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.

项目摘要/摘要 正常和疾病状态中基因表达精确控制的基础的调节机制 涉及多蛋白复合物，例如高度保守的SPT-ADA-GCN5乙酰转移酶（SAGA） 尽管已经确定了大多数传奇亚基，但虽然已经确定 功能是协调的，以精确调节基因表达。那是传奇综合体代表理想的 范式探索多蛋白复合物如何调节基因表达以及该项目的总体目标 是为传奇亚基函数的协调提供精确的机械和预测性理解。 传奇亚基被组织成“活动模块”。我们将专注于成熟的组蛋白 乙酰基转移酶（HAT），TATA结合蛋白（TBP）和Hisstone去泛素酶（DUB）活性模块 传奇，其中包含最佳特征和进化构成的传奇亚基，并已实施 在调节染色质结构（HAT）中，转录起始（TBP）和RNA导出（DUB）。我们的中心 假设是传奇亚基和模块一起起作用以精确协调基因的不同步骤 从染色质调节到RNA转录到RNA输出的表达。我们将研究渗透应力 诱导高渗透甘油（HOG1/p38）有丝分裂原激活蛋白激酶（MAPK）信号传导和基因 酵母中的表达研究基因表达的传奇亚基配位。重要的是，我们将使用新的 开发了动态单分子RNA的详细和集成的实验和计算分析 单个细胞中的表达（FISH）同时量化并模拟了基因调控中的每个步骤。 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 染色质状态和基因表达中随机性的动力学，但不调节基本转录， 转录启动或RNA降解。我们将确定特定的帽子模块亚基如何调节 这些过程的染色质结构和动力学（AIM 1）。我们将阐明转录的启动方式 由独特的TBP模块亚基（AIM 2）调节。我们将揭示特定的配音模块亚基 差异调节RNA导出（AIM 3）。为了实现这些目标，我们提出了一个严格的框架 与复杂数据分析和预测性集成的定量和动态单细胞实验 单细胞建模。这种创新的方法将通过医学上的机械剖析基因调节 相关和进化的构成多蛋白质蛋白综合体，提供了首次综合分析 单个实验中基因表达的多蛋白基因调节复合物协调。此外， 我们的研究将提供蓝图，以剖析其他多蛋白复合物如何调节基因表达。