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: 8451351
• 负责人: AN ZHOU
• 金额: $ 29.87万
• 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/8451351
• 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.

描述（由申请人提供）：短暂的“预适应”缺血产生对随后的长期缺血的“耐受性”，否则会导致脑损伤。宽容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机制有显着贡献。为了了解其中的原理，我们对耐受大脑的蛋白质组进行了表征（Stapels 等人，Sci Signaling，2010）。它富含组蛋白，尤其是多梳组 (PcG) 蛋白，其功能为转录抑制因子。因此，我们可能已经发现了耐受性中诱导转录抑制的机制。我们的结果表明表观遗传调控是由 PcG 蛋白介导的。此外，我们的结果表明，PcG 蛋白（以前被称为果蝇发育过程中的分割调节因子）在大脑中具有新颖的神经保护功能。我们关于体内和体外缺血耐受性的初步数据表明，缺血耐受性的发展依赖于PcG蛋白的表达：敲低消除耐受性，而过度表达产生耐受性。因此，我们提出以下目标来建立和定义 PcG 蛋白作为耐受促动器的作用。目标 1. 确定耐受诱导过程中 PcG 蛋白丰度和活性的早期差异变化。我们将描述不同多梳蛋白抑制复合物 (PRC) 内 PcG 蛋白表达的变化。随着时间的推移，我们还将描述小鼠模型耐受性发展过程中 PcG 蛋白介导的组蛋白修饰的特征。结果将确定哪些 PcG 蛋白和复合物参与耐受性；证明耐受开始时 PcG 蛋白丰度快速增加；并通过组蛋白修饰建立表观遗传调控作为缺血耐受的潜在机制。目标 2. 确定 PcG 蛋白在缺血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 蛋白的敲低或过度表达将分别通过使用小发夹 RNA (shRNA) 或重组 cDNA 在体内和体外实现。结果将证明 PcG 蛋白的丰度深刻影响缺血的结果。目标 3. 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 检测研究 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，将对培养的神经元、过度表达或表达不足的 PcG 蛋白进行电生理分析，以确定 PcG 蛋白对耐受效应子活性的影响。我们还将同时操作 PcG 蛋白和钾通道，并检查其对小鼠体内耐受诱导的影响。结果将表明，耐受效应基因和基因产物可以通过改变 PcG 蛋白丰度来调节。