Visualizing local and global chromatin architecture, and gene expression in the individual cell by structural (SUSHI) and temporal (3D-SMRT) single molecule imaging

通过结构 (SUSHI) 和时间 (3D-SMRT) 单分子成像可视化局部和整体染色质结构以及单个细胞中的基因表达

• 批准号: 9749351
• 负责人: David Grunwald
• 金额: $ 10万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-01 至 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9749351
• 关键词: 3' Untranslated Regions; Algorithmic Analysis; Architecture; Atlases; Binding Proteins; Biochemical; CRISPR/Cas technology; Cell Line; Cell Nucleus; Cell model; Cells; Chromatin; Chromatin Loop; Chromosome Mapping; Chronology; Code; Core Facility; Custom; DNA; Data; Detection; Development; Dimensions; Disease; ERG gene; Electron Microscopy; Elements; Enhancers; FOS Protein; FOS gene; Fluorescence; Gene Activation; Gene Expression; Genes; Genetic Transcription; Genome; Genomics; HeLa S3; Hour; Image; Image Analysis; Imaging Device; Imaging technology; Individual; Label; Light; Mammalian Cell; Manuscripts; Maps; Messenger RNA; Methodology; Methods; Microscope; Microscopy; Movement; Nuclear; Nuclear Pore; Nuclear Structure; Pathway interactions; Process; Proteins; Reaction; Resolution; Rest; Specific qualifier value; Stains; Structure; System; Techniques; Technology; Testing; Time; Translating; United States National Institutes of Health; Validation; base; cellular development; computerized data processing; cryogenics; design; experimental study; falls; gene product; genome-wide; high resolution imaging; image processing; image registration; imaging modality; imaging platform; interest; light microscopy; mRNA Export; mRNA Expression; mammalian genome; member; millisecond; molecular imaging; nanometer; nanometer resolution; novel; novel imaging technique; organizational structure; quantitative imaging; response; single molecule; spatiotemporal; stem; structural genomics; temporal measurement; tool

Project Summary: Responding to the NIH’s 4D Nucleome FOA for Imaging Tools, we propose here two novel imaging methods. When combined, these methods provide nanometer spatial resolution, and millisecond to hour temporal resolution of dynamic chromatin architecture rearrangement and its relation to cell activation and transcription. The first novel technique, termed SUSHI (SUb-zeroº-Stochastic-High-resolution-Imaging platform), enables quantitative, stochastic, single molecule imaging by combining intelligent labeling design with cryogenic fluorescence and emitter control using polarized excitation and depletion. This results in 1-5 nanometer isotropic structural resolution of nuclear chromatin, and the ability to discern DNA elements such as enhancers, suppressors or gene loci that can be mapped and tracked. The second method, termed 3D-SMRT Microscopy (three-dimensional Single-Molecule Real-Time microscopy, manuscript submitted), is an expansion on the recently described MFM (multi-focus microscopy). It provides real-time, simultaneous, multicolor, 30-80 nanometer-resolution tracking in the living cell at a millisecond to hour timescale. By implementing a well thought out labeling strategy, this method also allows for the detection of DNA elements and their nuclear movement in time and space. To be able to analyze hundreds of cells in different activation states, we also describe our streamlined image processing workflows for both SUSHI and 3D-SMRT, permitting the automated analysis of multiple loci of hundreds of single cells and many activation states. We will make the imaging platforms available to all members of the 4D Nucleome consortium by placing the microscopes in a core facility at UMMS, and will share all data processing techniques via code sharing. We focus on the FOS gene locus, as this “immediate-early response” gene has low to no expression of c-Fos mRNA and protein at rest. Upon activation, however, there is a rapid induction of c-Fos, which persists only for a few hours. Heat maps depicting intrachromosomal interaction matrices around the FOS gene indicate extensive looping at this locus. By labeling DNA in a living cell, we can resolve chromatin looping changes around the locus and its surrounding enhancers, determine potential gene locus positional movements toward the nuclear periphery and nuclear pores, and correlate this with mRNA expression and export, both at rest and upon activation. By collaborating with members of the 4D Nucleome consortium, we envision the final stage of this project to include direct correlation with biochemical, structural and genome-wide mapping derived data, thereby shedding light on how genomic information specifies proper execution of spatial and temporal gene expression at rest, upon activation, during cellular development and in diseased states.

项目概要： 为了响应 NIH 的 4D Nucleome FOA for Imaging Tools，我们在此提出两种新颖的成像方法。 结合起来，这些方法可提供纳米级的空间分辨率和毫秒到小时的时间分辨率 动态染色质结构重排的分辨率及其与细胞激活和转录的关系。 第一项新技术称为 SUSHI（亚零度随机高分辨率成像平台），可实现 通过将智能标记设计与低温相结合，实现定量、随机、单分子成像 使用偏振激发和耗尽的荧光和发射器控制。这导致 1-5 纳米 核染色质的各向同性结构分辨率，以及辨别 DNA 元件（例如增强子）的能力， 可以绘制和跟踪的抑制子或基因位点。 第二种方法，称为 3D-SMRT 显微镜（三维单分子实时显微镜） 显微镜（手稿已提交）是最近描述的 MFM（多焦点显微镜）的扩展。 它以 30-80 纳米分辨率在活细胞中提供实时、同步、多色跟踪 毫秒到小时的时间尺度。通过实施深思熟虑的标签策略，该方法还可以 DNA 元素及其核在时间和空间上的运动的检测。 为了能够分析数百个处于不同激活状态的细胞，我们还描述了我们的简化图像 SUSHI 和 3D-SMRT 的处理工作流程，允许自动分析多个位点 数百个单细胞和许多激活状态。 我们将把成像平台提供给 4D Nucleome 联盟的所有成员 UMMS 核心设施中的显微镜，并将通过代码共享来共享所有数据处理技术。 我们关注 FOS 基因位点，因为这种“立即早期反应”基因的 c-Fos 表达较低甚至没有 静止状态下的 mRNA 和蛋白质。然而，激活后，c-Fos 会快速诱导，仅持续一段时间 几个小时。描绘 FOS 基因周围染色体内相互作用矩阵的热图表明 在此位点处广泛循环。通过标记活细胞中的 DNA，我们可以解决染色质环的变化 在基因座及其周围增强子周围，确定潜在的基因位点位置移动 核外围和核孔，并将其与静止和静止状态下的 mRNA 表达和输出相关联 激活后。 通过与 4D Nucleome 联盟的成员合作，我们设想该项目的最后阶段 包括与生化、结构和全基因组图谱衍生数据的直接相关性，从而 揭示基因组信息如何指定空间和时间基因表达的正确执行 静止时、激活时、细胞发育期间和患病状态下。