Examining regulatory roles for long non-coding RNAs on Polycomb group functions

项目摘要/摘要 染色质结构调节对干细胞维持至关重要的基因表达模式 种群的分化以及它们分化成生物体的各种细胞类型。的地方建筑 染色质受到染色质修饰酶的影响，这些酶会改变核小体的紧致状态，从而 激活或沉默基因。多梳群(PcG)蛋白是染色质修饰物，在 抑制控制分化和干细胞自我更新之间平衡的发育基因。 PcG蛋白功能缺陷会导致发育进程缺陷，并与 增加罹患癌症的风险。该领域的主要问题之一是PcG蛋白是如何被招募到 哺乳动物基因组中的染色质。确定PcG复合体是如何招募的对于理解 发育调控的细节，并可能作为纠正异常细胞调控错误的靶点 类型。越来越多的长非编码RNA(LncRNAs)与PcG的调节有关 蛋白质的功能。关于lncRNAs在细胞中的功能作用，特别是它们是如何发挥作用的，人们知之甚少。 影响PcG活动。我们假设lncRNA相互作用是PcG蛋白募集到 特定的沉默区域，以影响发育进程。这项提议的目标是 确定染色质上lncRNAs和PcG蛋白之间的联系的机制细节 阐明这些相互作用如何影响多梳族蛋白的功能。为了实现这一目标，一个高- 吞吐量筛选要发现与PcG蛋白相互作用的一组lncRNA，将执行PRC1。 相互作用的lncRNAs将通过免疫沉淀后的rna样本的深度测序来鉴定。 来自人类间充质干细胞的复合体。一套仔细的选择标准将被实施到 建立相关的候选LncRNA，并将使用全面的功能验证策略来识别 涉及基因沉默的具有生物学意义的lncRNA相互作用。为了深入了解 LncRNAs在Prc1功能中的作用机制，控制Pc1功能的分子决定因素 LncRNAs和Prc1之间的相互作用将通过指导的结构/功能分析来确定。 总之，这些研究将为lncRNAs如何有序地与PcG复合体相互作用提供一个机制基础 让发育调控基因沉默。此外，揭示了lncRNA-PcG的机制细节 相互作用将为理解表观遗传调控建立一个新的框架，并最终可能提供 一种控制细胞命运和增殖的新靶点。

Project Summary/Abstract Chromatin structure regulates gene expression patterns important for the maintenance of stem cell populations and for their differentiation into the various cell types of an organism. The local architecture of chromatin is influenced by chromatin modifying enzymes that alter the compaction state of nucleosomes to activate or silence genes. Polycomb group (PcG) proteins are chromatin modifiers that have central roles in the silencing of developmental genes that control the balance between differentiation and stem cell self-renewal. Defects in PcG protein function lead to developmental progression defects and are associated with an increased risk for cancer. One of the major questions in the field is how PcG proteins are recruited to sites of chromatin in mammalian genomes. Determining how PcG complexes are recruited is crucial for understanding the details of developmental regulation and may serve as a target to correct misregulation in aberrant cell types. There is an increasing association of long non-coding RNAs (lncRNAs) with the regulation of PcG protein function. Little is known about the functional role of lncRNAs in the cell and in particular how they impact PcG activity. We hypothesize that lncRNA interactions are necessary for recruitment of PcG proteins to specific silencing regions in order to influence developmental progression. The goal of this proposal is to determine the mechanistic details of the association between lncRNAs and PcG proteins on chromatin and to elucidate how these interactions affect polycomb group protein function. To achieve this goal, a high- throughput screen to discover the set of lncRNAs that interact with the PcG protein, PRC1 will be performed. Interacting lncRNAs will be identified by deep sequencing of RNA samples after immunoprecipitation of PRC1 complexes from human mesenchymal stem cells. A careful set of selection criteria will be administered to establish relevant lncRNA candidates and a thorough functional validation strategy will be used to discern biologically meaningful lncRNA interactions involved in gene silencing. In order to gain insight into the mechanism of action employed by lncRNAs on PRC1 function, the molecular determinants that govern the interaction between lncRNAs and PRC1 will be determined through a guided structure/function analysis. Together, these studies will provide a mechanistic basis for how lncRNAs interact with PcG complexes in order to silence developmental regulatory genes. Furthermore, uncovering mechanistic details of lncRNA-PcG interactions will establish a new framework for understanding epigenetic regulation and may ultimately provide a novel target for controlling cell fate determination and proliferation.