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

项目摘要/摘要：中介蛋白复合体作为RNA的共激活因子发挥作用 聚合酶II(POL II)在真核细胞中的转录。Mediator接口POL II和 转录因子，有效地调节基因表达。它是整合转录信号的关键 与顺式调控染色质元件(增强子)结合的因子被认为与活性基因位点环路。 基因表达的错误调控会导致疾病，包括癌症和发育障碍。然而， 介体和POL II的组织，以及它们相互作用调节基因表达的机制是 到目前为止还不知道。我们的长期目标是剖析生物物理机制和原理 真核细胞中基因表达的高度调控。这笔赠款的目的是为了描述 活细胞中介体和POL II的时空组织。中心假设是Mediator和Pol II与其他转录因子在增强子DNA元件上形成功能簇，然后这些增强子 相关簇可以动态地与启动子DNA结合来激活基因表达。我们的理由是 对Mediator和Pol II相互作用的直接测量将提供对如何表达 真核细胞中的基因是受调控的。我们的具体目标将检验以下假设：(目标1)确定 介体和POL II的凝聚体影响基因表达的机制(目标2)以确定 Pol II和基因介体簇的暂态群体的动态和功能相关性 在活细胞中表达(目标3)以确定簇组成的变化如何导致不同的功能 和动态属性。最后，我们将了解Mediator和Pol II通过什么机制 集群，既有永久性凝集物(约占干细胞集群总数的10%)，也有瞬时集群 (90%的人在干细胞中)，调节活细胞中的基因表达。这一贡献是 意义重大，因为它将导致描述真核细胞中转录如何调控的新范式 对发展和疾病有重要影响。这项拟议的研究具有创新性，因为我们 研究介体和POL II之间的时空组织和相互作用机制 单细胞水平和超分辨率方法。洞察集群如何影响转录是有影响的 因为它为理解长程增强子-启动子的动态相互作用提供了新的途径 基因表达的控制，以及它的错误调控在疾病中是如何发生的。

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.