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

格劳乔的远程镇压机制：中央域的作用

• 批准号: 7546312
• 负责人: Wiam M Turki-Judeh
• 金额: $ 3万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-01 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7546312
• 关键词: Acetylation; Adult; Automobile Driving; Binding; Biochemical; Biological Assay; Bulla; C-terminal; Cell Fractionation; Cell Nucleus; Cells; Cytoplasm; Defect; Dependence; Development; Dorsal; Drosophila Proteins; Drosophila genus; Drug Delivery Systems; Embryo; Event; Family member; Gene Targeting; Genetic Transcription; Genomics; Glutamine; Heat-Shock Response; Immunofluorescence Immunologic; Investigation; LacZ Genes; Lead; Localized; Mediating; Molecular; Mutation; N-terminal; Nuclear; Nuclear Localization Signal; Nuclear Protein; Nuclear Proteins; Pattern; Personal Satisfaction; Phenotype; Play; Point Mutation; Protein Overexpression; Proteins; Range; Recruitment Activity; Regulation; Reporter; Reporter Genes; Repression; Role; Score; Series; Severities; Signal Pathway; Site; Staging; Structure; Tertiary Protein Structure; Testing; Thinking; Transgenic Organisms; Variant; Veins; WD Repeat; Wing; base; blind; cancer therapy; cancer type; chromatin immunoprecipitation; design; fly; gene repression; human HDAC1 protein; improved; insight; loss of function; mutant; promoter; research study; tumor; tumorigenesis

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.

描述（由申请人提供）：本提案的目的是通过研究果蝇蛋白Groucho（Gro）阐明与转录抑制相关的分子和生物化学事件。Gro是一种在后生动物的发育和信号通路中起重要作用的长距离辅阻遏物.它通过与许多DMA结合阻遏物（包括Brinker、Hairy和Dorsal）相互作用被募集到靶向启动子。Gro含有保守的N-末端富含谷氨酰胺（Q）和C-末端WD-重复结构域，这些结构域对于Gro介导的抑制是必需的。尽管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）尚未得到很好的表征。该项目旨在阐明的贡献，通过生成和分析多个Gro缺失缺乏一个或多个这些域的Gro介导的镇压的中央域。该提案有四个具体目标。首先，为了确定哪些结构域是阻遏所需的，将测定缺失变体在翼盘中错误表达时驱动Brinker靶标的异位阻遏的能力。其次，为了确定结构域在靶向ofGro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的距离依赖性的贡献，将使用一系列报告基因评估缺失变体，其中Gro募集位点和转录起始位点之间的距离已经系统地变化。除了检查靶基因活性的水平和模式外，还将通过染色质免疫沉淀（ChIP）试验评估靶基因的状态。Gro表达水平的增加与特定类型的癌症有关。因此，了解参与Gro抑制的因素可能有助于了解导致肿瘤形成的事件。