Single cell analysis of dynamic gene regulation

动态基因调控的单细胞分析

• 批准号: 10436385
• 负责人: Gregor Neuert
• 金额: $ 36.39万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10436385
• 关键词: Biological Process; Cell Fraction; Cell model; Cells; Chromatin; Chromatin Structure; Complex; Computer Analysis; Computer Models; Data; Data Analyses; Disease; Eukaryota; Gene Expression; Gene Expression Regulation; Genetic Transcription; Glycerol; Goals; Histones; Human; Immune System Diseases; Kinetics; Knowledge; Lead; Malignant Neoplasms; Measurement; Measures; Medical; Messenger RNA; Mitogen-Activated Protein Kinases; Modeling; Multiprotein Complexes; Mutate; Mutation; Neurologic; Nuclear; Osmolar Concentration; Pharmaceutical Preparations; Population; Process; RNA; RNA Degradation; Regulation; Regulator Genes; Role; SAGA; Signal Transduction; Spatial Distribution; Stress; TATA-Box Binding Protein; Testing; Time; Transcript; Transcription Initiation; Work; Yeasts; conditional mutant; developmental disease; experimental analysis; experimental study; gene induction; histone acetyltransferase; human disease; innovation; insight; member; mutant; novel; predictive modeling; single cell analysis; single molecule; spatiotemporal

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.

项目总结/摘要 在正常和疾病状态下精确控制基因表达的调控机制 涉及多蛋白复合物，如高度保守的Spt-Ada-Gcn 5乙酰转移酶（佐贺） 复杂.虽然大多数佐贺亚基已经被鉴定，但它们如何在细胞内表达仍然是未知的。 协调功能以精确调节基因表达。因此，佐贺复合物代表了理想的 范式来探索多蛋白复合物如何调节基因表达，以及本项目的总体目标 为佐贺亚基功能的协调提供精确的机制和预测性理解。 佐贺子单元被组织成“活动模块”。我们将重点关注已经确定的组蛋白 乙酰转移酶（HAT），TATA结合蛋白（TBP）和组蛋白去泛素化酶（DUB）活性模块， 佐贺，其中包含最好的特点和进化保守的佐贺亚基，并涉及 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起作用以精确地协调基因中不同步骤 从染色质调节到RNA转录再到RNA输出。我们将研究渗透压 高渗透压甘油（Hog 1/p38）丝裂原活化蛋白激酶（MAPK）信号传导和基因诱导 在酵母中表达，以研究佐贺亚基协调基因表达。重要的是，我们将使用新的 开发了详细的和综合的实验和计算分析的动态单分子RNA 在单个细胞中进行荧光原位杂交（FISH），以同时定量和建模基因调控中的这些步骤中的每一个。 令人兴奋的是，我们的初步研究表明，组蛋白乙酰转移酶Gcn 5 p增加了 染色质状态的动力学和基因表达的随机性，但不调节基础转录， 转录起始或RNA降解。我们将确定特定的HAT模块亚基如何调节 染色质结构和这些过程的动力学（目的1）。我们将阐明转录起始是如何 由独特的TBP模块亚基调节（Aim 2）。我们将揭示特定的DUB模块亚基 差异调节RNA输出（Aim 3）。为了实现这些目标，我们提出了一个严格的框架， 定量和动态单细胞实验，结合复杂的数据分析和预测 单细胞建模这种创新的方法将机械地解剖基因调控的医学 相关和进化保守的多蛋白佐贺复合物，提供了第一个全面的分析， 多蛋白基因调控复合物协调基因表达在一个单一的实验。此外，委员会认为， 我们的研究将为剖析其他多蛋白复合物如何调节基因表达提供蓝图。