IDENTIFICATION OF AN ERG-BOUND ENDOTHELIAL CELL RESTRICTED ENHANCER FOR IL1Α AND IL1Β

Abstract

The progression of atherosclerosis is characterized by plaque formation within the arterial walls that could manifest into coronary artery disease (CAD) leading to myocardial infarction or stroke. Our objective in this study is to identify regulatory elements in human aortic endothelial cells that are responsible for controlling the inflammatory environment. To do so we evaluated ERG’s binding landscape, an important transcription factor that suppresses inflammation, and among the topmost ERG-bound enhancer-like elements was a putative enhancer located between IL1α/β, genes. After deleting this candidate with CRISPR we found a reduction in the magnitude of the IL1α/β RNA upon TNFa treatment (p<0.05). We, therefore, concluded that this region functions as an enhancer for the transcription of the IL1-α and IL1-β genes. Deleting this genomic region also reduced leukocyte adhesion to endothelial cells demonstrating the inflammatory functional consequence of the enhancer. Next, we evaluated ERG’s function at the IL1αβ enhancer by knocking down and overexpressing ERG with siRNA transfections and lentiviral transduction, respectively. Findings suggested that ERG’s inflammatory function in regulating IL1-α and IL1-β is in combination with the enhancer element and that greater transcriptional regulation of the genes comes from the enhancer versus ERG alone. Within this region, we also identified an ERG motif and NFkB motif as being the source of the majority of regulation surrounding enhancer activity. By identifying the role performed by this IL1αβ enhancer and its regulatory network, new advanced strategies can be formulated to better target CAD such as those with therapeutic and preventative measures.