Repressor/co-repressor oligomerization and development

阻遏物/共阻遏物寡聚化和发育

• 批准号: 7988997
• 负责人: ALBERT J COUREY
• 金额: $ 10万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-12-17 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7988997
• 关键词: Address; Affinity; Alleles; Binding; Biological Assay; Cell Culture Techniques; Cell Nucleus; Cells; Chromatin; Chromosomal translocation; Cultured Cells; DHFR gene; DNA; DNA Binding; DNA Binding Domain; Defect; Development; 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; Receptor Protein-Tyrosine Kinases; Repression; Research; Resistance; Resolution; Role; SAM Domain; Signal Transduction; Structure; System; Tail; Testing; Transcription Repressor/Corepressor; 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; public health relevance; response

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发挥关键作用。许多这些因素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，并且能够在许多有丝分裂周期中存活下来，而在其他情况下，沉默是短暂的并且很容易逆转。沉默结构域由DNA中的顺式调控模块成核，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、扩散和沉默之间的联系尚不清楚。这里提出的研究将探索两种果蝇调节因子 Groucho (Gro) 和 Yan 的抑制机制，它们具有完善的寡聚功能。具体目标 1 是确定镇压是否需要 Gro 扩散。 Gro 是一种辅抑制因子，在胚胎和图像发育中具有多种重要作用 - 它的人类直系同源物在人类发育中也具有同样重要的作用。它包含一个高度保守的寡聚结构域，称为 Q 结构域。该结构域中阻止寡聚化的突变可防止抑制。此外，培养细胞和翼盘中的染色质免疫沉淀测定表明，Gro 抑制靶标与长度达数千碱基的大型 Gro 结合域相关。该提案旨在在 Gro 寡聚化、传播和镇压之间建立牢固的联系。将开发具有寡聚化特定缺陷的 Gro 救援构建体，并对携带这些突变等位基因的蝇系进行表型和染色质免疫沉淀分析，以确定 Gro 结合域的结构和功能。具体目标 2 是确定 Yan 寡聚化在抑制和 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 信号转导中发挥重要作用。 Ras 激活导致 MAP 激酶磷酸化 Yan，随后将 Yan 从细胞核输出，导致 Ras 途径靶基因去抑制。 Yan 含有一个 SAM 结构域，它形成了 S2 细胞抑制所需的头尾蛋白质聚合物，并且似乎还控制 Ras 信号的接收。将开发编码寡聚亲和力增强的蛋白质的新 yan 等位基因，并在培养细胞和胚胎中进行测试，以确定 Yan 聚合、抑制和 Ras 信号传导之间的机制联系。公共健康相关性：这里研究的两个因素 Gro 和 Yan 各自都有人类直向同源物，与人类癌症有许多联系。例如，Yan 的人类直系同源物 Tel 是白血病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，本研究将揭示的机制将与人类疾病相关。