Nuclear Organization and Dynamics of Mediator and RNA Polymerase II in Living Stem Cells

活干细胞中介质和 RNA 聚合酶 II 的核组织和动力学

• 批准号: 10392337
• 负责人: Jeff Gore
• 金额: $ 28.8万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-01 至 2023-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10392337
• 关键词: Affect; Biophysical Process; Biophysics; C-terminal; Cell Line; Cell Nucleus; Cells; ChIP-seq; Chromatin; Color; DNA; DNA Polymerase II; DNA-Directed RNA Polymerase; Data; Development; Developmental Disabilities; Disease; Distal; Drosophila genus; Elements; Embryo; Enhancers; Eukaryotic Cell; Gene Cluster; Gene Expression; Gene Silencing; Genes; Genetic Transcription; Genome; Goals; Grant; Health; Homeostasis; Human; Image; Imaging Techniques; In Vitro; Investigation; Label; Laboratories; Link; Liquid substance; Malignant Neoplasms; Mammalian Cell; Measurement; Mediator of activation protein; Messenger RNA; Methods; Microscopy; Molecular; Nuclear; Output; Phase; Phosphorylation; Population; Positioning Attribute; Property; Proteins; Research; Resolution; Role; Signal Transduction; Testing; Transcriptional Regulation; Work; base; beta Actin; cell growth regulation; cooking; density; design; developmental disease; genomic locus; genomic signature; high resolution imaging; imaging approach; improved; in vivo; innovation; insight; promoter; protein complex; recruit; spatiotemporal; stem cells; transcription factor

PROJECT SUMMARY/ABSTRACT: The Mediator protein complex functions as a coactivator of RNA Polymerase II (Pol II) transcription in eukaryotic cells. Mediator interfaces the interaction between Pol II and transcription factors to efficiently regulate gene expression. It is critical for integrating the signal of transcription factors bound at cis-regulatory chromatin elements (enhancers) that are believed to loop to active gene loci. Misregulation of gene expression leads to disease, including cancer and developmental disorders. Yet, the organization of Mediator and Pol II, and the mechanism by which they interact to regulate gene expression is unknown to date. Our long-term goal is to dissect the biophysical mechanisms and principles underlying the highly-regulated control of gene expression in eukaryotic cells. The objective of this grant is to characterize the spatiotemporal organization of Mediator and Pol II in live cells. The central hypothesis is that Mediator and Pol II form functional clusters with other transcription factors at enhancer DNA elements, then those enhancer associated clusters can dynamically associate with promoter DNA to activate gene expression. Our rationale is that direct measurements of Mediator and Pol II interaction will provide important insight into how expression of genes in eukaryotic cells is regulated. Our specific aims will test the following hypotheses: (Aim 1) to determine the mechanisms by which condensates of Mediator and Pol II affect gene expression (Aim 2) to determine the dynamics and functional relevance of the transient population of Pol II and Mediator clusters for gene expression in living cells (Aim 3) to determine how changes in cluster composition result in different functional and Dynamic Properties. Upon conclusion, we will understand the mechanisms by which Mediator and Pol II clusters, both the persistent condensates (~10% of cluster population in stem cells) and transient clusters (90% of the population in stem cells), act to regulate gene expression in living cells. This contribution is significant since it will lead to a new paradigm for describing how transcription is regulated in eukaryotic cells with important implications for development and disease. The proposed research is innovative because we investigate the spatiotemporal organization and mechanism of interaction between Mediator and Pol II at the single-cell level and with super-resolution methods. Insight into how clustering affect transcription is impactful as it provides new avenues for understanding how long-range enhancer-promoter interaction dynamically control of gene expression, and thus how its misregulation may occur in disease.

项目总结/摘要：介体蛋白复合物作为RNA的共激活因子发挥作用 真核细胞中的聚合酶II（Pol II）转录。Mediator接口Pol II和 转录因子来有效地调节基因表达。它是整合转录信号的关键 结合在顺式调节染色质元件（增强子）上的因子，这些元件被认为与活性基因位点成环。 基因表达的失调导致疾病，包括癌症和发育障碍。然而 Mediator和Pol II的组织，以及它们相互作用调节基因表达的机制， 未知日期我们的长期目标是剖析生物物理机制和原则的基础上， 真核细胞中基因表达的高度调节控制。这项补助金的目的是描述 活细胞中Mediator和Pol II的时空组织。中心假设是，调解人和波尔 II与其他转录因子在增强子DNA元件处形成功能簇，然后那些增强子 相关簇可以动态地与启动子DNA结合以激活基因表达。我们的理据是 直接测量Mediator和Pol II相互作用将为了解基因表达方式提供重要见解 真核细胞中的基因受到调控。我们的具体目标将测试以下假设：（目标1）确定 Mediator和Pol II的缩合物影响基因表达的机制（目的2），以确定 Pol II和Mediator基因簇瞬时群体的动力学和功能相关性 在活细胞中的表达（目的3），以确定簇组成的变化如何导致不同的功能性 动态属性。最后，我们将了解调解人和Pol II的机制， 簇，持久性凝聚物（干细胞中簇群的~10%）和瞬时簇 (90%的干细胞群体），用于调节活细胞中的基因表达。这种贡献 这一发现意义重大，因为它将为描述真核细胞中转录是如何调节的提供一个新的范例 对发育和疾病有重要意义。这项研究是创新的，因为我们 研究时空组织和中介人和Pol II之间的相互作用机制， 单细胞水平和超分辨率方法。深入了解聚类如何影响转录是有影响力的 因为它为理解长距离增强子-启动子相互作用如何动态提供了新的途径， 基因表达的控制，从而如何在疾病中发生失调。