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发生素如何启动导致动物发育过程中轴规范的复杂事件层次。所研究的形态发生素 Dorsal 是一种转录因子，分布在果蝇胚盘胚胎的背腹侧浓度梯度中。该因子既充当转录激活子又充当转录抑制子，以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阻遏物 Groucho。该提案的第一个具体目标是通过表征最近在背侧系统诱变中发现的两组突变来阐明背侧介导的激活机制。这两类突变定义了 RHD 上的两个不同表面。将采用亲和层析来鉴定介导由 II 类突变定义的表面功能的新型共激活剂。将采用遗传和生化方法来检验由 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镇压的机制。先前的研究提出了一种关于 Groucho 介导的远程抑制的假设，其中包括 Dorsal 招募 Groucho，随后 Groucho 招募组蛋白脱乙酰酶，最后沿着脱乙酰化模板传播 Groucho。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。 果蝇背腹模式的调节途径与脊椎动物免疫反应和脊椎动物模式形成的调节途径是同源的。因此，这些研究可能有助于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.