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

核小体重塑剂和长非编码 RNA 对染色质组织的控制

• 批准号: 8835656
• 负责人: Michael Jordan Rowley
• 金额: $ 5.07万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-02-01 至 2018-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8835656
• 关键词: Affect; Antibodies; Binding; Binding Sites; Biological Assay; Biological Models; Cell Nucleus; ChIP-seq; Chromatin; Chromatin Loop; Chromatin Structure; Communities; Complex; Dependence; Dependency; Development; Distant; Drosophila genus; Drosophila melanogaster; Elements; Enhancers; Exhibits; Gene Expression; Genes; Genome; Health; ISWI; Link; Maps; Mediating; NURF; Nuclear; Nucleosomes; Plants; Play; Positioning Attribute; Protein Binding; Proteins; Recruitment Activity; Research; Resources; Role; Series; Site; Testing; Transcriptional Regulation; Untranslated RNA; cell growth regulation; cellular development; genome-wide; histone modification; insight; member; promoter; research study; transcription factor

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.

描述(申请人提供)：核内染色质的三维组织支持基因组的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，对细胞健康和发育非常重要。总体而言，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。这种组织是由聚集在TAD边界的绝缘体蛋白介导的，并可能以不同的组合结合在增强剂上。绝缘体位置也可能取决于局部染色质环境，包括核小体占有率和长非编码RNA(LncRNA)。核小体重构体成分，如ISWI，特异性地定位于绝缘子结合部位，而绝缘体蛋白被认为更喜欢核小体无性区。此外，hsrω等lncRNA也参与了核小体重塑成分向染色质的募集。本提案的目的是研究核小体重构体和HSRωlncRNA在绝缘蛋白与染色质结合和染色质环路中的作用。这将通过检验以下假设来完成：ISWI依赖于HSRωlncRNA与染色质结合，并改变核小体的占有率，从而使绝缘蛋白可以结合。这一假说将通过以下目标进行验证：目的1：通过在核小体重构体(ISWI)和绝缘体蛋白基因敲除系中定位全基因组的核小体，确定ISWI在绝缘体功能中的作用。此外，ISWI对绝缘体蛋白结合染色质的依赖性，反之亦然，将通过在适当的敲除系中使用ISWI和绝缘体蛋白抗体进行一系列CHIP-SEQ实验来测试。目的：通过CHIRP-SEQ方法检测高铁ω基因的定位，确定高铁ω基因与绝缘子的关系。ω的S染色质相互作用对ω的依赖性也将以这种方式进行测试。我们还将对ω和绝缘蛋白进行芯片序列分析，以确定染色质结合对lncRNA的依赖性。目的：通过增强子阻断和HI-C分析来确定高铁ω对染色质组织的影响。建立的增强子阻断试验将用于测试高铁ω是否在与ISW相同的位点上表现出增强子阻断活性。高铁ω缺失系中的HI-C将用于检测高铁ω对全基因组染色质组织的影响。