Long Distance Enhancer-Promoter Interactions

描述（由申请人提供）：在后生动物发育过程中，转录激活数千个碱基外启动子的增强子对许多基因的表达至关重要。Chip蛋白是在一项基因筛选中发现的，该基因筛选的因素支持通过远端增强子激活果蝇切割基因。在人类中也发现了Chip，它通过远程增强子促进了包括eve在内的许多果蝇基因的激活。本方案的主要目标是确定Chip如何促进eve的激活。Chip与同型蛋白如Bicoid相互作用，增加Bicoid在体内的活性。主要的假设是Chip增加了Bicoid和其他同源蛋白与eve增强子的合作结合。另一个重要的想法是，Chip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-启动子之间的通信。吉普赛转座子插入可以阻断许多基因中增强子与其靶启动子之间的通信，包括eve和cut。Chip是gypsy绝缘子切割位点的遗传靶点，与结合gypsy、Su（Hw）和Mod(mdg4)-67.2的蛋白相互作用，表明Chip也是绝缘子的分子靶点。这项工作有四个目的：(1)通过体外dna结合试验和体内基因表达实验来确定Chip如何促进Bicoid活性；(2)利用染色质免疫沉淀法检测Chip在体内是否与eve直接相互作用，以及在eve中与之结合的位置；(3)使用体内位点特异性重组试验确定依赖芯片的远程eve增强子在激活过程中是否在物理上接近eve启动子；(4)通过体外蛋白相互作用分析和体内遗传实验，确定Su（Hw）和Mod(mdg4)-67.2绝缘子蛋白如何相互作用和Chip。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疾病的基本机制。

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.