Functional characterization of an enhancer RNA in cerebral ischemia

增强子 RNA 在脑缺血中的功能表征

基本信息

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
项目类别：
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

项目摘要

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到非编码RNA 以刺激依赖性方式转录本。越来越多的证据表明，这些增强子RNA（ERNAS）在策划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局。结果，Ernas成为重要的基因调节机械的组成部分。由于他们最近的发现，中风中的Ernas几乎是未知的。最近，我们应用了全基因组RNA-Seq和全基因组增强子映射使用ChIP-Seq识别多个中风诱导的ERNAS 小鼠大脑皮层中再灌注的时间点。我们的初步数据证实了增强子活性编码ERNAS的基因组基因座表明ERNAS定位于染色质，并揭示了一个一个这样的ERNA在调节中风后脑损伤和基因表达中的重要作用。这 Ernas对冲程后的分子相互作用，功能机制和性依赖性影响病理生理学是未开发的。在当前项目中，我们以初步数据为基础来评估ERNA 成年小鼠皮层中风期间的功能深入。具体目标1将使用交联 - 免疫沉淀，荧光原位杂交和转录状态分析与确定ERNAS的分子靶标是小鼠触摸后再灌注时间的函数皮质。特定的目标2将在体内采用ERNA功能丧失，然后使用细胞，生理，病理和神经学分析以评估ERNA在传播后击病理生理学中的作用。具体目标3将评估ERNA对女性的分子靶标和病理生理影响鼠标皮质。这项工作一起将阐明Ernas在大脑皮层中的重要性，并可能揭示中风中新型基因调节关系。这项工作将为未来的研究铺平道路探索对ERNAS的治疗操纵以改善中风后结果。