Repressor/co-repressor oligomerization and development

描述（由申请人提供）：转录抑制因子和辅抑制因子在发育过程中建立位置信息系统和介导细胞外信号反应中发挥关键作用。许多这些因素组织大的染色体结构域进入转录沉默状态。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下来，而在其他情况下，沉默是短暂的，很容易逆转。沉默结构域由DNA中的顺式调控模块成核，并可能以依赖于抑制子或辅抑制子寡聚化的方式从这些模块扩散。然而，抑制因子/辅抑制因子寡聚化、扩散和沉默之间的联系尚不清楚。本研究将探讨两种具有寡聚化功能的果蝇调控因子Groucho （Gro）和Yan的抑制机制。具体目标1是确定抑制是否需要Gro传播。Gro是一种辅助抑制因子，在胚胎和胚胎发育中具有多种重要作用，其人类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化结构域，称为Q结构域。这个区域的突变阻止了寡聚化，阻止了抑制。此外，在培养细胞和翅盘中进行的染色质免疫沉淀试验表明，Gro抑制靶点与长数千个碱基的Gro结合结构域相关。这一建议旨在建立Gro寡聚化、传播和抑制之间的牢固联系。将开发具有特定寡聚化缺陷的Gro拯救构建体，并对携带这些突变等位基因的果蝇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。具体目的2是确定Yan寡聚化在抑制和Ras信号传导中的作用。Yan及其人类同源基因Tel是DNA结合转录抑制因子，在Ras信号传导中起重要作用。Ras激活导致Yan被MAP激酶磷酸化，随后Yan从细胞核输出，导致Ras途径靶基因的抑制。Yan含有一个SAM结构域，该结构域形成了S2细胞中抑制所需的从头到尾的蛋白质聚合物，并且似乎也控制了Ras信号的接收。yan的新等位基因编码具有更高寡聚化亲和力的蛋白质，将在培养细胞和胚胎中进行开发和测试，以确定yan聚合、抑制和Ras信号传导之间的机制联系。公共卫生相关性：这里研究的两个因子，Gro和Yan，都与人类癌症有许多联系。例如，Yan的人类同源基因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，在这项研究中发现的机制将与人类疾病相关。

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.