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.

最近的研究报道了哺乳动物增强子广泛转录为非编码RNA 以刺激依赖的方式转录。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方面具有重要作用， 在发育和疾病过程中的表型结果。因此，eRNA正在成为一种重要的 基因调控机制的组成部分。由于最近的发现， eRNA在中风中的作用几乎是未知的。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定位，以在多个基因组中鉴定许多中风诱导的eRNA。 小鼠大脑皮层再灌注时间点。我们的初步数据证实了 编码eRNA的基因组位点，表明eRNA定位于染色质，并揭示了一种新的基因表达系统。 这种eRNA在调节中风后脑损伤和基因表达中的重要作用。的 eRNA在脑卒中后的分子相互作用、功能机制及性别依赖性作用 病理生理学尚未探索。在目前的项目中，我们基于我们的初步数据来评估eRNA 在成年小鼠脑皮层中风期间，具体目标1将使用以下组合 交联免疫沉淀、荧光原位杂交和转录状态分析， 确定eRNA的分子靶点作为小鼠中风后再灌注时间的函数 皮层特异性目标2将利用eRNA在体内的功能丧失，然后进行细胞、生理、病理学研究。 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eRNA在女性中的分子靶点和病理生理学作用 小鼠皮层总之，这项工作将阐明eRNAs在大脑皮层中的重要性， 揭示了中风中新的基因调控关系。这项工作将为今后的研究铺平道路， eRNA的治疗操作，以改善中风后的结果。