Noncoding RNAs as epigenomic modulators in Alzheimer's Disease

描述（由申请人提供）：我们实验室和其他人进行的大规模cDNA测序工作提供了强有力的证据，与早期的理解形成鲜明对比，大部分人类基因组被转录为非蛋白质编码RNA（ncRNA）。人类ncRNA转录是极其广泛和普遍的，对神经元功能具有潜在的广泛影响。短ncRNA，如microRNA（miRNA）和长ncRNA，如天然反义转录物（NAT）被报道为通过诱导DNA甲基化和染色质重塑的转录调节剂。本申请集中于ncRNA作为阿尔茨海默病的表观基因组调节剂的作用。我们最近的出版物（Nature Medicine 10.1038/nm.1784，2008年7月）表明ncRNA在阿尔茨海默病病理生理学中发挥关键作用。关于各种ncRNA家族在神经系统疾病中的作用的报告突出了这样一个事实，即ncRNA介导的表观基因组转录调节是普遍的，并且在神经系统中显得特别突出。ncRNA的几个功能，其中包括染色质结构和表观遗传记忆的调节，受到了广泛的关注。重要的是，由于许多组蛋白甲基转移酶（HMTase）复合物缺乏DNA结合结构域但具有RNA结合基序，因此提示了由ncRNA诱导的染色质结构的修饰。H3上的三甲基化K4（活跃转录的标记）和H3上的三甲基化K27（抑制染色质的标记）均与ncRNA相关，表明ncRNA在指导染色质修饰中的支架模型。在这里，我们描述了一种方法来创建一个全面的表观基因组库存的长和短的ncRNA与阿尔茨海默病。我们将通过采用NAT特异性siRNA文库以及深度测序方法来包括长和短ncRNA，以创建参与阿尔茨海默病发病机制的ncRNA的目录。通过应用染色质免疫沉淀（ChIP），以及MS 2 RNA标记和低温免疫沉淀（Cryo-IP），然后进行RNA组分的深度测序（ChIP-Seq）和蛋白质组分的质谱分析，我们将研究RNA-染色质，RNA-RNA和RNA-蛋白质相互作用。此外，我们将通过研究来自阿尔茨海默病患者和对照个体的不同人脑RNA集来验证阿尔茨海默病候选表观基因组调节剂的参与。在本申请中提出的实验有望产生显着的见解表观基因组机制，基因表达的控制。此外，我们假设ncRNA将被证明广泛参与阿尔茨海默病的发病机制。重要的是，对阿尔茨海默病相关ncRNA的详细研究将可能导致发现新的治疗靶点和/或生物标志物。 公共卫生相关性：阿尔茨海默氏病是一种破坏性的年龄相关性神经退行性疾病，其特征在于认知和短期记忆丧失的进行性损害。尽管做出了大量的研究努力，但研究人员距离揭示有助于诊断、预防和治疗该疾病的明确风险因素的最终目标还有很长的路要走。目前关于常见的晚发性阿尔茨海默病的发展理论的前提是阿尔茨海默病主要由遗传原因引起。然而，这些遗传原因至少部分是非典型的（非传统的），因此，我们认为，所谓的“表观遗传/表观基因组”的原因必须加以考虑。

DESCRIPTION (provided by applicant): Large-scale cDNA sequencing efforts conducted by our lab and others have provided strong evidence that, in sharp contrast to earlier understanding, much of the human genome is transcribed into non-protein-coding RNA (ncRNA). Human ncRNA transcription is extremely widespread and pervasive with potentially broad implications for neuronal function. Both short ncRNA, such as microRNA (miRNA) and long ncRNA, like natural antisense transcripts (NATs) are reported as modulators of transcription by induction of DNA methylation and chromatin remodeling. This application focuses on the role of ncRNA as epigenomic modulators of Alzheimer's disease. Our recent publication (Nature Medicine 10.1038/nm.1784,July 2008) demonstrates that ncRNA exert a pivotal role in Alzheimer's disease pathophysiology. The reports on the role of various families of ncRNA in neurological disorders highlight the fact that ncRNA-mediated epigenomic modulation of transcription is pervasive and appear particularly prominent in the nervous system. Several functions proposed for ncRNA, among them regulation of chromatin architecture and epigenetic memory, have received much attention. Importantly, modifications of chromatin structure induced by ncRNA are suggested as many histone methyl transferase (HMTase) complexes lack DNA-binding domains but possess RNA- binding motifs. Both trimethylated K4 on H3 (a mark of active transcription) and trimethylated K27 on H3 (a mark of repressed chromatin) were reported in association with ncRNAs, suggesting a scaffold model for the ncRNA in guiding chromatin modification. Here, we describe an approach to create a comprehensive epigenomic inventory of long and short ncRNA associated with Alzheimer's disease. We shall include both long and short ncRNAs by employing a NAT-specific siRNA library as well as a deep sequencing approach to create a catalog of ncRNA's involved in the pathogenesis of Alzheimer's disease. By applying Chromatin immunoprecipitation (ChIP), as well as MS2 RNA tagging and Cryogenic- immunoprecipitation, (Cryo-IP) followed by deep sequencing of RNA fractions (ChIP-Seq) and Mass-spectrometry of protein fractions, we will study RNA-chromatin, RNA-RNA and RNA- protein interactions. Further, we will validate involvement of candidate epigenomic modulators of Alzheimer's disease by studying different sets of human brain RNA derived from Alzheimer's disease patients and control individuals. The experiments proposed in this application promise to yield significant insights into epigenomic mechanisms by which gene expression is controlled. Furthermore we postulate that ncRNAs will prove to be broadly involved in the pathogenesis of Alzheimer's disease. Importantly, detailed studies on Alzheimer's disease-related ncRNAs will potentially lead to the discovery of novel therapeutic targets and/or biomarkers. PUBLIC HEALTH RELEVANCE: Alzheimer's disease is a devastating age-related neurodegenerative disorder characterized by progressive impairment of cognition and short-term memory loss. Despite considerable research efforts, researchers are still a long way from the ultimate goal of revealing clear risk factors that can help in the diagnosis, prevention and treatment of the disease. Current theories about the development of common late-onset Alzheimer's disease hinge on the premise that Alzheimer's disease arises mainly from heritable causes. However, these genetic causes are at least in part atypical (nonconventional), and we therefore argue that so-called "epigenetic/epigenomic" causes must be considered.