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

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.

序列特异性转录因子搜索和识别的真核基因控制位置的中心范式 在拥挤的核环境中定位其基因组靶标，通常以组合和合作的方式发挥作用 染色质中的调控 DNA 元件，用于招募和指导转录前启动的有序组装 由一般转录因子和RNA聚合酶组成的复合物虽然具有相同的身份和功能。 已知数百种转录相关蛋白，但关于时间尺度的信息很少 它们在转录过程中发挥作用，以及染色质结构的调节影响， 该提案旨在检验以下假设： 细胞核中转录起始蛋白的染色质结合在活细胞中以快速动力学发生，并且 我们将使用染色质重塑和修饰在调节转录蛋白动力学中发挥作用。 在模型生物芽殖酵母中进行活细胞单分子成像，以监测其扩散行为 这种“体内生物化学”方法有所不同。 来自 ChIP-Seq 技术，并且与 ChIP-Seq 技术互补，该技术可在全基因组范围内绘制转录因子占用情况 碱基对解析，但提供很少的关于结合动力学的信息来阐明碱基对的调节贡献。 染色质结构到转录起始蛋白动力学，我们将测量它们在酵母中的迁移率 有条件地耗尽染色质重塑和修饰酶的突变体。 我们将设计和功能验证 DNA 构建体编码代表的组件 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体 标记蛋白标签 (HaloTag)，允许使用细胞可渗透的有机荧光团 (Janelia Fluor) 进行标记。 单分子分辨率的荧光标记转录因子的细胞成像将测量蛋白质 扩散并区分染色质结合群体和无染色质群体并估计居住地 此外，我们将使用 6 个主要染色质重塑剂的条件去除和 组蛋白修饰剂揭示转录起始蛋白在一定条件下扩散参数的变化 染色质扰动的结果，以告知几个扩散参数中的哪些参数受到染色质控制。 通过将条件突变遗传学与活细胞单分子成像相结合，我们希望能够改变 了解染色质结构调节转录起始的动力学机制 处理和开发一种潜在的常规技术，以补充当前的全基因组分析 使酵母核和染色体生物学其他领域受益的技术，包括 DNA 复制的研究， 修复、重组。

项目成果

Kinetic principles underlying pioneer function of GAGA transcription factor in live cells.

• DOI: 10.1038/s41594-022-00800-z
• 发表时间: 2022-07
• 期刊: NATURE STRUCTURAL & MOLECULAR BIOLOGY
• 影响因子: 16.8
• 作者: Tang, Xiaona;Li, Taibo;Liu, Sheng;Wisniewski, Jan;Zheng, Qinsi;Rong, Yikang;Lavis, Luke D.;Wu, Carl
• 通讯作者: Wu, Carl

Spatiotemporal coordination of transcription preinitiation complex assembly in live cells.

• DOI: 10.1016/j.molcel.2021.07.022
• 发表时间: 2021-09-02
• 期刊: Molecular cell
• 影响因子: 16
• 作者: Nguyen VQ;Ranjan A;Liu S;Tang X;Ling YH;Wisniewski J;Mizuguchi G;Li KY;Jou V;Zheng Q;Lavis LD;Lionnet T;Wu C
• 通讯作者: Wu C