Dorsoventral Patterning Through Transcriptional Control

描述（由申请人提供）：拟议的研究旨在了解单个形态因子如何在动物发育过程中启动导致轴规范的复杂事件层次。被研究的形态原Dorsal是一种转录因子，在果蝇囊胚中以背腹侧浓度梯度分布。该因子作为转录的激活因子和抑制因子，沿背腹轴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 Cterminal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。一种对背侧介导的抑制至关重要的背侧相互作用蛋白是辅抑制蛋白Groucho。本提案的第一个具体目的是通过描述最近在背侧系统性突变中发现的两组突变来阐明背侧介导的激活机制。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II类突变定义的表面功能的新型共激活因子。遗传学和生物化学方法将被用来检验由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压抑机制。先前的研究提出了Groucho介导的远程抑制假说，其中包括Groucho通过Dorsal募集，Groucho随后募集组蛋白去乙酰化酶，最后Groucho沿着去乙酰化模板扩散。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。

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.