Functional characterization of an enhancer RNA in cerebral ischemia

增强子 RNA 在脑缺血中的功能表征

• 批准号: 10472073
• 负责人: Ashutosh Dharap
• 金额: $ 37.38万
• 依托单位: UNIVERSITY OF SOUTH FLORIDA
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-25 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10472073
• 关键词: Acetylation; Acute; Adult; Affect; Binding; Binding Sites; Biological; Biological Assay; Brain; Brain Injuries; Budgets; Cell Adhesion; Cell Cycle; Cell Death; Cerebral Ischemia; Cerebral cortex; ChIP-seq; Chromatin; Cyclic AMP; DNA; DNA Polymerase II; Data; Development; Disease; Down-Regulation; Enhancers; Estrogens; Extracellular Matrix; Female; Fluorescent in Situ Hybridization; Functional disorder; Future; Gene Expression; Gene Expression Regulation; Gene Proteins; Genes; Genetic Enhancer Element; Genetic Transcription; Genomics; Histones; Immunoprecipitation; Infarction; Investigation; Ischemia; Ischemic Stroke; Lysine; Maps; Mediating; Methods; Middle Cerebral Artery Occlusion; Molecular; Molecular Target; Mus; Neurologic; Neurons; Outcome; Pathologic; Pathway interactions; Phase; Phenotype; Physiological; Play; Proteins; RNA; Regulator Genes; Reperfusion Therapy; Reporter; Reporting; Role; Running; Sensorimotor functions; Signal Transduction; Stimulus; Stroke; Structure; TP53 gene; Testing; Therapeutic; Time; Transcript; Translations; Ubiquitination; Untranslated RNA; Western Blotting; Work; base; blood-brain barrier disruption; cdc Genes; cell type; crosslink; endoplasmic reticulum stress; experimental study; genome-wide; genomic locus; improved; in vivo; knock-down; loss of function; macrophage; male; new therapeutic target; novel; post stroke; programs; response; sex; stroke outcome; transcription factor; transcriptome sequencing; virtual

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.

最近的研究报告哺乳动物增强子广泛转录为非编码RNA 以刺激依赖性方式转录。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以促进基因表达和 发育和疾病期间的表型结果。因此，eRNA 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，表达和角色 中风中的 eRNA 几乎是未知的。最近，我们结合了全基因组RNA-seq和 使用 ChIP-seq 进行全基因组增强子作图，以识别多个中风诱发的 eRNA 小鼠大脑皮层再灌注的时间点。我们的初步数据证实了增强剂活性 编码 eRNA 的基因组位点，表明 eRNA 定位于染色质，并揭示了 其中一种 eRNA 在调节中风后脑损伤和基因表达中发挥着重要作用。这 eRNA 对中风后的分子相互作用、功能机制和性别依赖性影响 病理生理学尚未探索。在当前项目中，我们根据初步数据来评估 eRNA 成年小鼠皮层中风期间的深入功能。具体目标 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 eRNA 的分子靶标作为小鼠中风后再灌注时间的函数 皮质。具体目标 2 将采用体内 eRNA 功能丧失，然后进行细胞、生理、病理学分析 和神经学分析，以评估 eRNA 在传播中风后病理生理学中的作用。 具体目标 3 将评估女性 eRNA 的分子靶点和病理生理学效应 小鼠皮质。总之，这项工作将阐明 eRNA 在大脑皮层中的重要性，并可能 揭示中风中新的基因调控关系。这项工作将为未来的研究铺平道路 eRNA 的治疗操作可改善中风后的结果。