Polycomb-mediated epigenetic mechanisms in neurodegeneration and aging brain

描述（由申请人提供）：亨廷顿病（HD）是几种进行性和致死性神经退行性疾病之一。选择性神经元丢失是由遗传和环境因素驱动的，随着年龄的增长而加剧。转录失调是许多神经退行性疾病（其中包括HD）和正常衰老的核心。我们感兴趣的是神经元死亡的HD，一个过程中驱动的异常多聚谷氨酰胺在亨廷顿蛋白（Htt）的扩展。我们的研究已经证明了丝氨酸/苏氨酸激酶Rip 2在HD发病机制中的核心作用。Rip 2还驱动由其他凋亡刺激引起的神经元死亡，表明Rip 2激酶是多种神经损伤的常见介质。为了了解Rip 2促凋亡作用的分子机制，我们使用Rip 2作为诱饵进行酵母双杂交筛选。我们发现Rip 2直接结合EED，EED是主要表观遗传调节因子PRC 2的一个组成部分。PRC 2在赖氨酸27处使组蛋白H3三甲基化（H3-K27 me 3），从而重塑染色质并抑制基因转录。EED的这种活性表明Rip 2可能通过改变控制基因转录的表观遗传途径导致细胞死亡。我们将研究PRC 2表观遗传途径的分子调控，作为Rip 2影响HD和衰老的假定机制。我们设想，我们的研究结果将为HD的治疗提供新的靶点，并可能用于其他神经系统疾病和衰老。目的1：确定Rip 2如何调节含EED的组蛋白H3-K27甲基转移酶PRC 2。方法包括体外甲基转移酶和激酶测定、磷酸化的质谱分析、RNA干扰（RNAi）和蛋白质生物化学。目的2：研究PRC 2在突变型Htt和氧化应激诱导的神经细胞死亡中的作用。方法包括凋亡测定、RNAi、基因结构-功能分析和免疫细胞化学。目的3：筛选PRC 2和H3-K27 me 3在HD和老年脑中的基因靶点。方法包括靶基因染色质免疫沉淀（ChIP）和尖端ChIP测序，以在全基因组范围内鉴定基因靶。相关性：涉及组蛋白修饰的表观遗传转录变化在衰老和与年龄相关的神经退行性疾病中起关键作用。这项研究的结果将确定导致异常基因转录的特定信号通路。确定的途径将有助于开发新的治疗方法，这可能会延缓衰老和减缓神经退行性变。

DESCRIPTION (provided by applicant): Huntington's disease (HD) is one of several progressive and fatal neurodegenerative diseases. Selective neuronal loss is driven by genetic and environmental factors exacerbated by advancing age. Transcriptional dysregulation is central to many neurodegenerative diseases (HD among them) and to normal aging. We are interested in neuronal death in HD, a process driven by abnormal polyglutamine expansions in the huntingtin protein (Htt). Our studies have demonstrated a central role for the serine/threonine kinase Rip2 in HD pathogenesis. Rip2 also drives neuronal death elicited by other apoptotic stimuli, suggesting that Rip2 kinase is a common mediator of diverse neurological insults. To understand the molecular mechanism of Rip2's pro-apoptotic effects, we performed a yeast two-hybrid screen using Rip2 as bait. We found that Rip2 directly binds EED, a component of the major epigenetic regulator PRC2. PRC2 tri-methylates histone H3 at lysine 27 (H3-K27me3), thereby remodeling chromatin and repressing gene transcription. This activity of EED suggests that Rip2 may cause cell death by altering an epigenetic pathway controlling gene transcription. We will study the molecular regulation of the PRC2 epigenetic pathway as a putative mechanism by which Rip2 affects HD and aging. We envision that our findings will suggest novel targets for therapies for HD and potentially for other neurological diseases and aging. Aim 1: To determine how Rip2 regulates the EED-containing histone H3-K27 methyltransferase, PRC2. Approaches include in vitro methyltransferase and kinase assays, mass spectroscopic analysis of phosphorylation, RNA interference (RNAi), and protein biochemistry. Aim 2: To determine the role of PRC2 in neuronal cell death induced by mutant Htt and oxidative stress. Approaches include apoptosis assays, RNAi, gene structure-function analysis, and immunocytochemistry. Aim 3: To identify gene targets of PRC2 and H3-K27me3 in HD and aged brains. Approaches include target gene chromatin immunoprecipitation (ChIP) and cutting-edge ChlP-sequencing to identify gene targets on the genome-wide scale. RELEVANCE: Epigenetic transcriptional changes involving histone modification play a critical role in aging and age- associated neurodegenerative diseases. Findings in this proposal will identify specific signaling pathways that lead to aberrant gene transcription. The pathways identified will aid in the development of novel therapeutics, which may potentially retard aging and slow neurodegeneration.