Control of chromatin organization by nucleosome remodelers and long non-coding RNA

DESCRIPTION（由申请人提供）：细胞核内染色质的三维组织支持基因组线性距离部分之间的远程相互作用。染色质环有助于控制基因表达，在细胞健康和发育中起重要作用。总体而言，基因组组织包括拓扑关联域（TADs）和特异性增强子-启动子相互作用。这种组织是由聚集在TAD边界的绝缘子蛋白介导的，并可能以不同的组合结合在增强子上。绝缘体位点也可能取决于局部染色质环境，包括核小体占用和长链非编码RNA （lncRNA）。核小体重塑成分如ISWI特异性定位于绝缘体结合位点，绝缘体蛋白被认为更倾向于核小体自由区域。此外，lncRNA如hsrm，也参与将核小体重塑成分募集到染色质上。本研究旨在探讨核小体重塑子和hsrm lncRNA在绝缘体蛋白与染色质结合和染色质环中的作用。这将通过测试以下假设来完成：ISWI结合依赖于hsrm lncRNA的染色质，并改变核小体的占用，使绝缘体蛋白可以结合。目的1：通过在核小体重塑（ISWI）和绝缘子蛋白敲低系中绘制核小体全基因组图谱，确定ISWI在绝缘子功能中的作用。此外，ISWI结合染色质依赖绝缘子蛋白，反之亦然，将通过一系列ChIP-seq实验在适当的敲除系中使用ISWI和绝缘子蛋白抗体来测试。目的2：通过ChiRP-seq检测hsrω lncRNA的定位，确定hsrω lncRNA与ISWI、Insulators之间的关系。hsrω的染色质相互作用对ISWI的依赖性也将以这种方式进行测试。ChIP-seq还将用于hsrω耗损系的ISWI和绝缘子蛋白，以确定染色质结合对lncRNA的依赖性。目的3：通过增强子阻断和HI-C检测，确定hsrm lncRNA对染色质组织的影响。已建立的增强子阻断试验将用于检测hsrm是否在与ISWI相同的位点上表现出增强子阻断活性。hsrm损耗系的HI-C将用于检测hsrm如何影响全基因组的染色质组织。

DESCRIPTION (provided by applicant): The three-dimensional organization of chromatin within the nucleus supports long-range interactions between linearly distant portions of the genome. The chromatin loops help control gene expression and are important in cellular health and development. Overall, genome organization includes both Topologically Associating Domains (TADs) and specific enhancer-promoter interactions. This organization is mediated by insulator proteins which cluster at TAD boundaries, and may bind in varying combinations at enhancers. Insulator sites may also depend on the local chromatin context including nucleosome occupancy and long non-coding RNA (lncRNA). Nucleosome remodeler components such as ISWI specifically localize to insulator binding sites, and insulator proteins are thought to prefer nucleosome free regions. Additionally lncRNA such as hsrω, has been implicated in recruiting nucleosome remodeler components to chromatin. It is the purpose of this proposal to investigate the role of nucleosome remodelers and hsrω lncRNA in insulator protein binding to chromatin and in chromatin looping. This will be done by testing the following hypothesis: ISWI binds chromatin dependent on hsrω lncRNA, and alters nucleosome occupancy so that insulator proteins may bind. This hypothesis will be tested using the following aims: Aim1: Determine the role of ISWI in insulator function by mapping nucleosomes genome-wide in nucleosome remodeler (ISWI) and insulator protein knockdown lines. Additionally, the dependency on insulator proteins for ISWI to bind chromatin, and vice versa, will be tested by a series of ChIP-seq experiments using ISWI and insulator protein antibodies in the appropriate knockdown lines. Aim2: Determine the relationship between hsrω lncRNA, ISWI, and Insulators by testing localization of hsrω lncRNA by ChiRP-seq. Dependence of hsrω's chromatin interaction on ISWI will also be tested in this manner. ChIP-seq will also be performed for ISWI and insulator proteins in hsrω depletion lines to determine the dependency of chromatin binding on lncRNA. Aim3: Determine the effect of hsrω lncRNA on chromatin organization by using enhancer blocking and HI-C assays. Established enhancer blocking assays will be used to test if hsrω exhibits enhancer blocking activity at the same loci as ISWI. HI-C in hsrω depletion line will be used to detect how hsrω affects chromatin organization genome-wide.