Methods:
- Ligation-mediated PCR genomic footprinting was employed to analyze p53 binding within cell nuclei.
- DNase I protection assays were conducted on murine cells lacking p53 and cells with temperature-sensitive p53 (p53-Val135).
- Indirect Southern blot analysis was used to assess DNase I hypersensitivity at the MDM2 promoters.
Main Results:
- Wild-type p53-dependent protection was observed at p53 response elements (REs) and the TATA box of the MDM2 P2 promoter.
- Purified p53 showed less nucleotide sequence protection on naked DNA compared to p53-dependent protection within nuclei.
- Constitutive DNase I hypersensitivity at both MDM2 P1 and P2 promoters suggests a nucleosome-free state.
Conclusions:
- The MDM2 P2 promoter exists in a nucleosome-free conformation, pre-primed for transcriptional activation by p53.
- Intra-nuclear p53 binding to the MDM2 P2 promoter is specific and influences chromatin structure.