POLYCOMB GROUP PROTEINS AS EPIGENETIC MEDIATORS OF BRAIN ISCHEMIC TOLERANCE

描述（由申请人提供）：短暂的“预处理”缺血产生对随后长时间缺血的“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贡献。为了理解如何，我们表征了耐受性大脑的蛋白质组（Stapels等人，Sci Signaling，2010）。它富含组蛋白，并且显著地富含作为转录抑制因子的多梳组（polycomb group，PcG）蛋白。因此，我们可能已经发现了在耐受中诱导转录抑制的机制。我们的研究结果涉及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，以前被称为调节器的分割在果蝇的发展过程中，在大脑中有一个新的神经保护功能。我们在体内和体外关于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蛋白作为耐受性致动器的作用。目标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。我们将描述不同的polycomb蛋白抑制复合物（PRCs）内PcG蛋白表达的变化。我们还将表征在小鼠中建模的耐受性发展期间随时间推移的PcG蛋白介导的组蛋白修饰。结果将确定哪些PcG蛋白和复合物参与耐受;证明在耐受开始时PcG蛋白丰度快速增加;并通过组蛋白修饰建立表观遗传调节作为缺血耐受的基础机制。目标2.建立PcG蛋白在缺血耐受发展中的重要作用。我们将使用功能丧失和功能获得方法评估PcG蛋白表达对缺血结局的影响。PcG蛋白的敲低或过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蛋白的丰度深刻地影响缺血的结果。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。我们将研究PcG蛋白与耐受性下调基因启动子的相互作用，使用ChIP检测。当基因编码通道时，将进行培养的神经元的电生理学分析，过度表达或表达不足的PcG蛋白，以建立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。

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.