ABSTRACT
RNA plays a fundamental role in the organization of chromatin as well as the regulation of gene expression. Although the chromatin is pervasively attached by both coding and noncoding RNAs, the impact of these chromatin-associated RNAs (caRNAs) on gene expression and cellular functions and their underlying mechanisms have just begun to be unraveled. One approach to understand the potential mechanism of gene regulation by caRNAs is to identify the caRNA-associated genomic regions. Several groups have developed methods to capture RNA-chromatin interactions in either one RNA vs the whole genome, i.e., "one-to-all" or all RNAs vs the whole genome, i.e., "all-to-all" manner. In this chapter, we discuss several state-of-the-art methods highlighting the principles behind them, the experimental procedures, the advantages and limitations, and their applications. Our goal is to provide an overview and guide to researchers interested in exploring caRNAs using these techniques.
Subject(s)
Chromatin , RNA, Long Noncoding , Chromatin/genetics , RNA/genetics , RNA/metabolism , RNA, Untranslated/genetics , Genome , Gene Expression Regulation , RNA, Long Noncoding/geneticsABSTRACT
Impaired angiogenesis in diabetes is a key process contributing to ischemic diseases such as peripheral arterial disease. Epigenetic mechanisms, including those mediated by long noncoding RNAs (lncRNAs), are crucial links connecting diabetes and the related chronic tissue ischemia. Here we identify the lncRNA that enhances endothelial nitric oxide synthase (eNOS) expression (LEENE) as a regulator of angiogenesis and ischemic response. LEENE expression was decreased in diabetic conditions in cultured endothelial cells (ECs), mouse hind limb muscles, and human arteries. Inhibition of LEENE in human microvascular ECs reduced their angiogenic capacity with a dysregulated angiogenic gene program. Diabetic mice deficient in Leene demonstrated impaired angiogenesis and perfusion following hind limb ischemia. Importantly, overexpression of human LEENE rescued the impaired ischemic response in Leene-knockout mice at tissue functional and single-cell transcriptomic levels. Mechanistically, LEENE RNA promoted transcription of proangiogenic genes in ECs, such as KDR (encoding VEGFR2) and NOS3 (encoding eNOS), potentially by interacting with LEO1, a key component of the RNA polymerase II-associated factor complex and MYC, a crucial transcription factor for angiogenesis. Taken together, our findings demonstrate an essential role for LEENE in the regulation of angiogenesis and tissue perfusion. Functional enhancement of LEENE to restore angiogenesis for tissue repair and regeneration may represent a potential strategy to tackle ischemic vascular diseases.
Subject(s)
Diabetes Mellitus, Experimental , RNA, Long Noncoding , Humans , Mice , Animals , RNA, Long Noncoding/genetics , RNA, Long Noncoding/metabolism , Endothelial Cells/metabolism , Diabetes Mellitus, Experimental/genetics , Diabetes Mellitus, Experimental/metabolism , Muscle, Skeletal/metabolism , Neovascularization, Physiologic/genetics , Ischemia/genetics , Ischemia/metabolism , Mice, Knockout , Hindlimb , Mice, Inbred C57BLABSTRACT
Endothelial cells (ECs), uniquely localized and strategically forming the inner lining of vascular wall, constitute the largest cell surface by area in the human body. The dynamic sensing and response of ECs to mechanical cues, especially shear stress, is crucial for maintenance of vascular homeostasis. It is well recognized that different flow patterns associated with atheroprotective vs atheroprone regions in the arterial tree, result in distinct EC functional phenotypes with differential transcriptome profiles. Mounting evidence has demonstrated an integrative and essential regulatory role of non-coding genome in EC biology. In particular, recent studies have begun to reveal the importance of enhancers and enhancer-derived transcripts in flow-regulated EC gene expression and function. In this minireview, we summarize studies in this area and discuss examples in support of the emerging importance of enhancers and enhancer(-derived) long non-coding RNAs (elncRNAs) in EC mechanosensing, with a focus on flow-responsive EC transcription. Finally, we will provide perspective and discuss standing questions to elucidate the role of these novel regulators in EC mechanobiology.
Subject(s)
RNA, Long Noncoding , Endothelial Cells , Endothelium, Vascular , Humans , RNA, Long Noncoding/genetics , Stress, Mechanical , TranscriptomeABSTRACT
The homeostasis of vascular endothelium is crucial for cardiovascular health and endothelial cell (EC) aging and dysfunction could negatively impact vascular function. Leveraging transcriptome profiles from ECs subjected to various stimuli, including time-series data obtained from ECs under physiological pulsatile flow vs. pathophysiological oscillatory flow, we performed principal component analysis (PCA) to identify key genes contributing to divergent transcriptional states of ECs. Through bioinformatics analysis, we identified that a long non-coding RNA (lncRNA) RAMP2-AS1 encoded on the antisense of RAMP2, a determinant of endothelial homeostasis and vascular integrity, is a novel regulator essential for EC homeostasis and function. Knockdown of RAMP2-AS1 suppressed RAMP2 expression and caused EC functional changes promoting aging, including impaired angiogenesis and increased senescence. Our study demonstrates an integrative approach to quantifying EC aging based on transcriptome changes, which also identified a number of novel regulators, including protein-coding genes and many lncRNAs involved EC functional modulation, exemplified by RAMP2-AS1.
