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: 10557286
• 负责人: Carl Wu
• 金额: $ 2.92万
• 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/10557286
• 关键词: Address; Animal Model; Architecture; Area; Base Pairing; Behavior; Binding; Biochemical; Biochemistry; Biology; Cell Nucleus; Cells; ChIP-seq; Chromatin; Chromosomes; Complex; Crowding; DNA; DNA Binding; DNA biosynthesis; DNA-Directed RNA Polymerase; Diffuse; Diffusion; Elements; Engineering; Environment; Enzymes; Fluorescence Microscopy; Genes; Genetic; Genetic Recombination; Genetic Transcription; Genomics; Histones; Kinetics; Label; Maps; Measures; Modification; Monitor; Nuclear; Permeability; Population; Process; Protein Dynamics; Proteins; Resolution; Role; Saccharomyces cerevisiae; Saccharomycetales; Site; Techniques; Technology; Testing; Time; Transcription Initiation; Transcription Process; Transcriptional Regulation; Yeasts; chromatin modification; chromatin remodeling; combinatorial; conditional mutant; fluorescence imaging; fluorophore; genome-wide; in vivo; live cell imaging; molecular imaging; recruit; repaired; residence; single molecule; spatiotemporal; transcription factor; Address; Animal Model; Architecture; Area; Base Pairing; Behavior; Binding; Biochemical; Biochemistry; Biology; Cell Nucleus; Cells; ChIP-seq; Chromatin; Chromosomes; Complex; Crowding; DNA; DNA Binding; DNA biosynthesis; DNA-Directed RNA Polymerase; Diffuse; Diffusion; Elements; Engineering; Environment; Enzymes; Fluorescence Microscopy; Genes; Genetic; Genetic Recombination; Genetic Transcription; Genomics; Histones; Kinetics; Label; Maps; Measures; Modification; Monitor; Nuclear; Permeability; Population; Process; Protein Dynamics; Proteins; Resolution; Role; Saccharomyces cerevisiae; Saccharomycetales; Site; Techniques; Technology; Testing; Time; Transcription Initiation; Transcription Process; Transcriptional Regulation; Yeasts; chromatin modification; chromatin remodeling; combinatorial; conditional mutant; fluorescence imaging; fluorophore; genome-wide; in vivo; live cell imaging; molecular imaging; recruit; repaired; residence; single molecule; spatiotemporal; transcription factor

A central paradigm for eukaryotic gene control posits that sequence-specific transcription factors search for and locate their genomic targets in a crowded nuclear environment, often acting combinatorially and cooperatively at regulatory DNA elements in chromatin to recruit and direct the ordered assembly of a transcription pre-initiation complex composed of general transcription factors and RNA polymerase. Although the identities and functions of several hundred transcription-related proteins are known, there is little information on the timescales under which they operate in the transcription process, and the regulatory influence of chromatin architecture, remodeling and modification on transcription protein kinetics. This proposal aims to test the hypothesis that chromatin binding of transcription initiation proteins in the cell nucleus 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 in the model organism budding yeast to monitor the diffusive behavior of transcription initiation proteins at high spatio-temporal resolution. This ‘in vivo biochemistry’ approach differs from and is complementary to ChIP-Seq techniques that map transcription factor occupancy genome-wide at base pair resolution but provide little information on binding dynamics. To elucidate regulatory contributions of chromatin architecture to transcription initiation protein dynamics, we will measure their mobilities in yeast mutants conditionally depleted for chromatin remodeling and modification enzymes. 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 allows labeling with a cell-permeable organic fluorophore (Janelia Fluor). Live- cell imaging of fluorescently labeled transcription factors at single-molecule resolution will measure protein diffusion and distinguish between chromatin-bound and chromatin-free populations and estimate residence times of the bound population. Further, we will use conditional depletion of 6 major chromatin remodelers and histone modifiers to reveal changes in the diffusive parameters of transcription initiation proteins under conditions of chromatin perturbation to inform which among several diffusive parameters are subject to chromatin controls. By combining with conditional mutant genetics with live-cell single-molecule imaging, we hope to transform understanding of the kinetic mechanisms by which chromatin architecture regulates the transcription initiation process and develop a potentially routine technology complementary to current genome-wide analytical techniques to benefit other areas of yeast nuclear and chromosome biology, including studies of DNA replication, repair, and recombination.

真核基因控制的中央范式认为，序列特异性转录因子搜索和 在拥挤的核环境中找到其基因组靶标 染色质中的调节性DNA元素募集并指导转录预设的有序组装 由一般转录因子和RNA聚合酶组成的复合物。尽管身份和功能 在几百个与转录相关的蛋白质中，已知有关的蛋白质，几乎没有有关时间表的信息 他们在转录过程中运行的以及染色质结构的调节影响， 转录蛋白动力学的重塑和修饰。该建议旨在检验以下假设 细胞核中转录起始蛋白的染色质结合在活细胞中快速动力学发生，并且 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 模型生物体中活细胞的活细胞单分子成像，以监测 在高时空分辨率下的转录起始蛋白。这种“体内生物化学”方法有所不同 从并且已完成绘制转录因子占用基因组的芯片序列技术 基对分辨率，但很少提供有关绑定动力学的信息。阐明监管贡献 染色质结构到转录启动蛋白动力学，我们将测量其在酵母中的迁移率 有条件地耗尽染色质重塑和修饰酶的突变体。 我们将设计并在功能上验证编码组件的DNA构建体代表 一般转录因子和主要序列特异性DNA结合转录因子融合到自我 标记蛋白质标签（Halotag），该标签允许用可渗透的有机荧光团（Janelia Fluor）标记。居住- 单分子分辨率下荧光标记的转录因子的细胞成像将测量蛋白质 染色质结合和无染色质种群之间的扩散和区别以及估计住所 被约束人口的时间。此外，我们将使用6个主要的染色质远离曲霉的条件耗竭和 Hisstone修饰符在条件下揭示转录起始蛋白不同参数的变化 染色质扰动的信息，以告知几个不同参数之间受染色质对照的约束。 通过将有条件的突变遗传学与活细胞单分子成像结合，我们希望转换 了解染色质结构调节转录启动的动力学机制 处理并开发潜在的常规技术完成，以当前全基因组分析 受益于其他酵母核和染色体生物学的技术，包括对DNA复制的研究， 维修和重组。