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.
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