Abstract
The optimal expression of endothelial nitric oxide synthase (eNOS), the hallmark of endothelial homeostasis, is vital to vascular function. Dynamically regulated by various stimuli, eNOS expression is modulated at transcriptional, post-transcriptional, and post-translational levels. However, epigenetic modulations of eNOS, particularly through long non-coding RNAs (lncRNAs) and chromatin remodeling, remain to be explored. Here we identify an enhancer-associated lncRNA that enhances eNOS expression (LEENE). Combining RNA-sequencing and chromatin conformation capture methods, we demonstrate that LEENE is co-regulated with eNOS and that its enhancer resides in proximity to eNOS promoter in endothelial cells (ECs). Gain- and Loss-of-function of LEENE differentially regulate eNOS expression and EC function. Mechanistically, LEENE facilitates the recruitment of RNA Pol II to the eNOS promoter to enhance eNOS nascent RNA transcription. Our findings unravel a new layer in eNOS regulation and provide novel insights into cardiovascular regulation involving endothelial function.
Highlights
The optimal expression of endothelial nitric oxide synthase, the hallmark of endothelial homeostasis, is vital to vascular function
Among the 2054 long non-coding RNAs (lncRNAs) identified in the RNA-seq, we first filtered for those differentially regulated by pulsatile shear stress (PS) vs. oscillatory shear stress (OS) at the end point (i.e., “h 24”)
We took advantage of an integrative approach combining transcriptome and chromatin interactome profiling to identify lncRNA that enhances eNOS expression (LEENE), which is encoded by a distal enhancer region that forms proximity association with endothelial nitric oxide synthase (eNOS) locus; its RNA transcripts enhance RNA polymerase II (Pol II) binding to eNOS promoter and the consequent eNOS transcription
Summary
The optimal expression of endothelial nitric oxide synthase (eNOS), the hallmark of endothelial homeostasis, is vital to vascular function. Combining RNA-sequencing and chromatin conformation capture methods, we demonstrate that LEENE is co-regulated with eNOS and that its enhancer resides in proximity to eNOS promoter in endothelial cells (ECs). Endothelial nitric oxide synthase (eNOS), which is central to endothelial homeostasis and vascular function, is regulated at multiple levels[1], including post-translational modifications (such as phosphorylation and acetylation)[2, 3] and transcriptional regulation by transcription factors (TFs)[4]. Over 27,000 lncRNAs have been predicted/annotated in the human genome[10], but relatively little is known about their biological functions and the classification can be ambiguous due to the lack of functional characterization[11] Depending on their subcellular localization (i.e., in the nucleus or cytoplasm), lncRNAs can regulate gene expression through diverse mechanisms. Using multiple gain- or loss-of-function approaches, we provide evidence that LEENE promotes eNOS transcription, eNOSderived NO bioavailability, and endothelial function
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