Mechanism of long-range repression by Groucho: role of the central domains

描述（由申请人提供）：本提案的目的是通过对果蝇蛋白Groucho （Gro）的研究来阐明与转录抑制相关的分子和生化事件。Gro是一种远程协同抑制因子，在后生动物的发育和信号通路中起着许多重要作用。它通过与许多dma结合抑制因子（包括Brinker、Hairy和Dorsal）的相互作用而被招募到靶向启动子。Gro含有保守的n端富含谷氨酰胺(Q)和c端WD-repeat结构域，这是Gro介导的抑制所必需的。虽然Q和WD重复结构域的功能已被广泛研究，但该蛋白的中心区域（包括GP、CcN和SP结构域）尚未被很好地表征。该项目旨在通过生成和分析缺乏一个或多个这些结构域的多个Gro缺失来阐明中心结构域对Gro介导的抑制的贡献。该提案有四个具体目标。首先，为了确定哪些结构域是抑制所必需的，将分析删除变体在翼盘错误表达时驱动Brinker靶点异位抑制的能力。其次，为了确定这些结构域在gro靶向中的作用，将采用免疫荧光研究来检查每个缺失变体的亚细胞定位。第三，为了确定结构域在正常发育中的作用，将确定每个缺失变体能够挽救Gro功能丧失表型的程度。第四，为了确定每个结构域对抑制距离依赖性的贡献，将使用一系列报告器评估删除变体，其中Gro招募位点和转录起始位点之间的距离已经系统地改变。除了检测靶基因活性的水平和模式外，靶基因的状态将通过染色质免疫沉淀（ChIP）测定来评估。Gro表达水平的增加与特定类型的癌症有关。因此，了解Gro抑制的相关因素可能有助于深入了解导致肿瘤形成的事件。

DESCRIPTION (provided by applicant): The objective of this proposal is to elucidate the molecular and biochemical events associated with transcriptional repression through an investigation of the Drosophila protein Groucho (Gro). Gro is a long- range corepressor that plays many essential roles in metazoan development and signaling pathways. It is recruited to target promoters via interactions with numerous DMA-binding repressers including Brinker, Hairy, and Dorsal. Gro contains conserved N-terminal glutamine-rich (Q) and C-terminal WD-repeat domains that are essential for Gro-mediated repression. Although the functions of the Q and WD repeat domains have been studied extensively, the central region (which includes the GP, CcN,and SP domains) of the protein has not been well characterized. This project seeks to elucidate the contributions of the central domains to Gro-mediated repression by generating and analyzing multiple Gro deletions lacking one or more of the these domains. The proposal has four specific aims. First, to determine which domains are required for repression, the deletion variants will be assayed for their ability to drive ectopic repression of Brinker targets when misexpressed in the wing disc. Second, to determine the roles of the domains in the targeting ofGro, immunofluoresence studies will be employed to examine the subcellular localization of each deletion variant. Third, to determine the roles of the domains in normal development, the extent to which each deletion variant is able to rescue the Gro loss-of-function phenotype will be determined. Fourth, to determine the contribution of each domain to the distance dependence of repression, the deletion variants will be assessed using a series of reporters in which the distance between the site of Gro recruitment and the transcriptional start site has been systematically varied. In addition to examining levels and patterns of target gene activity, the state of the target genes will be assessed by chromatin immunoprecipitation (ChIP) assays. Increased expression levels of Gro have been associated with specific types of cancer. Therefore, understanding factors involved in repression by Gro may provide insight into the events leading to tumor formation.