Visualization of gene activity in the Drosophila embryo

项目摘要 远程转录增强子如何与其靶启动子通信以调节基因活性 一直是细胞和发育生物学的核心谜团之一。设想的经典模型 染色体环的形成导致增强子和启动子的直接物理结合。 然而，有新的证据表明，大的距离（~200 nm）分开的增强子和启动子 在转录激活过程中。此外，共享的增强子可以共激活顺式和非顺式连接的基因。 在同源染色体上的反式。这些观察结果显然与经典的 模型，并提出了另一种观点，“转录枢纽”，其中增强子和启动子， 募集转录激活因子的大簇或浓缩物。拟议研究的一个主要目标是 确定绝缘子DNA和拴系元件在转录的形成和功能中的作用 枢纽当插入远端增强器和靶之间时，绝缘体起到边界元件的作用 启动子它们还促进拓扑关联结构域（TADs）的形成和等位基因的配对 位于不同的同源染色体上。在果蝇中，束缚元件富含GAGA 促进长距离增强子-启动子相互作用的序列。 我们将采用基因组编辑，Micro-C XL测定和定量活体成像的组合 在活的果蝇胚胎中观察基因表达时间和水平的细微变化的方法， 由定义的绝缘子和束缚元件的突变、倒置和缺失引起的表达。 特别的努力将集中在复杂的遗传基因座内的远程增强子-启动子通信。 例如，我们将研究拴系元件在Scr转录激活中的作用， 远程增强子，必须绕过一个插入的Hox基因复合物内的插入。我们 还将探讨单独的绝缘体和拴系元件的贡献， 在knirps和Scylla基因座中通过共享增强子在75 kb和235 kb的距离上连锁基因， 分别最后，我们将研究拴系因素中的低复杂度序列 (e.g., Trithorax样）有助于稳定的增强子-促进环的形成成核 活化冷凝物。这些研究有可能解开当前关于 绝缘子，TADs和拴系元件在发育过程中基因调控的重要性。他们可能 还揭示了染色体环和转录枢纽形成之间的新联系。

Project Summary Abstract How remote transcriptional enhancers communicate with their target promoters to regulate gene activity persists as one of the central mysteries in cell and developmental biology. Classical models envisioned the formation of chromosomal loops that result in direct physical association of enhancers and promoters. However, there is emerging evidence that large distances (~200 nm) separate enhancers and promoters during transcription activation. Moreover, shared enhancers can co-activate linked genes, both in cis and in trans across homologous chromosomes. These observations are not obviously compatible with classical models and have prompted an alternate view, “transcription hubs”, whereby enhancers and promoters recruit large clusters or condensates of transcriptional activators. A major goal of the proposed study is to determine the role of insulator DNAs and tethering elements in the formation and function of transcription hubs. Insulators function as boundary elements when interposed between a distal enhancer and target promoter. They also foster the formation of topological associating domains (TADs) and paring of alleles located on different homologous chromosomes. In Drosophila, tethering elements are GAGA-rich sequences that facilitate long-range enhancer-promoter interactions. We will employ a combination of genome editing, Micro-C XL assays, and quantitative live imaging methods in living Drosophila embryos to visualize even subtle changes in the timing and levels of gene expression arising from mutations, inversions, and deletions of defined insulators and tethering elements. Particular efforts will focus on long-range enhancer-promoter communication within complex genetic loci. For example, we will examine the role of tethering elements in the activation of Scr transcription by a remote enhancer that must bypass an intervening TAD within the Antennapedia Hox gene complex. We will also explore the contributions of individual insulators and tethering elements for the co-regulation of linked genes by shared enhancers over distances of 75 kb and 235 kb in the knirps and Scylla loci, respectively. Finally, we will examine the possibility that low complexity sequences in tethering factors (e.g., Trithorax-like) contribute to the stability of enhancer-promoter loops by nucleating the formation of activation condensates. These studies have the potential to unravel current controversies regarding the importance of insulators, TADs, and tethering elements in gene regulation during development. They might also reveal new connections between chromosomal loops and the formation of transcriptional hubs.