Modulation of Brain Pathology by Enhancer RNAs in Cerebral Ischemia

Abstract

Recent studies have reported widespread stimulus-dependent transcription of mammalian enhancers into noncoding enhancer RNAs (eRNAs), some of which have central roles in the enhancer-mediated induction of target genes and modulation of phenotypic outcomes during development and disease. In cerebral ischemia, the expression and functions of eRNAs are virtually unknown. Here, we applied genome-wide H3K27ac ChIP-seq and genome-wide RNA-seq to identify enhancer elements and stroke-induced eRNAs, respectively, in the mouse cerebral cortex during transient focal ischemia. Following a 1-h middle cerebral artery occlusion (MCAO) and 6 h of reperfusion, we identified 77 eRNAs that were significantly upregulated in stroke as compared to sham, of which 55 were exclusively expressed in stroke. The knockdown of two stroke-induced eRNAs in the mouse brain resulted in significantly larger infarct volumes as compared to controls, suggesting that these eRNAs are involved in the post-stroke neuroprotective response. A preliminary comparison of eRNA expression in the male versus female cortices revealed sex-dependent patterns that may underlie the physiological differences in response to stroke between the two sexes. Together, this study is the first to illuminate the eRNA landscape in the post-stroke cortex and demonstrate the significance of an eRNA in modulating post-stroke cortical brain damage.

Keywords: Cerebral ischemia; Enhancer; Mouse; eRNA.

Fig. 1:. Stroke-induced eRNA transcription in the mouse cortex.

(a) Analysis pipeline for the identification of stroke-responsive eRNAs in the mouse cortex (left panel) and a representative overlay of the RNA-seq peaks, H3K27ac ChIP-seq peaks and active regions for eRNAs exclusively expressed in the stroke samples (right panel). (b) The genomic origins (left panel), size distribution (middle panel) and expression characteristics (right panel) of the 77 stroke-induced eRNAs. (c) Longitudinal expression profiles of the stroke-induced eRNAs from 6–24 h of reperfusion evaluated using RNA-seq datasets from our previously published studies[17, 18].

Fig. 2:. Subcellular localization of stroke-responsive eRNAs in the mouse cortex.

Subcellular localization of randomly selected stroke-induced eRNAs and 18s rRNA was evaluated in the mouse cortex using one-step real-time PCR. The data are expressed as mean ± SD from three independent experiments (p<0.05, unpaired t-test).

Fig. 3:. Effects of eRNA_06347 knockdown on brain pathology.

(a) Schematic showing the experimental timeline. (b) Real-time PCR evaluation of the expression of eRNA_06347 and eRNA_093384 in the cortices of animals undergoing targeted eRNA knockdown versus negative control (n=4/group; Welch’s t-test; data expressed as mean ± SD). The expression of the untargeted eRNA_04759 and eRNA_007108 in the respective groups remains unchanged. (c) Cresyl violet staining of whole-brain sections in sham, negative control (n=5/group) and respective eRNA knockdown groups (n=7/group). (d) Quantitation of the infarct volumes in the eRNA knockdown groups versus negative control (Welch’s t-test; data expressed as mean ± SD). KD: knockdown; Neg Control: negative control.

Fig. 4:. Expression of eRNAs in the post-stroke female cortex.

(a) Expression of four randomly selected eRNAs in the post-stroke female cortex versus sham as determined by real-time PCR. Values represent averages from three biological replicates tested in duplicate. Fold-changes between the groups were determined using the ΔΔCT method. All the eRNAs were significantly higher in the female cortex as compared to sham (p<0.05; fold-change >2.0), except eRNA_156140 which showed no significant difference between the groups. (b) Expression comparisons of the four eRNAs in the post-stroke female cortex as compared to the post-stroke male cortex. Three of the eRNAs showed significant expression differences between females and males during stroke, but the expression of eRNA_06347 was not significantly different between the two sexes.