■CALCRL(降钙素受体样)蛋白是内皮液切应力反应的重要介质,这与冠状动脉疾病的遗传风险有关。在这项研究中,我们在功能上鉴定了携带有单核苷酸多态性风险的冠状动脉疾病的非编码调控元件,并研究了它们在内皮细胞CALCRL表达调控中的作用.
■为了在功能上表征CALCRL基因周围的冠状动脉疾病单核苷酸多态性,我们应用了一种包含统计的综合方法,转录(RNA-seq),和表观遗传学(ATAC-seq,染色质免疫沉淀测定-定量聚合酶链反应,和电迁移变化测定)分析,除了荧光素酶报告基因测定,以及人主动脉内皮细胞中的靶向基因和增强子扰动(siRNA和成簇的规则间隔的短回文重复序列/成簇的规则间隔的短回文重复序列相关9)。
■我们证明,带有rs880890的调控元件表现出很高的增强子活性,并显示出明显的等位基因偏差。A等位基因优于G等位基因,特别是在剪切应力条件下,通过HSF1(热休克因子1)基序和结合的改变介导。rs880890增强子的CRISPR缺失导致CALCRL表达下调,而HSF1敲低导致rs880890增强子活性和CALCRL表达的显著降低。与单向流动相比,在受干扰的流动下观察到HSF1与内皮细胞中增强子区域的结合显着降低。CALCRL敲低和变异扰动实验表明CALCRL在介导eNOS(内皮NO合成酶)中的作用,APLN(apelin),血管生成素,前列腺素,和EDN1(内皮素-1)信号通路导致细胞增殖减少,管形成,没有生产。
■总的来说,我们的结果证明存在介导CALCRL表达的内皮特异性HSF(热休克因子)调节的转录增强子.更好地理解CALCRL基因调控和单核苷酸多态性在调节CALCRL表达中的作用可能为理解剪切应力信号反应的遗传调控提供重要步骤。
UNASSIGNED: CALCRL (calcitonin receptor-like) protein is an important mediator of the endothelial fluid shear stress response, which is associated with the genetic risk of coronary artery disease. In this study, we functionally characterized the noncoding regulatory elements carrying coronary artery disease that risks single-nucleotide polymorphisms and studied their role in the regulation of CALCRL expression in endothelial cells.
UNASSIGNED: To functionally characterize the coronary artery disease single-nucleotide polymorphisms harbored around the gene CALCRL, we applied an integrative approach encompassing statistical, transcriptional (RNA-seq), and epigenetic (ATAC-seq [transposase-accessible chromatin with sequencing], chromatin immunoprecipitation assay-quantitative polymerase chain reaction, and electromobility shift assay) analyses, alongside luciferase reporter assays, and targeted gene and enhancer perturbations (siRNA and clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9) in human aortic endothelial cells.
UNASSIGNED: We demonstrate that the regulatory element harboring rs880890 exhibits high enhancer activity and shows significant allelic bias. The A allele was favored over the G allele, particularly under shear stress conditions, mediated through alterations in the HSF1 (heat shock factor 1) motif and binding. CRISPR deletion of rs880890 enhancer resulted in downregulation of CALCRL expression, whereas HSF1 knockdown resulted in a significant decrease in rs880890-enhancer activity and CALCRL expression. A significant decrease in HSF1 binding to the enhancer region in endothelial cells was observed under disturbed flow compared with unidirectional flow. CALCRL knockdown and variant perturbation experiments indicated the role of CALCRL in mediating eNOS (endothelial nitric oxide synthase), APLN (apelin), angiopoietin, prostaglandins, and EDN1 (endothelin-1) signaling pathways leading to a decrease in cell proliferation, tube formation, and NO production.
UNASSIGNED: Overall, our results demonstrate the existence of an endothelial-specific HSF (heat shock factor)-regulated transcriptional enhancer that mediates CALCRL expression. A better understanding of CALCRL gene regulation and the role of single-nucleotide polymorphisms in the modulation of CALCRL expression could provide important steps toward understanding the genetic regulation of shear stress signaling responses.