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Influence of promoter DNA topology on sequence-specific DNA binding and transactivation by tumor suppressor p53
1Heinrich-Pette-Institut für Experimentelle Virologie und Immunologie an der Universität Hamburg, Martinistrasse 52, D-20251 Hamburg, Germany.
Abstract:
Transcriptional activation by the tumor suppressor p53 is regulated at multiple levels, including posttranslational modifications of the p53 protein, interaction of p53 with various regulatory proteins, or at the level of sequence-specific DNA binding to the response elements in p53's target genes. We here propose as an additional regulatory mechanism that the DNA topology of p53-responsive promoters may determine the interaction of p53 with its target genes. We demonstrate that sequence-specific DNA binding (SSDB) and transcriptional activation by p53 of the mdm2 promoter is inhibited when this promoter is present in supercoiled DNA, where it forms a non-B-DNA structure which spans the p53-responsive elements. Relaxation of the supercoiled DNA in vitro resulted in conversion of the non-B-DNA to a B-DNA conformation within the mdm2 promoter, and correlated with an enhanced SSDB of p53 and an elevated expression of a reporter gene. In contrast, sequence specific DNA binding and transcriptional activation of the p21 promoter were not inhibited by DNA supercoiling. We propose that conformational alterations within p53-responsive sites, which either promote or prohibit sequence specific DNA binding of p53, are an important feature in orchestrating the activation of different p53 responsive promoters.
Insights
DNA topology influences tumor suppressor p53
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Tumor suppressor p53 transcriptional activation is regulated by multiple mechanisms.
- These include posttranslational modifications, protein interactions, and DNA binding.
- The role of DNA topology in p53 regulation remains largely unexplored.
Purpose of the Study:
- To investigate DNA topology as an additional regulatory mechanism for p53 transcriptional activation.
- To determine if DNA supercoiling affects p53 binding and activation of target gene promoters.
Main Methods:
- Studied p53's sequence-specific DNA binding (SSDB) and transcriptional activation of the mdm2 promoter.
- Utilized supercoiled and relaxed DNA conformations in vitro.
- Assessed reporter gene expression to quantify transcriptional activation.
Main Results:
- DNA supercoiling inhibited p53 SSDB and transcriptional activation of the mdm2 promoter.
- Supercoiling induced a non-B-DNA structure in the mdm2 promoter, hindering p53 binding.
- Relaxation of DNA restored B-DNA conformation, enhancing p53 binding and activation.
- The p21 promoter's SSDB and activation were unaffected by DNA supercoiling.
Conclusions:
- DNA topology, specifically supercoiling, can regulate p53's interaction with target gene promoters.
- Conformational changes in p53-responsive elements modulate p53 binding affinity.
- This topological regulation contributes to orchestrating the activation of diverse p53 target genes.