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

批准号：
9306084
负责人：
David Grunwald
金额：
$ 33万
依托单位：
UNIV OF MASSACHUSETTS MED SCH WORCESTER
依托单位国家：
美国
项目类别：
财政年份：
2015
资助国家：
美国
起止时间：
2015-09-30 至 2020-06-30
项目状态：
已结题

项目摘要

DESCRIPTION (provided by applicant): 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.

描述（由应用程序提供）：对NIH的4D核心FOA响应成像工具，我们在这里提出了两种新颖的成像方法。合并后，这些方法可提供纳米空间分辨率，并将动态染色质结构重排的毫秒到小时临时分辨率及其与细胞激活和转录的关系。第一种新型技术称为寿司（零下 - 策略高分辨率成像平台），通过将智能标记设计与低温荧光和发射器控制结合使用偏光兴奋和消耗，从而实现了定量，随机的单分子成像。这导致1-5纳米各向同性的核染色质结构分辨率，并能力辨别可以映射和跟踪的增强剂，补充剂或基因基因座等DNA元素。第二种方法称为3D-SMRT显微镜（三维单分子实时显微镜，手稿提交），是对最近描述的MFM（多焦点显微镜）的扩展。它在活单元中以毫秒至小时的时间尺度提供实时，同时的，多色，30-80个纳米分辨率跟踪。通过实施经过深思熟虑的标签策略，该方法还允许检测DNA元素及其在时空中的核运动。为了能够分析不同激活状态的数百个细胞，我们还描述了寿司和3D-SMRT的流线型图像处理工作流，允许对单个单元格和许多激活状态的多个单位层进行自动分析。我们将通过将显微镜放置在UMMS的核心设施中，并通过代码共享将显微镜放置在UMMS的核心设施中，从而使4D核心财团的所有成员都可以使用成像平台。我们专注于FOS基因基因座，因为这种“立即响应”基因在静止时具有低至Cos mRNA和蛋白质的表达。但是，激活后，C-FOS迅速诱导，仅持续几个小时。热图描绘了该基因座的FOS基因指示器周围的鲜膜内相互作用物质。通过将DNA标记在活细胞中，我们可以解决该基因座及其周围增强子周围的染色质循环变化，确定潜在的基因基因座位置向核外周和核孔的位置运动，并将其与mRNA表达和导出相关，并在静止后和激活后将其相关。通过与4D核心财团的成员合作，我们设想了该项目的最后阶段，将其与生化，结构和全基因组映射的数据进行直接相关，从而阐明基因组信息指定如何正确执行空间和临时基因在静止状态，在激活状态，在激活状态和分裂状态下进行蜂窝状态。