Molecular Basis of Ecdysteroid Action in the Mosquito

描述(申请人提供)：在这个项目的前五年中，我们对20-羟基蜕皮酮(20E)在蚊子卵黄发生中作用的分子基础有了基本的了解。这项应用的目标是进一步表征在成年雌性蚊子中介导20E作用的遗传调控体系。我们克隆了埃及伊蚊20E基因序列中几个关键因子的异构体。在这个应用中，我们将利用为蚊子建立的RNAi敲除技术来系统地研究每一种异构体的作用。在蚊子卵黄发生过程中，蜕皮激素调控级联反应调控的靶基因的数量和特性知之甚少。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，我们将检验多条监管途径与20E监管级联整合的假设。20E靶效应基因的调节区，如编码卵黄蛋白前体的基因，含有ECR/USP和早期基因产物E74、E75和BR(广泛的复合体)的多个结合位点。我们将研究这些因素如何参与调节效应基因的转录活性。我们还将测试这一假设，即早期基因产物在与各自的DMA位点结合后，作为转录调节因子直接与ECR/USP相互作用，并以20E依赖的方式协同诱导效应基因的高水平表达。我们已经证明，在卵黄发生过程中，能力因子FTZ-F1是激活20E调控体系所必需的。在这个应用中，我们将通过为ECR/USP招募共激活因子(S)来检验FTZ-F1指导20E诱导的阶段特异性基因表达的假设。我们发现，USP与抑制子HR38和SVP或激活剂ECR的交替异二聚调节对20E的卵黄生成反应的周期性。在这个应用中，我们将检查在停滞状态下，ECR是否存在于与热休克蛋白或其他因素的复合体中。这项拟议的研究有望极大地提高我们对蚊卵发育关键调控级联的分子机制的了解，进而可用于开发针对这些最重要的人类疾病媒介的新控制方法。

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.