Abstract 174: Regulation of Post-Stroke Gene Expression and Pathophysiology by an Enhancer RNA

Abstract

Enhancer elements orchestrate genomic regulatory interactions to facilitate gene expression and phenotypic changes in development and disease. Recent studies have shown that mammalian enhancers are transcribed into noncoding enhancer RNAs (eRNAs) in a stimulus-dependent manner and some of these eRNAs have central roles in enhancer-mediated gene induction. Due to the very recent discovery of eRNAs, their expression and functions in cerebral ischemia are virtually unknown. Recently, we applied a combination of genome-wide H3K27ac ChIP-seq and genome-wide RNA-seq in ipsilateral cortices from adult male C57BL6/N mice following a 1 h middle cerebral artery occlusion (or sham surgery) and 6 h of reperfusion (n=6/group) to identify enhancer elements and stroke-induced eRNAs. We identified 77 eRNAs that were significantly upregulated (absolute fold-change ≥2.0; q<0.05) in stroke as compared to sham, of which 55 were exclusively expressed in stroke. All 77 eRNAs were novel, unspliced transcripts. Using luciferase reporter assays, we confirmed the enhancer capabilities of multiple eRNA-encoding genomic loci, and using cell fractionation experiments we determined that the eRNAs are preferentially localized to the chromatin, consistent with their eRNA status. Knockdown of one such eRNA in the mouse brain using antisense oligos, followed by a 1 h MCAO and 6 h of reperfusion, resulted in 49 downregulated genes and 106 upregulated genes (absolute fold-change ≥2.0; q<0.05) versus negative controls. The downregulated subset was enriched in several important genes with roles in mitochondrial activity, receptor signaling, ion channels and calcium homeostasis, and the upregulated subset was enriched in genes involved in inflammation and apoptosis. These changes were associated with worsened neurological scores and an 11.9% increase in infarct volume in the eRNA knockdown group versus negative control (p=0.004). This suggests an innate neuroprotective role for the eRNA during the stroke injury. Together, our study presents the first genome-wide identification of stroke-induced eRNAs in the adult cerebral cortex and illuminates the functional importance of one such eRNA in modulating post-stroke cortical gene expression and pathophysiology.