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

描述（由申请人提供）：核内染色质的三维组织支持基因组线性距离部分之间的长程相互作用。染色质环有助于控制基因表达，对细胞健康和发育非常重要。总的来说，基因组组织包括拓扑相关结构域（TADs）和特定的增强子-启动子相互作用。这种组织是介导的绝缘子蛋白簇的边界，并可能结合在不同的组合增强。绝缘子位点还可以取决于局部染色质环境，包括核小体占据和长非编码RNA（lncRNA）。核小体重塑组分如ISWI特异性定位于绝缘子结合位点，并且绝缘子蛋白被认为优选无核小体区域。此外，lncRNA如hsrω涉及将核小体重塑组分募集到染色质中。本研究的目的是探讨核小体重塑蛋白和hsrω lncRNA在绝缘子蛋白与染色质结合和染色质成环中的作用。这将通过检验以下假设来完成：ISWI依赖于hsrω lncRNA结合染色质，并改变核小体占有率，以便绝缘子蛋白可以结合。这一假设将使用以下目标进行测试：目标1：通过在核小体重塑（ISWI）和绝缘子蛋白敲低系中定位核小体全基因组，确定ISWI在绝缘子功能中的作用。此外，ISWI结合染色质对绝缘子蛋白的依赖性，反之亦然，将通过一系列ChIP-seq实验在适当的敲低系中使用ISWI和绝缘子蛋白抗体进行测试。目的2：通过ChiRP-seq检测hsr ω lncRNA的定位，确定hsr ω lncRNA、ISWI和绝缘子之间的关系。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。还将对hsrω耗竭系中的ISWI和绝缘子蛋白进行ChIP-seq，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分析确定hsrω lncRNA对染色质组织的影响。将使用已建立的增强子阻断试验来检测hsrω是否在与ISWI相同的基因座处表现出增强子阻断活性。将使用hsrω耗竭系中的HI-C来检测hsrω如何影响全基因组染色质组织。

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.