Long Distance Enhancer-Promoter Interactions

描述(由申请人提供)：在后生动物发育过程中，转录激活几千碱基以外的启动子的增强子对许多基因的表达至关重要。这种芯片蛋白是在基因筛查中发现的，目的是寻找支持远距离增强子激活果蝇CuT基因的因素。同样在人类身上发现的芯片，有助于远程增强子激活包括EVE在内的许多果蝇基因。这项建议的主要目标是确定芯片如何促进EVE的激活。CHIP能与同源蛋白相互作用，增加体内的双核蛋白活性。主要的假设是，芯片增加了双核和其他同源蛋白的协同结合，以增强EVE。另一个重要的想法是，芯片还帮助同源蛋白结合在EVE增强子和启动子之间，从而促进增强子和启动子之间的通信。吉普赛转座子的插入可以阻止许多基因中的增强子和它们的目标启动子之间的通信，包括Eve和Cut。ChIP是切割位点上吉普赛人绝缘子的遗传靶点，并与吉普赛人、SU(HW)和Mod(MDG4)-67.2结合的蛋白质相互作用，这意味着ChIP也是绝缘子的分子靶点。这项拟议的工作有四个目标：(1)通过体外DNA结合实验和体内基因表达实验来确定ChIP如何促进双功能蛋白的活性；(2)利用染色质免疫沉淀实验来测试ChIP是否在体内直接与Eve相互作用，以及它在Eve中的结合位置；(3)使用体内定点重组实验来确定依赖于ChIP的远程Eve增强子是否在物理上接近Eve启动子；以及(4)通过体外蛋白质相互作用分析和体内遗传学实验，确定SU(HW)和Mod(MDG4)-67.2绝缘蛋白如何相互作用和芯片。这些研究将增加对远程基因激活的理解，并阐明一些人类遗传病的基本机制。

DESCRIPTION (provided by applicant): Enhancers that transcriptionally activate promoters from several kilobases away are vital for expression of many genes during metazoan development. The Chip protein was discovered in a genetic screen for factors that support activation of the Drosophila cut gene by a distant enhancer. Chip, also found in humans, facilitates activation of many Drosophila genes, including eve, by remote enhancers. The primary goal of this proposal is to determine how Chip facilitates activation of eve. Chip interacts with homeoproteins such as Bicoid and increases Bicoid activity in vivo. The principal hypothesis is that Chip increases cooperative binding of Bicoid and other homeoproteins to eve enhancers. Another important idea is that Chip also helps homeoproteins bind between the eve enhancers and promoter and thereby facilitates enhancer-promoter communication. Gypsy transposon insertions can block communication between enhancers and their target promoters in many genes, including eve and cut. Chip is a genetic target of the gypsy insulator at the cut locus, and interacts with proteins that bind gypsy, Su(Hw) and Mod(mdg4)-67.2, implying that Chip is also a molecular target of the insulator. The proposed work has four aims: (1) to determine how Chip promotes Bicoid activity using in vitro DNA-binding assays and in vivo gene expression experiments; (2) to test if Chip interacts directly with eve in vivo, and where in eve it binds using chromatin immunoprecipitation assays; (3) to determine if a remote eve enhancer that is Chip-dependent comes physically close to the eve promoter during activation using an in vivo site-specific recombination assay; and (4) to define how the Su(Hw) and Mod(mdg4)-67.2 insulator proteins interact with each other and Chip using in vitro protein interaction assays, and in vivo genetic experiments. These studies will increase understanding of long-range gene activation and illuminate fundamental mechanisms underlying some human genetic diseases.