Dorsoventral Patterning Through Transcriptional Control

描述（由申请方提供）：拟定研究旨在了解动物发育期间单个形态原如何启动导致轴规格的复杂事件层级。研究中的形态原Dorsal是一种转录因子，在果蝇胚盘胚胎中呈背腹浓度梯度分布。该因子作为转录的激活因子和抑制因子发挥作用，以沿背腹轴沿着建立多个基因活性域。Dorsal的多种功能是由Dorsal rel同源结构域（RHD）、Dorsal C末端结构域（CTD）和许多其他调节因子之间的相互作用网络介导的。一种Dorsal相互作用蛋白是Dorsal介导的阻遏所必需的，它是辅阻遏物Groucho。本提案的第一个具体目的是阐明Dorsal介导的激活机制，通过表征在最近的Dorsal系统诱变中发现的两组突变。这两类突变在RHD上定义了两个不同的表面。亲和色谱法将用于鉴定介导由II类突变定义的表面功能的新型共活化剂。将采用遗传和生物化学方法来检验由I类突变定义的表面调节CTD活性的假设。第二个具体目标是确定格劳乔的抑制机制。以前的研究已经导致了一个假设，格劳乔介导的远程抑制，涉及招聘格劳乔Dorsal，随后招聘的组蛋白脱乙酰酶的格劳乔，最后格劳乔沿着的脱乙酰化模板的蔓延。遗传分析，结构研究和染色质免疫沉淀实验将结合起来，严格检验这一假设。 调节果蝇背腹图案形成的途径与调节脊椎动物免疫应答和脊椎动物图案形成的途径是同源的。因此，这些研究可能会导致更好地了解人类免疫和发育障碍。

DESCRIPTION (provided by applicant): The proposed studies are aimed at understanding how a single morphogen initiates the complex hierarchy of events leading to axis specification during animal development. The morphogen under investigation, Dorsal, is a transcription factor that is distributed in a dorsoventral concentration gradient in the Drosophila blastoderm embryo. This factor functions as both an activator and a repressor of transcription to establish multiple domains of gene activity along the dorsoventral axis. The diverse functions of Dorsal are mediated by a network of interactions between the Dorsal rel homology domain (RHD), the Dorsal Cterminal domain (CTD), and many additional regulatory factors. One Dorsal-interacting protein that is essential for Dorsal-mediated repression is the corepressor Groucho. The first specific aim of this proposal is to illuminate the mechanism of Dorsal-mediated activation by characterizing two sets of mutations discovered in a recent systematic mutagensis of Dorsal. These two classes of mutations define two distinct surfaces on the RHD. Affinity chromatography will be employed to identify novel coactivators that mediate the function of the surface defined by the class II mutations. Both genetic and biochemical approaches will be employed to test the hypothesis that the surface defined by the class I mutations modulates the activity of the CTD. The second specific aim is to determine the mechanism of repression by Groucho. Previous studies have led to a hypothesis for Groucho-mediated long-range repression that involves the recruitment of Groucho by Dorsal, the subsequent recruitment of a histone deacetylase by Groucho, and finally the spreading of Groucho along the deacetylated template. Genetic analysis, structural studies, and chromatin immunoprecipitation experiments will be combined to test this hypothesis rigorously. The pathway regulating dorsoventral patterning in Drosophila is homologous to pathways regulating the vertebrate immune response and vertebrate pattern formation. Thus, these studies may lead to a better understanding of human immunological and developmental disorders.