Determination of a Novel Epigenetic Silencing Mechanism

 描述(申请人提供)：表观基因组调控生物体何时、何地和如何使用储存在其基因组中的遗传信息。它在许多细胞过程中都是必不可少的，如基因表达的调节、基因组组织和细胞命运的决定。它还管理着增长、发展和最终的人类健康。异染色质代表沉默的染色质域，通过表观遗传机制组装和维持。广泛的研究集中在RNA干扰(RNAi)途径介导的着丝粒异染色质组装上。最近的研究，包括我们的研究，表明外切体，一个RNA质量控制和监测复合体，与RNAi平行地发挥作用，介导表观遗传沉默。虽然RNAi和外体途径都是由RNA介导的，并参与将长的非编码RNA(NcRNAs)加工成小RNA，但外体途径如何参与异染色质组装尚不清楚。是否其他RNAi独立的RNA加工途径参与表观遗传沉默也是未知的。我们最近在表观遗传沉默中发现了一种RNA处理机制，它独立于RNAi和外切体工作。Dhp1是一种保守的5‘到3’外切核糖核酸酶，是发芽酵母Rat1和Metazoan Xrn2的同源基因，除了在转录终止中已确定的作用外，它在表观遗传沉默中发挥着迄今未知的作用。遗传学、细胞生物学和基因组学方法将被用来研究：1)Dhp1在异染色质区域的定位；2)Dhp1结合蛋白；以及3)Dhp1的酶活性和在表观遗传沉默中潜在的共激活因子。这些结果将阐明各种RNA加工途径如何共同或独立地作用于真核生物基因组的表观遗传调控，这是基因表达的基本机制。这项研究将在裂解酵母中进行，裂殖酵母是研究表观遗传沉默的主要模型，因为它具有保守的表观遗传成分，适合于强大的组合实验方法和本科研究。这个项目意义重大，具有创新性。它将在表观遗传沉默中定义一种新的RNA处理机制，该机制独立于RNAi和外切体工作。此外，这将是第一次探索Dhp1/Rat1/Xrn2在染色质沉默中的作用。

 DESCRIPTION (provided by applicant): The Epigenome regulates when, where and how an organism uses the genetic information stored in its genome. It is essential to many cellular processes, such as the regulation of gene expression, genome organization and cell-fate determination. It also governs growth, development, and ultimately human health. Heterochromatin represents silenced chromatic domains, which are assembled and maintained through epigenetic mechanisms. Extensive studies have focused on the assembly of heterochromatin at centromeres mediated by the RNA interference (RNAi) pathway. Recent studies, including ours, indicate that the exosome, an RNA quality control and surveillance complex, performs in a parallel pathway with RNAi to mediate epigenetic silencing. Although both RNAi and the exosome pathways are RNA-mediated and involved in processing long noncoding RNAs (ncRNAs) into small RNAs, exactly how the exosome pathway participates in heterochromatin assembly is not been demonstrated. Whether other RNAi-independent RNA processing pathways participate in epigenetic silencing is also unknown. We have recently identified a RNA-processing mechanism in epigenetic silencing that works independently of both RNAi and the exosome. The proposed research aims are designed to test the hypothesis that Dhp1, a conserved 5' to 3' exoribonuclease and ortholog of budding yeast Rat1 and metazoan Xrn2, plays a heretofore unknown role in epigenetic silencing, beyond its established role in transcription termination. Genetic, cell biology, and genomic approaches will be employed to investigate: 1) the localization of Dhp1 at heterochromatic regions; 2) Dhp1 binding proteins; and 3) the enzymatic activity of Dhp1 and potential co-activators in epigenetic silencing. Results will clarify how various RNA-processing pathways, acting together or independently, contribute to epigenetic regulation of the eukaryotic genome, a fundamental mechanism of gene expression. This study will be carried out in fission yeast, Schizosaccharomyces pombe (S. pombe), a premier model for studying epigenetic silencing because of its conserved epigenetic components and suitability for powerful combinatory experimental methods as well as undergraduate research. This project is significant and innovative. It will define a novel RNA-processing mechanism in epigenetic silencing that works independently of both RNAi and the exosome. Additionally, it will be the first exploration into how Dhp1/Rat1/Xrn2 functions in chromatin-based silencing.