Abstract 12662: A Genome-Wide Analysis of eNOS-Binding DNA Regions Reveals Unprecedented Regulatory Elements Important for Estrogen Transcriptional Response in Human Endothelial Cells
INTRODUCTION: eNOS has been identified as a nuclear co-factor of Estrogen Receptors alpha and beta 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 17beta-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; about 10% in the promoter region of known genes and about 33% falling in extra-genic regions. 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 estrogen-dependent and -independent epigenetic effects on gene expression.
- © 2011 by American Heart Association, Inc.