LONG DISTANCE ENHANCER-PROMOTER INTERACTIONS

长距离增强子-启动子相互作用

• 批准号: 6180708
• 负责人: Dale L Dorsett
• 金额: $ 9.88万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-07-01 至 2000-09-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6180708
• 关键词: DNA binding protein; Drosophilidae; crosslink; developmental genetics; enhancer binding protein; gene deletion mutation; genetic enhancer element; genetic promoter element; genetic regulation; immunoprecipitation; laboratory rabbit; protein structure function; transfection; yeast two hybrid system

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.

激活启动子几千碱基的增强剂对 许多发展重要基因的表达。我们最近 在屏幕中发现了支持激活的因素 果蝇切割基因中的远程增强子。 芯片无处不在 整个发育过程中调节多种基因的染色体蛋白。 芯片是胚胎分割所必需的，并增强激活 通过均匀搭配（EVE）的所有远程条纹增强器 基因。 我们的目标是了解芯片如何调节夏娃增强器。 芯片是相互作用的新发现的小鼠蛋白的同源物 具有LIM结构域和同源域（HD）蛋白。 LIM域支持 多种蛋白质蛋白质相互作用。 我们假设芯片调节 激活因子与绑定前夕的阻遏物之间的相互作用 增强剂。 芯片也可能促进高清蛋白之间的相互作用 与散布在整个夏娃基因的位点结合到 在结构上有助于增强器促销。 增强器结合 与CHIP相互作用的蛋白质将通过体外鉴定 互动测定。 双聚体HD蛋白，该蛋白调节多个夏娃 在试验实验中，增强剂与CHIP相互作用。 评估 相互作用的功能，体外所需的芯片域将会 与体内夏娃表达所需的域进行比较。 转基因将用于探索夏娃控制区域的结构 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器上起作用，则带有 单一夏娃增强器将用于测试芯片阻止或 刺激激活。 如果芯片似乎起着结构性的作用 体内交联和染色质免疫沉淀将用于 探索像某些HD蛋白一样结合芯片的可能性 几个站点分散在整个基因中。 的基础 芯片与毛翼抑制剂之间的体内对抗 绝缘蛋白（SUHW）阻止增强剂促销通信 将通过测试芯片和SUHW是否具有共同的相互作用来探索 合作伙伴。 这些实验将有助于定义芯片的作用 增强增强剂活动，并增加对远程的理解 增强剂调节转录。