LONG DISTANCE ENHANCER-PROMOTER INTERACTIONS

激活启动子的增强子在几个酶之外是至关重要的， 许多发育重要基因的表达。我们最近 在支持激活的因素的屏幕中发现CHIP， 果蝇cut基因的远程增强子 CHIP是一个无处不在的 在整个发育过程中调节不同基因的染色体蛋白质。 CHIP是胚胎分割所必需的，并增强激活 通过偶数跳过（EVE）对规则中的所有远程条带增强器 基因 我们的目标是了解CHIP如何调节Eve增强子。 CHIP是新发现的小鼠蛋白质的同源物， LIM结构域和同源结构域（HD）蛋白。 LIM域支持 不同的蛋白质-蛋白质相互作用。 我们假设CHIP调节 激活子和抑制子之间的相互作用， 增强剂。 CHIP还可以促进HD蛋白之间的相互作用 与分散在整个Eve基因中的位点结合， 结构上有助于增强子-启动子通讯。 增强子结合 与CHIP相互作用的蛋白质将通过体外鉴定， 相互作用测定。 BICOID HD蛋白，调节多种细胞因子， 增强子，在试验实验中与CHIP相互作用。 评价 由于相互作用的功能，体外所需的CHIP结构域将 与EVE体内表达所需的结构域进行比较。 转基因将用于探索eve控制区结构如何 影响增强剂对CHIP的依赖性。 这将有助于确定是否 CHIP在单个增强子中发挥作用，或者在细胞内起结构作用。 整个基因。 如果CHIP在单个增强子处起作用，则具有 将使用单个增强子来测试CHIP是否阻断阻遏或 刺激激活。 如果CHIP似乎发挥了结构性作用， 体内交联和染色质免疫沉淀将用于 探索CHIP像某些HD蛋白质一样， 几个位点分散在整个基因中。 的基础 CHIP与Hairy-wing抑制因子之间的体内拮抗作用 阻断增强子-启动子通讯的绝缘子蛋白（SUHW 将通过测试CHIP和SUHW是否有共同的相互作用来探索 伙伴 这些实验将有助于确定CHIP在以下方面的作用： 增强增强子活性，并增加了解如何远程 增强子调节转录。

Enhancers that activate promoters several kilobases away are vital for the expression of many developmentally important genes. We recently discovered CHIP in a screen for factors that support activation by a remote enhancer in the Drosophila cut gene. CHIP is a ubiquitous chromosomal protein that regulates diverse genes throughout development. CHIP is required for embryonic segmentation and potentiates activation by all the remote stripe enhancers in the even-skipped (eve) pair-rule gene. Our goal is to understand how CHIP regulates the eve enhancers. CHIP is a homologue of newly-discovered mouse proteins that interact with LIM domain and homeodomain (HD) proteins. LIM domains support diverse protein-protein interactions. We postulate that CHIP regulates interactions between activators and repressors that bind the eve enhancers. CHIP may also promote interactions between HD proteins binding to sites scattered throughout the entire eve gene to structurally aid enhancer-promoter communication. Enhancer-binding proteins that interact with CHIP will be identified by in vitro interaction assays. The BICOID HD protein, which regulates multiple eve enhancers, interacts with CHIP in trial experiments. To evaluate the functions of the interactions, the CHIP domains required in vitro will be compared to the domains required for eve expression in vivo. Transgenes will be used to explore how eve control region structure affects an enhancer's dependence on CHIP. This will help determine if CHIP plays roles at individual enhancers, or a structural role in the entire gene. If CHIP functions at individual enhancers, transgenes with single eve enhancers will be used to test if CHIP blocks repression or stimulates activation. If CHIP appears to play a structural role, in vivo crosslinking and chromatin immunoprecipitation will be used to explore the possibility that CHIP, like certain HD proteins, binds several sites dispersed throughout the entire gene. The basis for the in vivo antagonism between CHIP and the suppressor of Hairy-wing insulator protein (SUHW) that blocks enhancer-promoter communication will be explored by testing if CHIP and SUHW have common interaction partners. These experiments will help define the roles of CHIP in potentiating enhancer activity, and increase understanding of how remote enhancers regulate transcription.