Coronary Artery Disease Risk Variant Dampens the Expression of CALCRL by Reducing HSF Binding to Shear Stress Responsive Enhancer in Endothelial Cells In Vitro

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 tha...

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Published in:	Arteriosclerosis, thrombosis, and vascular biology Vol. 44; no. 6; pp. 1330 - 1345
Main Authors:	Selvarajan, Ilakya, Kiema, Miika, Huang, Ru-Ting, Li, Jin, Zhu, Jiayu, Pölönen, Petri, Örd, Tiit, Õunap, Kadri, Godiwala, Mehvash, Golebiewski, Anna Kathryn, Ravindran, Aarthi, Mäklin, Kiira, Toropainen, Anu, Stolze, Lindsey K, Arce, Maximiliano, Magnusson, Peetra U, White, Stephen, Romanoski, Casey E, Heinäniemi, Merja, Laakkonen, Johanna P, Fang, Yun, Kaikkonen-Määttä, Minna
Format:	Journal Article
Language:	English
Published:	United States 01-06-2024
Subjects:	coronary artery disease coronary vessels gene expression genome-wide association study polymorphism single nucleotide coronary artery disease coronary vessels gene expression genome-wide association study polymorphism, single nucleotide
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Summary:	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 expression in endothelial cells. To functionally characterize the coronary artery disease single-nucleotide polymorphisms harbored around the gene , we applied an integrative approach encompassing statistical, transcriptional (RNA-seq), and epigenetic (ATAC-seq, 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. 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 expression, whereas HSF1 knockdown resulted in a significant decrease in rs880890-enhancer activity and expression. A significant decrease in HSF1 binding to the enhancer region in endothelial cells was observed under disturbed flow compared with unidirectional flow. knockdown and variant perturbation experiments indicated the role of CALCRL in mediating eNOS (endothelial NO synthase), APLN (apelin), angiopoietin, prostaglandins, and EDN1 (endothelin-1) signaling pathways leading to a decrease in cell proliferation, tube formation, and NO production. Overall, our results demonstrate the existence of an endothelial-specific HSF (heat shock factor)-regulated transcriptional enhancer that mediates expression. A better understanding of gene regulation and the role of single-nucleotide polymorphisms in the modulation of expression could provide important steps toward understanding the genetic regulation of shear stress signaling responses.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	1079-5642 1524-4636 1524-4636
DOI:	10.1161/ATVBAHA.123.318964