Functional Specificity of Drosophila homeoprotein DNA binding complexes

描述（由申请人提供）：发育过程涉及编码DMA结合蛋白的基因组之间的调控级联，DMA结合蛋白指导所有生命阶段正常功能所需的全部细胞行为。关于DMA结合蛋白保守家族成员如何协同作用以协调基因表达模式，仍有许多有待了解。该项目将研究这些家族中最高度保守的家族之一，同源结构域蛋白，已知其参与发育异常和癌症/白血病进展，以及其他重要的疾病过程。这些研究的广泛，长期目标是了解DMA结合蛋白如何相互作用及其在基因组中的靶位点，以实现发育过程中的功能特异性。Engrailed同源结构域蛋白与同一DNA结合超家族的其他几个成员相互作用，以帮助它找到并调节适当的靶基因。该建议是使用体外DNA结合分析和果蝇中可用的遗传和转基因方法的组合来研究潜在的机制，包括报告转基因、产生异位蛋白表达的转基因和模拟内源性表达模式的拯救转基因，每种转基因用于野生型和突变遗传背景。具体目标是：1）通过体外结合研究和体内转基因分析的组合，鉴定和剖析直接靶基因中鉴定的靶增强子内的Engrailed的结合位点;和2）剖析Engrailed与那些靶位点上的辅因子合作所必需的蛋白结构域，并测试那些结构域对于体内功能的要求。公共卫生相关性：细胞过程机制的基础知识已被证明对于开发真正新颖的方法来诊断和干预各种疾病过程至关重要，特别是在癌症发展中，随着时间的推移积累的遗传变化现在被理解为导致导致癌症的异常细胞行为。这些研究将通过研究从果蝇到人类高度保守的相互作用的DNA结合蛋白家族来扩展我们对基因如何调节其他基因的理解。

DESCRIPTION (provided by applicant): Developmental processes involve regulatory cascades among groups of genes that encode DMA binding proteins, which direct the full range of cellular behaviors required for normal function at all life stages. Much remains to be learned about how members of conserved families of DMA binding proteins act in concert to orchestrate patterns of gene expression. This project will investigate one of the most highly conserved of these families, the homeodomain proteins, which are known to be involved in developmental abnormalities and cancer/leukemia progression, among other important disease processes. The broad, long-term goal of these studies is to understand how DMA binding proteins interact with each other and their target sites in the genome to achieve functional specificity during development. The Engrailed homeodomain protein interacts with several other members of the same DNA binding superfamily to help it find and regulate the appropriate target genes. This proposal is to investigate underlying mechanisms using a combination of in vitro DNA binding analysis and the genetic and transgenic methods available in Drosophila, including reporter transgenes, transgenes that generate ectopic protein expression, and rescuing transgenes that mimic the endogenous pattern of expression, each used in both wildtype and mutant genetic backgrounds. The specific aims are: 1) to identify and dissect binding sites for Engrailed within an identified target enhancer in the direct target gene sloppy-paired through a combination of in vitro binding studies and in vivo transgenic analysis; and 2) to dissect the protein domains necessary for Engrailed to cooperate with its cofactors on those target sites, and test the requirements for those domains for in vivo function. PUBLIC HEALTH RELEVANCE: Fundamental knowledge of mechanisms that underlie cellular processes has proven to be essential for developing truly novel approaches to diagnosis and intervention in disease processes of all kinds, most notably in cancer development, where genetic changes accumulating over time are now understood to result in the abnormal cellular behaviors that lead to cancer. These studies will extend our understanding of how genes regulate other genes by studying a family of interacting DNA binding proteins that are highly conserved from Drosophila to humans.