Single cell analysis of dynamic gene regulation

项目摘要/摘要 在正常和疾病状态下精确控制基因表达的调控机制 涉及多蛋白复合体，如高度保守的SPT-Ada-Gcn5乙酰基转移酶(SAGA) 很复杂。虽然大多数佐贺亚基已经被确定，但本质上仍然不清楚它们是如何 功能协调，以精确地调节基因表达。因此，传奇情结代表了一种理想 探索多蛋白复合体如何调控基因表达的范式，以及本项目的总体目标 是为SAGA亚单位功能的协调提供精确的机械性和预测性的理解。 SAGA亚单位被组织成“活动模块”。我们将关注久负盛名的组蛋白 乙酰转移酶(HAT)、TATA结合蛋白(TBP)和组蛋白脱泛素酶(DUB)活性模块 SagA，包含最具特征和进化上保守的SagA亚基，并被牵连 在染色质结构(HAT)、转录起始(TBP)和RNA输出(DUB)的调节中。我们的中央 假设佐贺亚基和模块一起工作，以精确协调基因的不同步骤 表达从染色质调控到RNA转录再到RNA输出。我们将研究渗透胁迫 高渗透压甘油(Hog1/p38)丝裂原活化蛋白激酶(MAPK)信号和基因的诱导 在酵母中表达，研究SAGA亚基与基因表达的协调性。重要的是，我们将使用一个新的 对动态单分子RNA进行了详细而完整的实验和计算分析 在单个细胞中表达(FISH)，以同时对基因调控中的这些步骤中的每一个步骤进行量化和建模。 令人兴奋的是，我们的初步研究表明，组蛋白乙酰转移酶Gcn5p增加了 染色质状态的动态和基因表达的随机性，但不调节基础转录， 转录启动，或RNA降解。我们将确定特定的HAT模块亚单位是如何调节的 染色质结构和这些过程的动力学(目标1)。我们将阐明转录启动是如何进行的 由独特的TBP模块亚基调节(目标2)。我们将揭示特定的配音模组亚基是如何 差别化监管RNA输出(目标3)。为了实现这些目标，我们提出了一个严格的框架 与复杂的数据分析和预测相结合的定量和动态单细胞实验 单细胞建模。这种创新的方法将机械地剖析医学上的基因调控 相关和进化保守的多蛋白佐贺复合体，提供了第一个全面的分析 多蛋白基因调控复合体在单个实验中协调基因表达。此外， 我们的研究将为剖析其他多蛋白复合体如何调控基因表达提供蓝图。

PROJECT SUMMARY/ABSTRACT Regulatory mechanisms underlying the precise control of gene expression in normal and disease states involve multiprotein complexes such as the highly conserved Spt-Ada-Gcn5 Acetyltransferase (SAGA) complex. Although most of the SAGA subunits have been identified, it remains essentially unknown how their functions are coordinated to precisely regulate gene expression. Thus, the SAGA complex represents an ideal paradigm to explore how multiprotein complexes regulate gene expression, and the overall goal of this project is to provide a precise mechanistic and predictive understanding for the coordination of SAGA subunit function. SAGA subunits are organized into “activity modules”. We will focus on the well-established histone acetyltransferase (HAT), TATA-binding protein (TBP), and histone deubiquitinase (DUB) activity modules in SAGA, which contain the best characterized and evolutionarily conserved SAGA subunits, and are implicated in the regulation of chromatin structure (HAT), transcription initiation (TBP) and RNA export (DUB). Our central hypothesis is that SAGA subunits and modules function together to precisely coordinate different steps in gene expression from chromatin regulation to RNA transcription to RNA export. We will investigate osmotic stress induction of high osmolarity glycerol (Hog1/p38) mitogen-activated protein kinase (MAPK) signaling and gene expression in yeast to study SAGA subunit coordination of gene expression. Importantly, we will use a newly developed detailed and integrated experimental and computational analysis of dynamic single-molecule RNA expression (FISH) in single cells to simultaneously quantify and model each of these steps in gene regulation. Excitingly, our preliminary studies have revealed that the histone acetyltransferase Gcn5p increases the dynamics of chromatin states and stochasticity in gene expression but does not regulate basal transcription, transcription initiation, or RNA degradation. We will determine how the specific HAT module subunits regulate chromatin structure and the kinetics of these processes (Aim 1). We will elucidate how transcription initiation is regulated by unique TBP module subunits (Aim 2). And we will reveal how the specific DUB module subunits differentially regulate RNA export (Aim 3). To accomplish these aims, we propose a rigorous framework of quantitative and dynamic single-cell experiments integrated with sophisticated data analysis and predictive single-cell modeling. This innovative approach will mechanistically dissect gene regulation by the medically relevant and evolutionary conserved multiprotein SAGA complex, providing the first comprehensive analysis of multiprotein gene regulatory complex coordination of gene expression within a single experiment. Furthermore, our studies will provide a blueprint to dissect how other multiprotein complexes regulate gene expression.