A Genome-wide Analysis of eNOS-binding DNA Regions Reveals Unprecedented Regulatory Elements Important for Estrogen Transcriptional Response in Human Endothelial Cells.

INTRODUCTION: In prior work we identified eNOS as a nuclear co-factor of Estrogen Receptors ? and ? in different cellular contexts, i.e. endothelial and prostate cancer cells. To elucidate the nuclear role of eNOS and clarify its involvement in the basal and/or hormone-dependent transcriptional regulation, a genome-wide profiling, by Chromatin ImmunoPrecipitation/Sequencing (ChIP-Seq), of protein-DNA interacting sites was performed after eNOS nuclear enrichment following estrogen stimulation. METHODS AND RESULTS: ChIP-Seq experiments were performed using a anti-eNOS antibody in HUVEC before and after treatment with 17?-estradiol (E2, 10-7M for 45 minutes). ChIP-Seq data analysis revealed a wide distribution across the genome of eNOS-associated DNA sites containing putatively active transcription complexes: specifically about 10,683 DNA-binding peaks were observed in untreated cells. Upon E2 treatment the peaks not only decreased to 3,701 but changed location, indicating a specific hormone-dependent re-distribution of the protein along the genome. Interestingly, the peak distribution in annotated regions was similar in both experimental conditions with 57% of total peaks being localized in intragenic DNA regions (6,148 peaks out of 10,683 in untreated (NT) and 2,101 out of 3,701 in E2-treated cells (E2); about 10% in the promoter regions of known genes (1,075/10,683 in NT and 374/3,701 in E2) and about 33% falling in extra-genic regions (3,456/10,683 in NT and 1,222/3,701 in E2). Based on this analysis, numerous potentially novel eNOS-targeted genes were identified in both untreated and E2-treated samples suggesting that eNOS may participate in the regulation of basal transcription of large gene sets. In this regard, only a minority of E2-induced peaks were localized on known E2-target genes (e.g. hTERT). Thus eNOS-targets in E2-stimulated cells may constitute a new category of estrogen/eNOS-dependent gene families. Validation of a subset of newly identified eNOS-positive regions was obtained by quantitative PCR. Four different promoter regions, overlapping with eNOS-peaks were analysed and the eNOS protein was found physically associated to them. CONCLUSION: The evidence that eNOS is in the nucleus and is associated to chromatin is highly suggestive that it may exert important regulatory effects on gene expression in estrogen-dependent and -independent manner.