Visualization of gene activity in the Drosophila embryo

果蝇胚胎中基因活性的可视化

• 批准号: 10445268
• 负责人: Michael Steven Levine
• 金额: $ 66.39万
• 依托单位: PRINCETON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-23 至 2026-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10445268
• 关键词: Alleles; Biological Assay; Boundary Elements; Bypass; Cellular biology; Chromosomes; Communication; Complex; DNA; DNA Polymerase II; Development; Developmental Biology; Distal; Drosophila genus; Elements; Embryo; Enhancers; Fostering; Gene Expression; Gene Expression Regulation; Genes; Genetic Transcription; Goals; Hi-C; Homeobox Genes; Individual; Insulator Elements; Link; Maps; Modeling; Mutation; RNA; Regulation; Role; Testing; Transcription Coactivator; Transcriptional Activation; Visualization; genome editing; genomic locus; imaging modality; promoter; quantitative imaging; recruit; transcription factor

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.

项目摘要摘要 远程转录增强子如何与其目标启动子沟通以调节基因活性 一直是细胞和发育生物学的中心谜团之一。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，大距离(~200 nm)将增强子和启动子分开 在转录激活期间。此外，共享的增强子可以在顺式基因和顺式基因中共同激活相连的基因。 在跨越同源染色体的情况下。这些观察结果显然与经典的 模型，并引发了另一种观点，“转录中枢”，即增强子和启动子 招募转录激活剂的大簇或凝聚体。拟议研究的一个主要目标是 确定绝缘体DNA和系链元件在转录形成和功能中的作用 集线器。当绝缘体插入远端增强剂和靶之间时，绝缘体起到边界元件的作用 推动者。它们还促进了拓扑关联结构域(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，系留元素富含Gaga 促进长程增强子-启动子相互作用的序列。 我们将采用基因组编辑、Micro-C XL分析和定量实时成像的组合 方法在活的果蝇胚胎中观察基因的时间和水平的细微变化 由定义的绝缘子和系链元件的突变、倒置和缺失引起的表达。 特别的努力将集中在复杂遗传位点内的远程增强子-启动子通信上。 例如，我们将研究拴系元件在激活scr转录中的作用。 必须绕过触角线虫Hox基因复合体中的TAD的远程增强子。我们 还将探讨单个绝缘子和系留元件对共同调节 在Knirps和Scylla基因座中，通过75kb和235kb的距离共享增强子将基因连接在一起， 分别进行了分析。最后，我们将研究低复杂度序列在系留因素中的可能性 (例如，类三胸)通过成核形成增强子-启动子环来促进增强子-启动子环的稳定性 活化冷凝物。这些研究有可能解开目前关于 绝缘体、TADS和系留元件在发育过程中基因调控中的重要性。他们可能会 还揭示了染色体环和转录中心的形成之间的新联系。