LONG DISTANCE ENHANCER-PROMOTER INTERACTIONS

在几千碱基之外激活启动子的增强剂对 许多发育重要基因的表达。我们最近 在屏幕中发现芯片，以寻找支持由 果蝇CuT基因中的远程增强子。芯片是无处不在的 在整个发育过程中调节不同基因的染色体蛋白。 芯片是胚胎分割所必需的，并增强了激活 通过偶数跳过(EVE)对规则中的所有远程条带增强器 吉恩。我们的目标是了解CHIP如何调控EVE增强子。 芯片是新发现的相互作用的小鼠蛋白质的同源物 含有LIM结构域和同源结构域(HD)蛋白。LIM域支持 不同的蛋白质之间的相互作用。我们假设芯片可以调节 结合夏娃的激活物和抑制物之间的相互作用 增强剂。芯片也可能促进HD蛋白之间的相互作用 结合到散布在整个Eve基因上的位点 在结构上有助于增强启动子之间的沟通。增强子结合 与芯片相互作用的蛋白质将通过体外鉴定 相互作用分析。调节多个EVE的双核HD蛋白 增强剂，在试验实验中与芯片相互作用。要评估 相互作用的功能，体外所需的芯片结构域将 与EVE在体内表达所需的结构域进行比较。 转基因将被用来探索Eve如何控制区域结构 影响增强剂对芯片的依赖。这将有助于确定是否 芯片在单个增强器中扮演角色，或者在 整个基因。如果芯片在单个增强子上发挥作用，转基因与 单个EVE增强器将用于测试芯片是否阻止抑制或 刺激激活。如果芯片似乎扮演着结构性的角色，那么在 体内交联和染色质免疫沉淀将用于 探索CHIP与某些HD蛋白结合的可能性 有几个位置分散在整个基因中。的基础是 CHIP与毛翅抑制因子的体内拮抗作用 阻断增强子-启动子通信的绝缘蛋白(SUHW) 将通过测试芯片和SUHW是否具有共同的交互来进行探索 合伙人。这些实验将有助于确定芯片在 增强增强剂活性，并增加对远程 增强剂调节转录。

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.