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

• 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
• 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

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.

细胞的寿命受基因组信息从DNA到RNA的调节转录的控制 酶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 从遗传和生化研究的数十年中阐明了其身份和功能的核。 但是，对于转录相关蛋白搜索和靶标的时间尺度知之甚少 活细胞中的染色质位点，以及这种动态关联如何受染色质的修饰影响 建筑学。该建议旨在通过应用超分辨率荧光来解决这些问题 显微镜，直接可视化主要转录起始蛋白在单个的扩散运动 在染色质结构的正常和变化状态下，萌芽酵母中的分子分辨率。具体来说，我们 将检验以下假设：转录起始蛋白的染色质缔合蛋白质与快速动力学发生 活细胞，染色质重塑和修饰在调节转录蛋白动力学中起作用。 我们将使用活细胞单分子成像来监视转录起始蛋白的扩散行为 在酵母核中高时空分辨率下。这种“体内生物化学”方法与 补充绘制稳态占领的芯片序列技术，但几乎没有提供 有关绑定动力学的信息。我们将设计并在功能上验证编码的DNA构建体 代表一般转录因子和主要序列特异性DNA结合的组成部分 转录因子融合到自标记蛋白标签（Halotag）的转录因子，该蛋白质标签（Halotag）可以与可渗透的细胞共价反应 有机荧光团（Janelia Fluor）。荧光标记的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 人群，并估计约束人口的停留时间。此外，我们将使用有条件的耗竭 6个主要的染色质重塑剂和组蛋白修饰剂，以揭示转录迁移率的变化 引发蛋白并告知几个扩散参数中的蛋白质受染色质控制。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 用于转录启动的动力学机制，并为其他酵母核方面提供了新的方法 和染色体生物学，包括对DNA复制，修复和重组的研究。