Regulation of transcriptional bursting in the Drosophila embryo

项目摘要 转录是一个关键而复杂的过程，需要精确的调控才能确保正常发育。 基因组中大量的分子和调控元件应以组合方式相互作用 来决定一个基因需要在何时何地开启和关闭。如此复杂的过程 这使得理解转录调控的全面机制具有挑战性。最近 利用最先进的实时成像技术进行的研究提供了明确的证据， 动态的，由一系列顺序和随机的突发组成。虽然有人认为，转录 爆发是许多物种的基因表达的一般特性，转录的根本原因是， 爆炸仍然是未知的。提案中描述的实验探讨了 不稳定的增强子-启动子相互作用作为转录爆发的原因。 本研究计划拟测试增强子-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来自不稳定的增强子- 启动子相互作用虽然一些对培养细胞的研究表明，爆发可能源于不稳定的细胞， 组装前起始复合物或竞争性结合的核小体，还没有努力 研究增强子在这一过程中的作用。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，并且实时成像允许详细的可视化。 在发育过程中从头转录。该系统非常适合于测试假设，随机 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。 第二，传统的循环模型的增强子-启动子相互作用将被探讨。一般 假设与其同源激活子结合的增强子以某种方式环到靶启动子。这 成环过程募集前起始复合物并允许Pol II的释放以开始转录。的 然而，初步结果表明，这种物理循环可能不是转录所必需的。一 使用其中两个靶启动子由单个共享增强子调节的质粒构建体来显示 这两个启动子表现出协调的转录爆发，以类似的动力学打开和关闭。这 提供了一个反例，以传统的循环模型，其中两个报告基因将显示 由于单独的增强子-启动子成环相互作用而连续爆发。结合转基因， 活成像和光遗传学，该研究项目提出，迫使单个增强子的相互作用， 启动子环，并用定量图像处理分析对转录动力学的影响。的 这些研究的完成将为增强子-启动子的性质提供一个动态的视角。 通讯以及它如何有助于真核生物的转录调控。

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.