Long Distance Enhancer-Promoter Interactions

描述（由申请人提供）：转录激活启动子的增强子来自几个启动子酶，在后生动物发育过程中对许多基因的表达至关重要。Chip蛋白是在一次基因筛选中发现的，该筛选旨在寻找支持通过远距离增强子激活果蝇cut基因的因子。在人类中也发现了芯片，通过远程增强子促进许多果蝇基因的激活，包括eve。这项提案的主要目标是确定芯片如何促进夏娃的激活。Chip与同源异型蛋白如Bicoid相互作用并增加体内Bicoid活性。主要假设是Chip增加了Bicoid和其他同源异型蛋白与Eve增强子的协同结合。另一个重要的观点是，Chip还帮助同源异型蛋白在增强子和启动子之间结合，从而促进增强子-启动子的通讯。Gypsy转座子插入可以阻断许多基因中增强子和它们的靶启动子之间的通讯，包括eve和cut。Chip是gypsy绝缘子在切割位点的遗传靶点，并与结合gypsy的蛋白质Su（Hw）和Mod（mdg 4）-67.2相互作用，这意味着Chip也是绝缘子的分子靶点。本研究的目的有四个：（1）通过体外DNA结合实验和体内基因表达实验确定Chip如何促进Bicoid活性;（2）通过染色质免疫沉淀实验检测Chip是否与eve在体内直接相互作用，以及在eve中与何处结合;（3）使用体内位点特异性重组测定来确定在激活期间Chip依赖性的远程eve增强子是否在物理上接近eve启动子;和（4）使用体外蛋白质相互作用测定和体内遗传实验来确定Su（Hw）和Mod（mdg 4）-67.2绝缘子蛋白如何彼此相互作用和Chip相互作用。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.