Dynamic association of transcription initiation proteins with chromatin at single-molecule resolution in living yeast

活酵母中转录起始蛋白与染色质在单分子分辨率下的动态关联

基本信息

批准号：
10403495
负责人：
Carl Wu
金额：
$ 44.6万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-08-01 至 2023-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10403495
关键词：
Address Animal Model Architecture Area Base Pairing Behavior Binding Biochemical Biochemistry Biology Cell Nucleus Cells ChIP-seq Chimeric Proteins Chromatin Chromosomes Complex Custom DNA DNA Binding DNA Polymerase II DNA Repair DNA biosynthesis DNA-Directed RNA Polymerase Detection Diffuse Diffusion Engineering Environment Enzymes Fluorescence Fluorescence Microscopy Genetic Genetic Recombination Genetic Transcription Genome Genomics Histones ISWI Image Kinetics Label Lasers Life Maps Measures Mediator of activation protein Microscope Modification Molecular Genetics Monitor Motion Movement Nuclear Output Permeability Phenotype Population Process Protein Dynamics Protein Engineering Proteins Proteome RNA Resolution Role Saccharomyces cerevisiae Saccharomycetales Site Source System Techniques Testing Time Transcription Factor TFIIA Transcription Initiation Transcription Process Transcriptional Regulation Visualization Yeasts chromatin modification chromatin remodeling computerized data processing conditional mutant fluorescence imaging fluorophore genome-wide image processing imaging modality imaging platform improved in vivo insight live cell imaging molecular imaging movie novel strategies promoter protein function repaired residence single molecule spatiotemporal transcription factor

项目摘要

The life of a cell is governed by the regulated transcription of genomic information from DNA to RNA by the enzyme RNA polymerase. Transcriptional control occurs through the actions of hundreds of proteins in the cell nucleus whose identities and functions have been elucidated from decades of genetic and biochemical studies. However, little is known about the timescales under which transcription-related proteins search for and target chromatin sites in living cells, and how such dynamic associations are influenced by modifications of chromatin architecture. This proposal aims to address these questions by applying super-resolution fluorescence microscopy to directly visualize the diffusive movements of major transcription initiation proteins at single- molecule resolution in budding yeast, under normal and altered states of chromatin architecture. Specifically, we will test the hypothesis that chromatin association of transcription initiation proteins occurs with rapid kinetics in live cells, and that chromatin remodeling and modification has a role in regulating transcription protein dynamics. We will use live-cell single-molecule imaging to monitor the diffusive behavior of transcription initiation proteins at high spatio-temporal resolution in the yeast nucleus. This `in vivo biochemistry' approach differs from and is complementary to ChIP-Seq techniques that map steady-state occupancies genome-wide but provide little information on binding dynamics. We will engineer and functionally validate DNA constructs encoding components representative of the general transcription factors and major sequence-specific DNA binding transcription factors fused to a self-labeling protein tag (HaloTag) that can react covalently with a cell-permeable organic fluorophore (Janelia Fluor). Live-cell imaging of fluorescently labeled transcription factors at single- molecule resolution measures diffusion coefficients, distinguishes between chromatin-bound and chromatin-free populations, and estimates residence times for the bound population. Further, we will use conditional depletion of 6 major chromatin remodelers and histone modifiers to reveal changes in the mobilities of transcription initiation proteins and inform which among several diffusive parameters are subject to chromatin controls. This combination of conditional mutant genetics and live-cell single-molecule imaging may transform understanding of the kinetic mechanisms for transcription initiation and offer a new approach to other areas of yeast nuclear and chromosome biology, including studies of DNA replication, repair, and recombination.

细胞的生命受到基因组信息从 DNA 到 RNA 的调节转录的控制。 RNA聚合酶。转录控制是通过细胞中数百种蛋白质的作用发生的数十年的遗传和生化研究已经阐明了其身份和功能。然而，人们对转录相关蛋白搜索和靶向的时间尺度知之甚少。活细胞中的染色质位点，以及染色质修饰如何影响这种动态关联建筑学。该提案旨在通过应用超分辨率荧光来解决这些问题显微镜直接观察主要转录起始蛋白在单点的扩散运动在正常和改变的染色质结构状态下，芽殖酵母中的分子分辨率。具体来说，我们将检验转录起始蛋白的染色质关联以快速动力学发生的假设活细胞，染色质重塑和修饰在调节转录蛋白动态中发挥作用。我们将使用活细胞单分子成像来监测转录起始蛋白的扩散行为酵母细胞核中的高时空分辨率。这种“体内生物化学”方法不同于并且是与 ChIP-Seq 技术互补，该技术可绘制全基因组稳态占据图谱，但提供的信息很少有关结合动力学的信息。我们将设计 DNA 构建体并对其进行功能验证代表一般转录因子和主要序列特异性 DNA 结合的成分与自标记蛋白标签 (HaloTag) 融合的转录因子，可与细胞渗透性共价反应有机荧光团（Janelia Fluor）。荧光标记转录因子的活细胞成像分子分辨率测量扩散系数，区分染色质结合和无染色质人口，并估计绑定人口的停留时间。此外，我们将使用条件耗尽 6 个主要染色质重塑剂和组蛋白修饰剂，揭示转录迁移率的变化起始蛋白并告知几个扩散参数中哪些受染色质控制。这条件突变遗传学和活细胞单分子成像的结合可能会改变理解转录起始的动力学机制，并为酵母核的其他领域提供了一种新方法和染色体生物学，包括 DNA 复制、修复和重组的研究。