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

 描述（由申请人提供）：响应NIH的成像工具4D Nucleome FOA，我们在此提出了两种新型成像方法。当组合时，这些方法提供纳米空间分辨率，以及动态染色质结构重排及其与细胞活化和转录的关系的毫秒至小时的时间分辨率。第一种新技术，称为SUSHI（Sub-zeroº-Stochastic-High-resolution-Imaging platform），通过将智能标记设计与低温荧光和使用偏振激发和耗尽的发射器控制相结合，实现定量，随机，单分子成像。这导致核染色质的1-5纳米的各向同性结构分辨率，并且能够 以辨别DNA元件，如增强子、抑制子或可以定位和跟踪的基因位点。第二种方法，称为3D-SMRT显微镜（三维单分子实时显微镜，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在毫秒到小时的时间尺度。通过实施深思熟虑的标记策略，该方法还允许检测DNA元件及其在时间和空间中的核运动。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SUSHI和3D-SMRT的简化图像处理流程，允许自动分析数百个单细胞和许多激活状态的多个位点。我们将通过将显微镜放置在UMMS的核心设施中，使4D Nucleome联盟的所有成员都可以使用成像平台，并将通过代码共享共享所有数据处理技术。我们专注于FOS基因位点，因为这种“立即早期反应”基因在静息时c-Fos mRNA和蛋白质的表达很低甚至没有。然而，一旦激活，c-Fos的快速诱导仅持续几个小时。热图描绘了FOS基因周围的染色体内相互作用矩阵，表明该基因座存在广泛的环。通过标记活细胞中的DNA，我们可以解析基因座及其周围增强子周围的染色质循环变化，确定潜在的基因座向核周边和核孔的位置移动，并将其与mRNA表达和输出（静止和激活时）相关联。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，结构和全基因组映射衍生数据的直接相关性，从而阐明基因组信息如何指定在静止时，激活时，细胞发育期间和疾病状态下正确执行空间和时间基因表达。

 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.