Repressor/co-repressor oligomerization and development

阻遏物/共阻遏物寡聚和发育

• 批准号: 7590269
• 负责人: ALBERT J COUREY
• 金额: $ 36.06万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-08-07 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7590269
• 关键词: Address; Affinity; Alleles; Binding; Biological Assay; Cell Nucleus; Cells; Chromatin; Chromosomal translocation; Cultured Cells; DHFR gene; DNA; DNA Binding; DNA Binding Domain; Defect; Development; Dihydrofolate Reductase; Dorsal; Drosophila genus; Ectoderm; Ectopic Expression; Embryo; Escherichia coli; Filament; Gene Targeting; Genomics; Head; Homo; Human; Human Development; Investigation; Length; Life; Link; Lung Adenocarcinoma; Malignant Neoplasms; Mediating; Mitogen-Activated Protein Kinases; Mitotic; Mutation; N-terminal; Nucleosomes; Orthologous Gene; Pathway interactions; Pattern; Phosphorylation; Play; Point Mutation; Polymers; Progress Reports; Proteins; Public Health; Range; Receptor Protein-Tyrosine Kinases; Repression; Research; Resistance; Resolution; Role; SAM Domain; Signal Transduction; Structure; System; Tail; Testing; Transcription Repressor/Corepressor; Transcriptional Activation; Up-Regulation; Wing; base; chromatin immunoprecipitation; depolymerization; derepression; design; dimer; embryonic cell culture; extracellular; fly; gene repression; human disease; in vivo; leukemia; morphogens; mutant; particle; polymerization; prevent; response; size

DESCRIPTION (provided by applicant): Transcriptional repressors and corepressors play key roles in establishing systems of positional information and in mediating responses to extracellular signals during development. Many of these factors organize large chromosomal domains into transcriptionally silent states. In some cases, these states are self-propagating and survive many mitotic cycles, while in other cases, silencing is short lived and readily reversed. Silent domains are nucleated by cis-regulatory modules in the DNA and may spread from these modules in a manner that is dependent upon repressor or corepressor oligomerization. However, the connections between repressor/corepressor oligomerization, spreading, and silencing are not well understood. The research proposed here will explore the mechanisms of repression by two Drosophila regulatory factors, Groucho (Gro) and Yan, with well-established oligomerization functions. Specific aim 1 is to determine if Gro spreading is required for repression. Gro is a corepressor with multiple essential roles in embryonic and imaginal development - its human orthologs have equally important roles in human development. It contains a highly conserved oligomerization domain termed the Q domain. Mutations in this domain that prevent oligomerization prevent repression. Furthermore, chromatin immunoprecipitation assays in cultured cells and wing discs demonstrate that Gro repression targets are associated with large Gro-bound domains many kilobases in length. This proposal seeks to establish firm links between Gro oligomerization, spreading, and repression. Gro rescue constructs with specific defects in oligomerization will be developed, and fly lines carrying these mutant alleles will be subjected to phenotypic and chromatin immunoprecipitation analysis to determine the structure and function of Gro-bound domains. Specific aim 2 is to determine the role of Yan oligomerization in repression and Ras signaling. Yan and it human ortholog Tel are DNA binding transcriptional repressors with essential roles in Ras signaling. Ras activation leads to the phosphorylation of Yan by MAP kinase and the subsequent export of Yan from the nucleus resulting in derepression of Ras pathway target genes. Yan contains a SAM domain, which forms a head to tail protein polymer that is required for repression in S2 cells and also appears to control reception of the Ras signal. New alleles of yan that encode proteins with increased oligomerization affinity will be developed and tested in both cultured cells and the embryo to determine the mechanistic connections between Yan polymerization, repression, and Ras signaling. PUBLIC HEALTH RELEVANCE: The two factors under investigation here, Gro and Yan, each have human orthologs with many connections to human cancer. For example, Tel, the human ortholog of Yan, is a frequent target of chromosomal translocations in leukemia, while up-regulation of mammalian Gro has been linked to lung adenocarcinomas. Therefore, the mechanisms to be uncovered in this research will be relevant to human disease.

描述（由申请人提供）：转录阻遏物和辅阻遏物在建立位置信息系统和介导发育过程中对细胞外信号的反应中起关键作用。这些因素中的许多将大的染色体结构域组织成转录沉默状态。在某些情况下，这些状态是自我繁殖的，并在许多有丝分裂周期中存活，而在其他情况下，沉默是短暂的，并容易逆转。沉默结构域由DNA中的顺式调节模块成核，并且可以以依赖于阻遏物或辅阻遏物寡聚化的方式从这些模块扩散。然而，阻遏物/辅阻遏物寡聚化，扩散和沉默之间的联系还没有很好地理解。本研究将探讨果蝇调控因子Groucho（Gro）和Yan的抑制机制，这两个因子具有成熟的寡聚化功能。具体目标1是确定是否需要抑制Gro扩散。Gro是一种辅阻遏物，在胚胎和成虫发育中具有多种重要作用-其人类直系同源物在人类发育中具有同样重要的作用。它含有一个高度保守的寡聚化结构域，称为Q结构域。该结构域中阻止寡聚化的突变阻止阻遏。此外，在培养的细胞和翼盘染色质免疫沉淀试验表明，Gro阻遏目标与大的Gro结合域长度的许多酶。该建议旨在建立Gro寡聚化，扩散和抑制之间的紧密联系。将开发在寡聚化中具有特定缺陷的Gro拯救构建体，并且将对携带这些突变等位基因的果蝇品系进行表型和染色质免疫沉淀分析以确定Gro结合结构域的结构和功能。具体目标2是确定Yan寡聚化在阻遏和Ras信号传导中的作用。Yan及其人类直系同源物Tel是DNA结合转录抑制因子，在Ras信号传导中具有重要作用。Ras激活导致MAP激酶对Yan的磷酸化，随后Yan从细胞核输出，导致Ras途径靶基因的去阻遏。Yan包含SAM结构域，其形成在S2细胞中抑制所需的头到尾蛋白聚合物，并且似乎还控制Ras信号的接收。将在培养的细胞和胚胎中开发和测试编码具有增加的寡聚化亲和力的蛋白质的新的Yan等位基因，以确定Yan聚合、阻遏和Ras信号传导之间的机械联系。公共卫生相关性：这里调查的两个因素，Gro和Yan，每个都有与人类癌症有许多联系的人类直系同源物。例如，Tel，Yan的人类直系同源物，是白血病中染色体易位的常见靶标，而哺乳动物Gro的上调与肺腺癌有关。因此，这项研究中发现的机制将与人类疾病相关。