Regulation of transcriptional bursting in the Drosophila embryo

果蝇胚胎转录爆发的调控

• 批准号: 9257592
• 负责人: Bomyi Lim
• 金额: $ 5.67万
• 依托单位: PRINCETON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-01-01 至 2018-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9257592
• 关键词: Affect; Binding; Biological Models; Communication; Competitive Binding; Complex; Consensus; Cultured Cells; DNA Binding; DNA Polymerase II; Detection; Development; Drosophila genus; Elements; Embryo; Enhancers; Ensure; Eukaryota; Exhibits; Frequencies; Gene Expression; Gene Transfer Techniques; Genes; Genetic Transcription; Genome; Goals; Image; Imagery; Imaging Techniques; Label; Light; Link; Mediating; Messenger RNA; Methods; Modeling; Monitor; Nature; Nucleosomes; Organism; Output; Phase; Plasmids; Process; Property; Proteins; RNA; Recruitment Activity; Regulation; Regulatory Element; Reporter Genes; Research; Research Project Grants; Role; Series; Site; System; Testing; Transcript; Transcriptional Regulation; Work; base; combinatorial; experimental study; fly; image processing; imaging modality; in vivo; insight; optogenetics; promoter; quantitative imaging; transcription factor

Project Summary Transcription is a critical yet a complex process that requires precise regulation to ensure normal development. A large number of molecules and regulatory elements in the genome should interact in combinatorial manner to determine when and where a gene needs to be turned on and off. Such complex nature of the process makes it challenging to understand the comprehensive mechanisms of transcriptional regulation. Recent studies with state-of-the-art live imaging techniques provide clear evidence that transcription activity is dynamic, consisting of a series of sequential and stochastic bursts. While it is suggested that transcriptional bursting is a general property of gene expression across many species, the underlying cause of transcriptional bursting remains largely unknown. The experiments described in the proposal explore the potential role of unstable enhancer-promoter interactions as a cause of the transcriptional bursting. This research project proposes to test two aspects of the relationship between enhancer-promoter interactions and transcription. First, it will be determined whether transcriptional bursts arise from unstable enhancer- promoter interactions. While some studies with cultured cells suggested that bursts might arise from unstable assembly of pre-initiation complex or competitive binding of nucleosomes, there has been no effort to investigate the role of enhancers in this process. Drosophila embryos will be used as a model system, since extensive studies have identified hundreds of enhancers, and live imaging permits the detailed visualization of de novo transcription in development. The system is ideally suited for testing the hypothesis that stochastic enhancer-promoter interactions influence the frequency, duration, or amplitudes of transcriptional bursts. Second, the traditional looping models of enhancer-promoter interactions will be explored. It is generally assumed that enhancers that are bound to their cognate activators somehow loop to the target promoter. This looping process recruits the pre-initiation complex and allows the release of Pol II to start transcription. The preliminary results, however, suggest that this physical looping might not be required for transcription. A plasmid construct where two target promoters are regulated by a single shared enhancer was used to show that both promoters exhibit coordinated transcriptional bursts, turning on and off with similar dynamics. This provides a counter-example to the traditional looping models, whereby two reporter genes would display sequential bursts due to separate enhancer-promoter looping interactions. With combination of transgenesis, live imaging, and optogenetics, this research project proposes to force interactions of single enhancer- promoter loops and analyze the effect on transcription dynamics with quantitative image processing. The accomplishment of the proposed studies will provide a dynamic view of the nature of enhancer-promoter communication and how it can contribute to transcriptional regulation in eukaryotes.

项目概要 转录是一个关键而复杂的过程，需要精确的调控以确保正常发育。 基因组中的大量分子和调控元件应以组合方式相互作用 以确定何时何地需要打开和关闭基因。该过程的复杂性 使得理解转录调控的综合机制变得具有挑战性。最近的 使用最先进的实时成像技术进行的研究提供了明确的证据，表明转录活性 动态的，由一系列顺序和随机突发组成。虽然有人建议转录 爆发是许多物种基因表达的普遍特性，是转录的根本原因 爆裂仍然很大程度上未知。提案中描述的实验探索了潜在的作用 不稳定的增强子-启动子相互作用是转录爆发的原因。 该研究项目旨在测试增强子-启动子相互作用之间关系的两个方面 和转录。首先，将确定转录爆发是否由不稳定的增强子引起—— 启动子相互作用。虽然一些针对培养细胞的研究表明，爆发可能是由不稳定引起的 预起始复合物的组装或核小体的竞争性结合，尚未做出努力 研究增强子在此过程中的作用。果蝇胚胎将被用作模型系统，因为 广泛的研究已经确定了数百种增强子，并且实时成像可以实现增强子的详细可视化 发育中的从头转录。该系统非常适合检验随机假设 增强子-启动子相互作用影响转录爆发的频率、持续时间或幅度。 其次，将探索增强子-启动子相互作用的传统循环模型。一般是 假设与其同源激活子结合的增强子以某种方式循环至目标启动子。这 循环过程招募预起始复合物并允许释放 Pol II 以开始转录。这 然而，初步结果表明，转录可能不需要这种物理循环。一个 使用其中两个目标启动子由单个共享增强子调节的质粒构建体来显示 两个启动子都表现出协调的转录爆发，以相似的动态开启和关闭。这 提供了传统循环模型的反例，其中两个报告基因将显示 由于单独的增强子-启动子循环相互作用而导致的连续爆发。结合转基因技术， 实时成像和光遗传学，该研究项目建议强制单个增强子的相互作用- 启动子循环并通过定量图像处理分析对转录动力学的影响。这 拟议研究的完成将为增强子-启动子的性质提供动态视图 通讯及其如何促进真核生物的转录调控。