Abstract 16136: RPA-dependent Melting of Triplex DNA at NOS2a Gene Promoter is Indispensible for p53-mediated NOS2a Synthesis and Cardioprotection
Nitric oxide (NO) is a critical signal transduction molecule involved in the protection and survival of the ischemic myocardium. NO is generated by a family of enzymes, the nitric oxide synthase (NOS), essentially the inducible nitric oxide (iNOS) isoform plays crucial role in reducing infarct size post-myocardial infarction. Recently we have identified a p53-dependent pathway of cardioprotection via upregulation of p53-induced transcriptional activation of the NOS family members (NOS3). In the present study we observed that p53 has a response element (RE) in the NOS2a promoter 3451 base pairs (bp) upstream of the +1 transcription start site (TSS)(Fig A). However, the in vitro transcription assay at the 4 kbp NOS2a promoter resulted in the absence of p53-mediated transcription. Analysis of the NOS2a promoter showed the presence of a triplex forming penta-nucleotide sequence (CCTTT)n at 2753 bp upstream of +1 TSS and downstream p53 RE. We hypothesized that the melting of this triplex DNA structure by replication protein-A (RPA) results in p53-dependent transcription at the NOS2a promoter. We have presented evidence using p53 and RPA knockout models of the human cardiomyocytes that both p53 and RPA are must for transcription at the NOS2a promoter (Fig B&C). Essentially, the RPA protein melts the triplex DNA structure and facilitates p53-mediated transcription at the NOS2a gene promoter and NOS2a expression in human cardiomyocytes. The understanding of this novel molecular mechanism of p53- and RPA-mediated transcription at NOS2a gene promoter will enable designing strategies to achieve cardioprotection.
- © 2012 by American Heart Association, Inc.