ENGRAILED PARTNERS AND TRANSCRIPTIONAL REPRESSION

本项目研究了含有同源结构域的转录抑制因子Engraated(EN)和Even-Skiped(Eve)的功能。EN是如何在细胞核内定位其靶基因的？要做到这一点，它似乎与HOX蛋白辅因子牙外和同胸相互作用。这些辅因子中的每一个都被证明对EN在抑制果蝇胚胎中的一个直接靶基因的活动中起重要作用。该项目将确定体内这些功能相互作用的机制基础。识别目标基因sloppy配对中的直接结合位点将有助于研究DNA上的合作相互作用。为了实现其功能，EN已被证明与辅阻遏物相互作用，包括Groucho(Gro)。和EN一样，Eve既有Gro相互作用区，也有Gro非依赖性抑制域。为什么在这两个发育调节因子中都发现了两个具有不同功能特性的抑制域？构建了一种能够完全挽救Eve零突变体的转基因。由于EVE在胚胎发育的几个阶段中起着决定细胞命运的重要作用，并且靶基因已经被很好地描述，因此该转基因将被用于研究体内不同类别的阻遏结构域之间的功能差异。EN还与哪些其他伙伴蛋白相互作用以实现其功能？已鉴定出几种与EN的特定功能结构域相互作用的蛋白质。一种蛋白与EN中的第二类抑制域相互作用，遗传学研究表明这两种蛋白在体内具有密切的功能关系。EV还与哪些其他伙伴蛋白相互作用以实现其功能？已鉴定出几种与EN的特定功能结构域相互作用的蛋白质。一种蛋白与EV中的第二类阻遏结构域相互作用，遗传学研究表明，这两种蛋白在体内具有密切的功能关系。有趣的是，它是细胞周期调节蛋白E。这种关系可能被证明是转录调控和细胞周期之间的重要联系。对相互作用的分析将作为检验这一假设的模型。这个项目将利用遗传学、细胞培养和生化手段来研究这种相互作用和其他新的相互作用。对于那些代表新基因的相互作用的蛋白质，将识别和分析特定的突变对EN的靶基因以及一般发育的影响。因此，这些研究将有助于更好地理解同源结构域蛋白在发育和疾病中的调节机制。

This project investigates the function of the homeodomain-containing transcription repressors Engrailed (En) and Even-skipped (Eve). How does En locate its target genes within the nucleus? To do so, it appears to interact with the Hox protein cofactors Extradenticle and Homothorax. Each of these cofactors has been shown to be important for the activity of En in repressing a direct target gene in Drosophila embryos. The project will identify the mechanistic basis of these functional interactions in vivo. Studies of cooperative interactions on the DNA will be facilitated by the identification of direct binding sites in the target gene sloppy paired. In order to carry out its function, En has been shown to interact with corepressors, including Groucho (Gro). Like En, Eve has both a Gro interaction region and a Gro-independent repression domain. Why are two repression domains with different functional properties found in both of these developmental regulators? A transgene capable of completely rescuing eve null mutants has been constructed. Since Eve plays a major role in establishing cell fates during several stages of embryogenesis, and target genes have been well characterized, this transgene will be used to study functional distinctions among different classes of repression domain in vivo. What other partner proteins does En interact with to carry out its function? Several proteins have been identified that interact with specific functional domains of En. One interacts with the second class of repression domain in En, and genetic studies indicate that the two proteins have a close functional relationship in vivo. What other partner proteins does Ev interact with to carry out its function? Several proteins have been identified that interact with specific functional domains of En. One interacts with the second class of repression domain in Ev, and genetic studies indicate that the two proteins have a close functional relationship in vivo. Intriguingly, it is the cell cycle regulator cyclin E. This relationship may prove to be an important connection between regulation of transcription and the cell cycle. Analysis of the interaction will serve as a model for testing this hypothesis. This project will utilize genetic, cell culture, and biochemical means to study this and other novel interactions. For those interacting proteins that represent new genes, specific mutations will be identified and analyzed for their effects on the target genes of En, as well as on development generally. These studies will thus lead to a greater understanding of the regulatory mechanisms of homeodomain protein function in development and disease.