The insulator complex in higher-order chromatin organization

高阶染色质组织中的绝缘体复合体

• 批准号: 8133813
• 负责人: Edward Ramos
• 金额: $ 13.41万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-12 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8133813
• 关键词: Affect; Beryllium; Binding Proteins; Biochemical; Biological Assay; Biology; Carcinoma; Cell Cycle Regulation; Cell Nucleus; Cell physiology; Cells; Cessation of life; Chromatin; Chromatin Fiber; Communication; Communities; Complex; DNA; DNA Sequence; Data; Development; Disease; Doctor of Philosophy; Drosophila genus; Elements; Eukaryotic Cell; Event; Faculty; Fiber; Gel; Gene Expression; Genes; Genetic; Gypsies; Health; Higher Order Chromatin Structure; Human; Individual; Insulator Elements; Lead; Letters; Loss of Heterozygosity; Maintenance; Mutate; Neoplasm Metastasis; Nucleosome Core Particle; Nucleosomes; Organism; Postdoctoral Fellow; Principal Investigator; Proteins; RNA Interference; Regulation; Research; Role; Testing; Topoisomerase II; Transcriptional Regulation; Vertebrates; Yeasts; base; career development; cell growth; genetic analysis; graduate student; induced pluripotent stem cell; insight; member; mutant; novel; null mutation; prevent; professor; programs; promoter; research study; scaffold; yeast two hybrid system

DESCRIPTION (provided by applicant): Our genetic information is contained on DNA and the proper organization of this DNA within the nucleus is important for our development and individual cell health. Disorder of DNA within the nucleus can lead to aberrant expression of genes critical to cell cycle regulation and ultimately can facilitate cell metastasis. This organization requires varying degrees of packaging beginning with the nucleosome core. Multiple cores are then further condensed to form chromatin fibers. The proper organization of the chromatin fibers within the nucleus is essential for eukaryotic gene expression but the mechanism for packaging the chromatin within the nucleus is not well understood. Data suggests the organization and compartmentalization of chromatin fibers into higher-order chromatin domains can involve insulator elements. These insulator elements and their binding proteins have been shown to cluster in the nucleus to form insulator bodies. Clustering of insulator elements into insulator bodies can be a means of organizing chromatin. In this way, insulators may provide a strategy by which cells control the establishment and maintenance of independent transcriptional domains. Organizing the chromatin into loops may be a way of making certain DNA sequences more accessible to transcriptional machinery and in essence dictating which genes can be turned on and off. This proposal seeks to better define the mechanism by which insulator components regulate higher-order chromatin organization. To this end, I will conduct a detailed analysis of one newly identified protein that regulates insulator complexes as well as identify additional proteins involved in insulator body formation. I have identified Topo II as a potent regulator of insulator complexes and higher-order chromatin structure. Topo II will be characterized and its genetic and biochemical basis for interaction with other insulator components will be assayed using gel-shift, co-IPs, and yeast two-hybrids. In addition, the enzymatic and scaffolding functions of Topo II will be individually tested for their role in insulator body formation by inhibiting each function in separate assays. Finally, an RNAi cell based screen will be conducted to identify novel proteins involved in insulator body formation and regulation. From these studies I hope to gain insight as to the precise mechanism by which insulators and accessory proteins establish and regulate higher-order chromatin domains and how these domains affect transcriptional control and cell function.

描述（由申请人提供）：我们的遗传信息包含在DNA上，该核内该DNA的适当组织对我们的发育和个体细胞健康很重要。细胞核内的DNA障碍会导致对细胞周期调节至关重要的基因的异常表达，并最终可以促进细胞转移。该组织需要从核小体核心开始的不同程度的包装。然后将多个核进一步凝结以形成染色质纤维。核中染色质纤维的正确组织对于真核基因表达至关重要，但是将染色质包装在核中的机制尚不清楚。数据表明，将染色质纤维的组织和分室化为高阶染色质结构域可能涉及绝缘体元件。这些绝缘子元素及其结合蛋白已被证明在核中聚集以形成绝缘体。绝缘子元件将其聚集到绝缘体中可以是组织染色质的一种手段。这样，绝缘子可以提供一种策略，通过该策略控制独立转录域的建立和维护。将染色质组织成环可能是使某些DNA序列更容易被转录机械访问的方式，从本质上讲，可以打开和关闭哪些基因。该建议旨在更好地定义绝缘体组件调节高阶染色质组织的机制。为此，我将对一种新鉴定的蛋白质进行详细分析，该蛋白质调节绝缘子复合物，并确定与绝缘体形成有关的其他蛋白质。我已经确定Topo II是绝缘子复合物和高阶染色质结构的有效调节剂。 TOPO II将被表征，其与其他绝缘体组件相互作用的遗传和生化基础将使用凝胶移位，Co-ips和酵母两杂化物分析。此外，Topo II的酶促和脚手架功能将通过在单独的测定中抑制每个功能来单独测试它们在绝缘体体形中的作用。最后，将进行基于RNAi细胞的筛选，以鉴定与绝缘体体形和调节有关的新型蛋白质。从这些研究中，我希望对绝缘剂和辅助蛋白建立和调节高阶染色质结构域以及这些结构域如何影响转录控制和细胞功能的确切机制获得洞察力。