Visualization of gene activity in the Drosophila embryo

 描述（由申请人提供）：在过去的25-30年里，MIRA计划中正在考虑合并的两项赠款（GM 46638和GM 34431）为动物发育期间基因表达控制的潜在机制提供了许多见解。具体的亮点包括证据表明，Hox蛋白作为序列特异性转录因子的功能，复杂的夏娃条纹2增强子的表征，并阐明了由te Dorsal（NF-κ B）梯度差异调节基因表达的亲和力阈值模型。这些研究建立了早期果蝇胚胎作为研究动物发育中基因活性的首要系统。MIRA计划的目标是利用新成像技术的出现来揭示基因控制的新机制。在过去的一年中，我们已经开始使用实时成像方法来可视化果蝇胚胎发育过程中基因表达的动态调节。这些研究已经导致了一些引人注目的观察，我们希望在即将到来的资助期间跟进，包括eve stripe 2表达的转录爆发，阴影增强子的非加性活动，转录记忆和等位基因通讯。我们将确定转录爆发是否是由于不稳定的增强子-启动子循环相互作用，并研究在给定的遗传位点的两个等位基因以某种方式通信，以确保在发展过程中的“平衡”表达水平的可能性。该研究计划包括四个具体目标：1）研究单个增强子激活两个不同靶启动子的动力学; 2）探索时间精度取决于“增强子转换”的可能性，即具有重叠活性的增强子在发育过程中以顺序方式工作; 3）确定转录记忆是否依赖于特定的组蛋白修饰，如甲基化; 4）通过检测活胚胎中的各种“致敏”转基因，证实了我们关于等位基因通讯的初步证据。

 DESCRIPTION (provided by applicant): During the past 25-30 years the two grants under consideration for merger in the MIRA program (GM46638 and GM34431) have provided numerous insights into the mechanisms underlying the control of gene expression during animal development. Specific highlights include evidence that Hox proteins function as sequence-specific transcription factors, the characterization of the complex eve stripe 2 enhancer, and the elucidation of the affinity threshold model for the differential regulation of gene expression by te Dorsal (NF-kB) gradient. These studies have established the early Drosophila embryo as a premiere system for the study of gene activity in animal development. It is the goal of the proposed MIRA grant to exploit the advent of new imaging technologies to uncover novel mechanisms of gene control. During the past year we have begun to use live imaging methods to visualize the dynamic regulation of gene expression during Drosophila embryogenesis. These studies have led to a number of striking observations that we wish to follow-up during the upcoming funding period, including transcriptional bursts of eve stripe 2 expressions, the non-additive activities of shadow enhancers, transcriptional memory, and allelic communication. We will determine whether transcriptional bursting is due to unstable enhancer-promoter looping interactions, and examine the possibility that the two alleles at a given genetic locus somehow communicate to ensure "balanced" levels of expression during development. The research plan includes four specific aims: 1) investigate the dynamics by which a single enhancer activates two different target promoters; 2) explore the possibility that temporal precision depends on "enhancer switching", whereby enhancers with overlapping activities work in a sequential manner during development; 3) determine whether transcriptional memory depends on specific histone modifications such as methylation; and 4) substantiate our preliminary evidence for allele communication by examining a variety of "sensitized" transgenes in living embryos.