Repressor/co-repressor oligomerization and development

描述(申请人提供)：转录抑制物和辅阻遏物在建立位置信息系统和调节发育过程中对细胞外信号的反应中发挥关键作用。其中许多因子将大的染色体结构域组织成转录沉默状态。在某些情况下，这些状态是自我传播的，并在许多有丝分裂周期中幸存下来，而在另一些情况下，沉默是短暂的，很容易逆转。沉默结构域由DNA中的顺式调节模块成核，并可能以依赖于抑制子或辅阻遏子齐聚的方式从这些模块扩散。然而，抑制子/辅抑制子齐聚、扩散和沉默之间的联系还不是很清楚。这项研究将探索果蝇调节因子Groucho(Gro)和Yen的抑制机制，这两个调节因子具有成熟的寡聚功能。具体目标1是确定压制是否需要GRO扩散。GRO是一种核心抑制因子，在胚胎和想象发育中具有多种重要作用--其人类同源基因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。这个结构域中防止寡聚的突变可以防止抑制。此外，在培养细胞和翼盘中的染色质免疫沉淀分析表明，GRO抑制靶与大的GRO结合结构域相关，长度为许多千碱基。这项提议寻求在GRO齐聚、扩散和抑制之间建立牢固的联系。将开发具有寡聚特定缺陷的GRO救援构建体，并将对携带这些突变等位基因的苍蝇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。特异目的2是确定严寡聚在抑制和RAS信号转导中的作用。Yen和它的人类同源基因TEL是DNA结合的转录抑制因子，在RAS信号转导中起着重要作用。RAS的激活导致MAP激酶对Yen的磷酸化，进而导致Yen从细胞核输出，从而导致RAS途径靶基因的去抑制。Yen含有一个SAM结构域，它形成了一种从头到尾的蛋白质聚合物，这是抑制S2细胞所必需的，似乎也控制着RAS信号的接收。Yen的新等位基因将被开发出来，并在培养细胞和胚胎中进行测试，以确定Yen聚合、抑制和RAS信号之间的机制联系。公共卫生相关性：这里正在调查的两个因素Gro和Yen，每个都有与人类癌症有许多联系的人类同源基因。例如，TELL，人类Yen的直系同源基因，经常成为白血病染色体易位的目标，而哺乳动物GRO的上调与肺腺癌有关。因此，这项研究中揭示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.