Regulation of transcriptional bursting in the Drosophila embryo

项目摘要 转录是一个关键但复杂的过程，需要精确的调控来确保正常发育。 基因组中的大量分子和调控元件应该以组合的方式相互作用 以确定基因需要在何时何地开启和关闭。这一过程如此复杂 这使得理解转录调控的全面机制变得具有挑战性。近期 用最先进的实时成像技术进行的研究提供了明确的证据，证明转录活性是 动态的，由一系列连续和随机的突发组成。虽然有人建议转录 猝发是许多物种基因表达的一种普遍特征，是转录的根本原因 爆炸在很大程度上仍是未知的。提案中描述的实验探索了 不稳定的增强子-启动子相互作用是转录爆发的一个原因。 这项研究计划从两个方面测试增强子-启动子相互作用之间的关系。 和抄写。首先，将确定转录猝发是否源于不稳定的增强子- 启动子相互作用。虽然一些对培养细胞的研究表明，猝发可能是由于不稳定 组装预引发复合体或核小体的竞争结合，一直没有努力 研究增强剂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剂，而实时成像可以详细地可视化 新的转录正在开发中。该系统非常适合于检验随机性假设 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。它一般都是 假设与其同源激活子结合的增强子以某种方式环到目标启动子。这 环过程招募预启动复合体，并允许POL II的释放开始转录。这个 然而，初步结果表明，这种物理循环可能不是转录所必需的。一个 其中两个靶启动子由单个共享增强子调控的质粒构建用于展示 这两个启动子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，在该模型中，将显示两个报告基因 由于单独的增强子-启动子环相互作用而导致的顺序猝发。结合转基因技术， 实时成像和光遗传学，这项研究项目建议强制单个增强子- 启动子循环，并用定量图像处理分析其对转录动力学的影响。这个 拟议研究的完成将提供对增强子-启动子性质的动态看法 通讯以及通讯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.