POLYCOMB GROUP PROTEINS AS EPIGENETIC MEDIATORS OF BRAIN ISCHEMIC TOLERANCE

多梳蛋白作为脑缺血耐受的表观遗传介质

• 批准号: 8297177
• 负责人: AN ZHOU
• 金额: $ 30.23万
• 依托单位: MOREHOUSE SCHOOL OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-04-01 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8297177
• 关键词: Affect; Biochemical; Biological; Biological Assay; Brain; Brain Injuries; Brain Ischemia; Cells; Cellular biology; Complementary DNA; Complex; Data; Development; Drosophila genus; Energy Metabolism; Epigenetic Process; Exhibits; Gene Expression; Gene Targeting; Genes; Genomics; Histone H2A; Histones; In Vitro; Injury; Ischemia; Literature; Mediating; Mediator of activation protein; Modeling; Modification; Molecular; Molecular Biology; Mus; Neurons; Outcome; Pathway interactions; Physiological; Polycomb; Potassium Channel; Protein Biosynthesis; Proteins; Proteome; Proteomics; Publishing; Recombinants; Regulation; Repressor Proteins; Resistance; Role; Signal Transduction; Stroke; Techniques; Testing; Therapeutic; Time; Transcription Repressor/Corepressor; gain of function; gene repression; histone modification; in vivo; knock-down; loss of function; neuroprotection; novel; overexpression; preconditioning; prevent; promoter; protein complex; protein expression; protein function; response; small hairpin RNA

DESCRIPTION (provided by applicant): Brief "preconditioning" ischemia produces "tolerance" to subsequent prolonged ischemia that would otherwise cause brain injury. The genomic signature of the tolerant brain is transcriptional suppression. The development of tolerance, however, requires new protein synthesis, indicating that changes in protein expression contribute significantly to the mechanism of tolerance. To understand how, we characterized the proteome of the tolerant brain (Stapels et al. Sci Signaling, 2010). It is enriched in histone proteins and, remarkably, in polycomb group (PcG) proteins, which function as transcriptional suppressors. Thus, we may have discovered the mechanism that induces transcriptional suppression in tolerance. Our results implicate epigenetic regulation mediated by PcG proteins. Further, our results show that PcG proteins, previously known as regulators of segmentation during development in Drosophila, have a novel neuroprotective function in the brain. Our preliminary data on ischemic tolerance in vivo and in vitro show that the development of ischemic tolerance is dependent upon the expression of PcG proteins: knockdown ablates tolerance, and over- expression produces tolerance. Accordingly, we offer the following aims to establish and define PcG proteins role as actuators of tolerance. Aim 1. To identify early, differential changes in PcG protein abundance and activity during the induction of tolerance. We will characterize changes in the expression of PcG proteins within different polycomb protein repressive complexes (PRCs). We will also characterize PcG protein- mediated histone modifications during the development of tolerance modeled in mice, over time. The results will define which PcG proteins and complexes participate in tolerance; demonstrate a rapid increase in PcG protein abundance at the initiation of tolerance; and establish epigenetic regulation through histone modi- fication as a mechanism underlying ischemic tolerance. Aim 2. To establish an essential role for PcG proteins in the development of ischemic tolerance. We will evaluate the effect of PcG protein expression on the outcome of ischemia using loss-of-function and gain-of-function approaches. Knockdown or over'expression of PcG proteins will be achieved by using small hairpin RNA (shRNA) or recombinant cDNA, respectively, both in vivo and in vitro. The results will demonstrate that the abundance of PcG proteins profoundly affects the outcome of ischemia. Aim 3. To demonstrate that PcG proteins control the expression of genes that are suppressed in ischemic tolerance. We will investigate the interaction of PcG proteins with the promoters of genes downregulated in tolerance using ChIP assays. When genes encode channels, electrophysiological analyses of cultured neurons, over- or under-expressing PcG proteins, will be performed to establish the effect of PcG proteins on the activity of tolerance effectors. We will also manipulate PcG proteins and potassium channels simultaneously and examine the effect on tolerance induction in mice in vivo. The results will show that tolerance effector genes and gene products can be modulated by alterations in PcG protein abundance. PUBLIC HEALTH RELEVANCE: The brain can be made "tolerant" to stroke, which is often caused by brain ischemia, when the brain is exposed to a small, non-lethal ischemia prior to a prolonged, otherwise injurious ischemia. Understanding the molecular mechanisms that underlie brain ischemic tolerance is extremely important in our efforts to develop new treatment for stroke. In the proposed study, we will investigate a novel mechanism that may be able to induce ischemic tolerance in the brain, a mechanism that involves a special group of proteins called epigenetic regulators (proteins that control the way genes in the brain respond to ischemia), by using various advanced neuroanatomical, biochemical, cell biology, molecular biology and physiological techniques.

描述（由申请人提供）：短暂的“预处理”缺血对随后的长时间缺血产生“耐受性”，否则会导致脑损伤。耐受性大脑的基因组特征是转录抑制。然而，耐受性的发展需要新的蛋白质合成，这表明蛋白质表达的变化对耐受性的机制有重要作用。为了理解其中的原因，我们对耐受性大脑的蛋白质组进行了表征（Stapels等）。Sci Signaling, 2010)。它富含组蛋白，值得注意的是，在多梳组（PcG）蛋白中，其功能是转录抑制因子。因此，我们可能已经发现了诱导耐受性转录抑制的机制。我们的研究结果暗示了PcG蛋白介导的表观遗传调控。此外，我们的研究结果表明，PcG蛋白在果蝇发育过程中被称为片段调节因子，在大脑中具有新的神经保护功能。我们在体内和体外对缺血耐受的初步数据表明，缺血耐受的发展依赖于PcG蛋白的表达：敲低会降低耐受性，过表达会产生耐受性。因此，我们提出以下目标来建立和定义PcG蛋白作为耐受性驱动因子的作用。目的1。在诱导耐受性过程中，早期识别PcG蛋白丰度和活性的差异变化。我们将描述不同多梳蛋白抑制复合物（PRCs）中PcG蛋白表达的变化。随着时间的推移，我们还将表征PcG蛋白介导的组蛋白修饰在小鼠耐受性模型的发展过程中。结果将确定哪些PcG蛋白和复合物参与了耐受性；在耐受性开始时，PcG蛋白丰度迅速增加；并通过组蛋白修饰建立表观遗传调控作为缺血耐受的机制。目标2。建立PcG蛋白在缺血性耐受性发展中的重要作用。我们将使用功能丧失和功能获得的方法来评估PcG蛋白表达对缺血结果的影响。PcG蛋白的敲低或过表达将分别通过小发夹RNA （shRNA）或重组cDNA在体内和体外实现。结果表明，PcG蛋白的丰度对缺血的预后有深远的影响。目标3。为了证明PcG蛋白控制在缺血耐受中被抑制的基因的表达。我们将使用ChIP法研究PcG蛋白与耐受下调基因启动子的相互作用。当基因编码通道时，将对培养神经元进行电生理分析，过高或过低表达PcG蛋白，以确定PcG蛋白对耐受效应物活性的影响。我们还将同时操纵PcG蛋白和钾通道，并在小鼠体内研究对耐受性诱导的影响。结果表明，耐受性效应基因和基因产物可以通过改变PcG蛋白丰度来调节。