The insulator complex in higher-order chromatin organization

描述（由申请人提供）：我们的遗传信息包含在DNA中，细胞核内DNA的正确组织对我们的发育和个体细胞健康至关重要。细胞核内DNA的紊乱可导致对细胞周期调控至关重要的基因的异常表达，并最终可促进细胞转移。这种组织需要从核小体核心开始的不同程度的包装。然后多个核进一步凝聚形成染色质纤维。核内染色质纤维的适当组织对于真核基因表达是必不可少的，但是核内包装染色质的机制还不清楚。数据表明染色质纤维的组织和区室化成更高级的染色质结构域可能涉及绝缘子元件。这些绝缘子元件和它们的结合蛋白已被证明聚集在细胞核中形成绝缘体。绝缘子元件聚集成绝缘子体可以是组织染色质的一种手段。通过这种方式，绝缘子可以提供一种策略，细胞通过该策略控制独立转录结构域的建立和维持。将染色质组织成环状可能是使某些DNA序列更容易被转录机制所接近的一种方式，本质上决定了哪些基因可以被打开和关闭。该提案旨在更好地定义绝缘子组件调节高阶染色质组织的机制。为此，我将对一种新发现的调节绝缘子复合物的蛋白质进行详细分析，并确定参与绝缘子体形成的其他蛋白质。我已经确定拓扑II作为一个有效的调节绝缘子复合物和高阶染色质结构。拓扑结构II的特点和它的遗传和生物化学基础与其他绝缘子组件的相互作用将使用凝胶位移，共-IP和酵母双杂交检测。此外，将通过在单独的试验中抑制Topo II的酶和支架功能，单独测试其在绝缘体形成中的作用。最后，将进行基于RNAi细胞的筛选以鉴定参与绝缘体形成和调节的新蛋白。从这些研究中，我希望能够深入了解绝缘子和辅助蛋白建立和调节高阶染色质结构域的精确机制，以及这些结构域如何影响转录控制和细胞功能。

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.