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

基本信息

项目摘要

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: 响应NIH的4D Nucleome FOA成像工具,我们在这里提出了两种新的成像方法。 当结合使用时,这些方法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解析及其与细胞活化和转录的关系。 第一种新技术,称为SUSHI(零以下随机高分辨率成像平台), 通过将智能标记设计与低温结合的定量、随机、单分子成像 使用偏振激发和耗尽的荧光和发射体控制。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元件如增强子的能力, 抑制基因或基因位点,可以映射和跟踪。 第二种方法,称为3D-SMRT显微镜(三维单分子实时成像)。 显微镜,手稿提交),是最近描述的MFM(多焦点显微镜)的扩展。 它提供实时,同步,可调,30-80纳米分辨率的活细胞跟踪, 毫秒到小时的时间尺度。通过实施经过深思熟虑的标记策略,该方法还允许 检测DNA元素及其核在时间和空间中的运动。 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流线型图像 SUSHI和3D-SMRT的处理流程,允许自动分析多个基因座, 数百个单细胞和许多激活状态。 我们将通过将4D Nucleome联盟的所有成员都能使用成像平台, 在UMMS的核心设施,显微镜,并将通过代码共享共享所有的数据处理技术。 我们关注FOS基因位点,因为这种“立即早期反应”基因的c-Fos表达很低甚至没有 静止时的mRNA和蛋白质。然而,一旦激活,c-Fos的快速诱导,其仅持续24小时。 几个小时描绘FOS基因周围的染色体内相互作用矩阵的热图表明, 在这个位点形成了大量的环状通过标记活细胞中的DNA,我们可以解决染色质循环变化 围绕基因座及其周围的增强子,确定潜在的基因座位置移动, 核周边和核孔,并将其与mRNA表达和输出相关,无论是在休息和 在激活时。 通过与4D Nucleome联盟的成员合作,我们设想该项目的最后阶段, 包括与生物化学、结构和全基因组作图数据直接相关,从而 阐明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。

项目成果

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David Grunwald其他文献

David Grunwald的其他文献

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{{ truncateString('David Grunwald', 18)}}的其他基金

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:
    9306084
  • 财政年份:
    2015
  • 资助金额:
    $ 10万
  • 项目类别:
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:
    9918495
  • 财政年份:
    2015
  • 资助金额:
    $ 10万
  • 项目类别:

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