Replication protein A1 reduces transcription of the endothelial nitric oxide synthase gene containing a -786T-->C...

We recently reported that a mutation (-786T-->C) in the promoter region of the endothelial nitric oxide synthase (eNOS) gene reduced transcription of the gene and was strongly associated with coronary spastic angina and myocardial infarction. To elucidate the molecular mechanism for the reduced eNOS gene transcription, we have now purified a protein that specifically binds to the mutant allele in nuclear extracts from HeLa cells. The purified protein was identical to replication protein A1 (RPA1), known as a single-stranded DNA binding protein essential for DNA repair, replication and recombination. In human umbilical vein endothelial cells, inhibition of RPA1 expression using antisense oligonucleotide restored transcription driven by the mutated promoter sequence, whereas, conversely, overexpression of RPA1 further reduced it. RPA1 was similarly detected in placenta and eNOS mRNA levels in placentas carrying the -786T-->C mutation were significantly lower than in placentas without it. The functional importance of the diminished eNOS expression was revealed by the finding that serum nitrite/nitrate levels among individuals carrying the -786T-->C mutation were significantly lower than among those without the mutation. RPA1 thus apparently functions as a repressor protein in the -786T-->C mutation-related reduction of eNOS gene transcription associated with the development of coronary artery disease.