CRISPR‐mediated deletion of a regulatory element downstream of the EDN1 gene increases expression of a long non‐coding RNA, EDN1‐AS

Abstract

Endothelin‐1 (ET‐1) is a peptide hormone that functions as a vasoconstrictor in the vasculature whereas in the collecting duct of the kidney it exerts blood pressure‐lowering effects via natriuretic actions. Aberrant ET‐1 signaling is associated with several pathological states including hypertension and chronic kidney disease. ET‐1 expression is regulated largely through transcriptional control of the gene that encodes ET‐1, Edn1. We recently discovered a long, non‐coding RNA (lncRNA) that appears to be antisense to the Edn1 gene, called EDN1‐AS. Because EDN1‐AS represents a potential novel mechanism to regulate ET‐1 expression, we examined the molecular regulation of EDN1‐AS expression and action. Using the ENCODE database and the UCSC genome browser, we located a putative glucocorticoid receptor response (GR) element upstream of the predicted transcription start site for EDN1‐AS. Preliminary chromatin immunoprecipitation results indicated that both GR and the circadian clock protein PER1 interact with this region of DNA in human renal proximal tubule HK‐2 cells. Using CRISPR, we deleted the region containing this potential regulatory element. Two homozygous deletion clones were identified following selection with puromycin (A11 and H3). An HK‐2 clone that went through the same selection procedure but was negative for the deletion served as the control cell cline. This deletion resulted in a significant increase in the expression of EDN1‐AS (2 fold, n=3–4, p<0.05). This increase in EDN1‐AS expression was associated with increased secretion of ET‐1 peptide from HK‐2 cells (KO H3=1.68 pg/mg vs. control line F1=1.53pg/mg, n=4, p= 0.004). Phenotypic characterization of these CRISPR clones revealed a difference in cell growth rates. Using a standard growth assay, we determined that the A11 clone exhibited a 3 fold increase in growth over 8 days compared to control cells (n=4, P<0.0001) and the H3 clone exhibited a 2 fold increase (n=4, p<0.001). These results support a role for EDN1‐AS as a novel regulatory mechanism of ET‐1 expression and cellular proliferation.Support or Funding InformationNIH T32DK104721 (LGD), NIH R21AG052861 (MLG), The Gatorade Trust through the UF Department of MedicineThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.