POLYCOMB GROUP PROTEINS AS EPIGENETIC MEDIATORS OF BRAIN ISCHEMIC TOLERANCE

描述(申请人提供)：短暂的“预适应”缺血产生对随后的长时间缺血的“耐受性”，否则将导致脑损伤。耐受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重要贡献。为了了解如何，我们描述了耐受脑的蛋白质组(Stapels等人)。SCI信号，2010)。它富含组蛋白，值得注意的是，它还富含作为转录抑制因子的多梳蛋白(PcG)。因此，我们可能已经发现了诱导耐受中转录抑制的机制。我们的结果暗示了PcG蛋白介导的表观遗传调控。此外，我们的结果表明，PcG蛋白，以前被认为是果蝇发育过程中分段的调节器，在大脑中具有一种新的神经保护功能。我们在体内和体外关于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：敲除耐受消除耐受，过度表达产生耐受。因此，我们提供了以下目标来建立和定义PcG蛋白作为耐受执行因子的作用。目的1.鉴定耐受诱导过程中PcG蛋白丰度和活性的早期差异变化。我们将表征PcG蛋白在不同多梳蛋白抑制复合体(PRCs)中表达的变化。我们还将表征PcG蛋白介导组蛋白修饰在小鼠耐受发展过程中随时间的推移。这些结果将确定哪些PcG蛋白和复合体参与耐受；证明在耐受开始时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。目的2.确定PcG蛋白在缺血耐受形成中的重要作用。我们将使用功能丧失和功能获得两种方法评估PcG蛋白表达对缺血结局的影响。无论是在体内还是在体外，分别利用小发夹RNA(ShRNA)和重组cDNA可以实现PcG蛋白的低表达和高表达。结果将证明，PcG蛋白的丰度深刻地影响着缺血的结局。目的3.证实PcG蛋白调控缺血耐受抑制基因的表达。我们将利用芯片技术研究PcG蛋白与耐受性下调基因启动子之间的相互作用。当基因编码通道时，将对培养的神经元进行电生理分析，高表达或低表达PcG蛋白，以确定PcG蛋白对耐受效应器活性的影响。我们还将同时操纵PcG蛋白和钾通道，并在体内检测其对小鼠耐受诱导的影响。结果表明，耐受效应基因和基因产物可以通过PcG蛋白丰度的变化来调节。 与公共卫生相关：当大脑在长时间的、否则有害的缺血之前暴露于小的、非致命性的缺血时，可以使大脑对中风“耐受”，中风通常是由脑缺血引起的。了解脑缺血耐受的分子机制对于我们开发新的中风治疗方法是极其重要的。在这项拟议的研究中，我们将利用各种先进的神经解剖学、生化、细胞生物学、分子生物学和生理学技术，研究一种可能能够在大脑中诱导缺血耐受的新机制，该机制涉及一组特殊的蛋白质，称为表观遗传调节蛋白(控制大脑中基因对缺血的反应方式的蛋白质)。

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.