Functional characterization of an enhancer RNA in cerebral ischemia

最近的研究报道了哺乳动物增强子广泛转录成非编码的rna。 以依赖刺激的方式进行转录。越来越多的证据表明，这些增强子RNA(ERNA) 在协调高阶染色质相互作用中发挥重要作用，以促进基因表达和 发育和疾病期间的表型结果。因此，eRNAs正在成为一种重要的 基因调控机制的组成部分。由于他们最近的发现，它的表达和作用 中风中的eRNAs几乎是未知的。最近，我们应用了全基因组RNA-seq和 使用CHIP-SEQ进行全基因组增强子作图以识别多个卒中诱导的eRNA 小鼠大脑皮层再灌流的时间点。我们的初步数据证实了该蛋白的增强活性 编码eRNAs的基因组位点，表明eRNAs定位于染色质，并揭示了 一个这样的Erna在调节中风后脑损伤和基因表达方面发挥了重要作用。这个 ERNAs在卒中后的分子相互作用、作用机制及性别依赖效应 病理生理学尚未被研究。在当前的项目中，我们基于我们的初步数据来评估Erna 中风期间成年小鼠大脑皮质功能的深入研究。《特定目标1》将结合使用 交联免疫沉淀、荧光原位杂交和转录状态分析 确定eRNAs的分子靶点作为小鼠卒中后再灌注时间的函数 大脑皮层。具体目标2将在体内使用Erna功能丧失，随后是细胞、生理、病理 以及神经学分析，以评估ERNA在传播卒中后病理生理方面的作用。 特异性目标3将评估ERNA在女性中的分子靶点和病理生理效应 小鼠皮质。总之，这项工作将阐明eRNAs在大脑皮层中的意义，并可能 揭示中风中新的基因调控关系。这项工作将为未来探索 对eRNAs的治疗操作以改善中风后的预后。

Recent studies have reported widespread transcription of mammalian enhancers into noncoding RNA transcripts in a stimulus-dependent manner. Growing evidence shows that these enhancer RNAs (eRNAs) have essential roles in orchestrating higher-order chromatin interactions to facilitate gene expression and phenotypic outcomes during development and disease. As a result, eRNAs are emerging as an important component of the gene regulatory machinery. Due to their very recent discovery, the expression and roles of eRNAs in stroke is virtually unknown. Recently, we applied a combination of genome-wide RNA-seq and genome-wide enhancer mapping using ChIP-seq to identify a number of stroke-induced eRNAs at multiple time-points of reperfusion in the mouse cerebral cortex. Our preliminary data confirmed enhancer activity of the genomic loci encoding the eRNAs, showed that the eRNAs are localized to the chromatin, and revealed an important role for one such eRNA in modulating post-stroke brain damage and gene expression. The molecular interactions, functional mechanisms and sex-dependent effects of eRNAs on the post-stroke pathophysiology are unexplored. In the current project, we build upon our preliminary data to evaluate eRNA functionality in-depth during stroke in the adult mouse cortex. Specific Aim 1 will use a combination of crosslinking-immunoprecipitation, fluorescence in situ hybridization and transcriptional-state analysis to determine the molecular targets of the eRNAs as a function of post-stroke reperfusion time in the mouse cortex. Specific Aim 2 will employ eRNA loss-of-function in vivo followed by cellular, physiological, pathological and neurological analyses to evaluate the role of the eRNA in propagating the post-stroke pathophysiology. Specific Aim 3 will evaluate the molecular targets and pathophysiological effects of the eRNA in the female mouse cortex. Together, this work will illuminate the significance of eRNAs in the cerebral cortex and may reveal novel gene regulatory relationships in stroke. This work will pave the way for future studies exploring the therapeutic manipulation of eRNAs to improve post-stroke outcomes.