Molecular Basis of Ecdysteroid Action in the Mosquito

描述（由申请人提供）：在本项目的前五年中，我们对20-羟基蜕皮激素（20 E）在蚊子卵黄发生中作用的分子基础有了基本的了解。本申请的目的是进一步表征成年蚊子雌性中介导20 E作用的遗传调控层次结构。我们克隆了埃及伊蚊的20 E遗传层次的几个关键因素的亚型。在这项应用中，我们将利用RNAi敲除技术，建立了蚊子，系统地研究每种亚型的作用。在蚊子卵黄发生过程中，蜕皮激素调控级联反应所调控的靶基因的数目和身份知之甚少。我们将使用基因组微阵列方法结合特定基因的RNAi敲除来描绘新的基因组网络和靶基因。然后，我们将测试的假设，多个调节途径与20 E调节级联集成。20 E靶效应基因的调控区，如编码卵黄蛋白前体的调控区，含有EcR/USP和早期基因产物E74、E75和BR（宽复合物）的多个结合位点。我们将研究这些因子是如何参与调节效应基因的转录活性。我们还将测试的假设，即早期的基因产物，在其结合到各自的DMA网站，作为转录调节因子，直接与EcR/USP相互作用，并协同引发高水平的表达的效应基因在20 E-依赖的方式。我们已经表明，在卵黄发生过程中的20 E调节层次的激活所需的能力因子FTZ-F1。在本申请中，我们将检验FTZ-F1通过募集EcR/USP的共激活因子来指导阶段特异性20 E诱导的基因表达的假设。我们已经发现，替代异源二聚体的USP无论是与阻遏物HR 38和SVP或与激活剂EcR调节卵黄反应的周期性20 E。在这个应用程序中，我们将检查是否存在于一个复杂的EcR与热休克蛋白或其他因素在逮捕的状态。这项拟议的研究预计将大大推进我们对蚊子卵发育中关键调控级联的分子机制的了解，这反过来又可用于开发针对这些最重要的人类疾病载体的新控制方法。

DESCRIPTION (provided by applicant): During the previous five years of this project, we achieved a fundamental understanding of the molecular basis of 20-hydroxyecdysone (20E) action in mosquito vitellogenesis. The goal of this application is to further characterize the genetic regulatory hierarchy mediating 20E action in the adult mosquito female. We cloned isoforms for several key factors of the 20E genetic hierarchy from Aedes aegypti. In this application, we will take advantage of the RNAi knockout technique, well established for mosquitoes, to systematically study the role of each isoform. Little is known about number and identities of target genes regulated by the ecdysteroid regulatory cascade during mosquito vitellogenesis. We will use the genomic microarray approach in combination with RNAi knockouts of specific genes to delineate novel genomic networks and target genes. We will then test the hypothesis that multiple regulatory pathways are integrated with the 20E regulatory cascade. Regulatory regions of 20E target effector genes, such as those encoding for yolk protein precursor, contain multiple binding sites for EcR/USP and the early gene products E74, E75, and BR (broad complex). We will study how these factors are involved in regulating the transcriptional activity of effector genes. We will also test the hypothesis that the early gene products, upon their binding to respective DMA sites, serve as transcriptional regulators that directly interact with the EcR/USP and cooperatively elicit a high level of expression of an effector gene in a 20E-dependent manner. We have shown that the competence factor FTZ-F1 is required for activation of the 20E regulatory hierarchy during vitellogenesis. In this application, we will test the hypothesis that FTZ-F1 directs stage-specific, 20E-induced gene expression by recruiting co-activator(s) for EcR/USP. We have discovered that alternative heterodimerization of USP either with the repressors HR38 and SVP or with an activator EcR regulates the cyclicity of vitellogenic response to 20E. In this application, we will examine whether EcR exists in a complex with heat shock proteins or other factors during the state of arrest. The proposed research is expected to advance significantly our knowledge concerning molecular mechanisms governing the key regulatory cascade in mosquito egg development, which in turn could be used for developing novel control methods against these most important vectors of human diseases.