Noncoding RNAs as epigenomic modulators in Alzheimer's Disease

描述(申请人提供)：我们实验室和其他实验室进行的大规模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的是，人类基因组的大部分转录成非蛋白质编码的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，如天然反义转录本(NAT)，都被认为是通过诱导DNA甲基化和染色质重塑来调节转录的。这项应用侧重于ncRNA作为阿尔茨海默病表观基因组调节器的作用。我们最近的出版物(自然医学10.1038/nm.1784,2008年7月)表明，ncRNA在阿尔茨海默病的病理生理学中发挥着关键作用。关于不同家族的ncRNA在神经系统疾病中的作用的报道强调了这样一个事实，即ncRNA介导的表观基因组转录调节是普遍存在的，并且在神经系统中显得尤为突出。对于ncRNA提出的几个功能，其中包括对染色质结构的调节和表观遗传记忆，受到了极大的关注。重要的是，由于许多组蛋白甲基转移酶(HMTase)复合体缺乏DNA结合结构域，但具有RNA结合基序，因此建议由ncRNA引起染色质结构的改变。H3上的三甲基化K4(一个活跃转录的标志)和H3上的三甲基化K27(一个抑制染色质的标志)都与ncRNAs有关，这表明ncRNA在指导染色质修饰方面是一种支架模型。在这里，我们描述了一种方法来创建与阿尔茨海默病相关的长和短ncRNA的全面表观基因组清单。我们将通过使用NAT特定的siRNA文库以及深度测序方法来包括长ncRNA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MS2 RNA标记和低温免疫沉淀(Cryo-IP)以及随后的RNA组分深度测序(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-蛋白质的相互作用。此外，我们将通过研究来自阿尔茨海默病患者和对照组的不同组人类大脑RNA来验证阿尔茨海默病候选表观基因组调节剂的参与。这项申请中提出的实验有望对控制基因表达的表观基因组机制产生重要的见解。此外，我们假设ncRNAs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的详细研究可能会导致发现新的治疗靶点和/或生物标记物。 公共卫生相关性：阿尔茨海默病是一种破坏性的与年龄相关的神经退行性疾病，其特征是进行性认知障碍和短期记忆丧失。尽管做出了相当大的研究努力，但研究人员距离揭示有助于诊断、预防和治疗这种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氏症的发展理论建立在阿尔茨海默氏症主要由遗传原因引起的前提下。然而，这些遗传原因至少部分是非典型的(非常规)，因此我们认为必须考虑所谓的“表观遗传学/表观遗传学”原因。

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.