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(申请人提供)：针对美国国立卫生研究院的4D核组FOA成像工具，我们在此提出两种新的成像方法。当这些方法结合在一起时，可以提供纳米级的空间分辨率，以及毫秒到小时的动态染色质结构重排及其与细胞激活和转录的关系的时间分辨率。第一项新技术名为SUHI(亚零值随机高分辨率成像平台)，通过将智能标记设计与低温荧光相结合，以及使用偏振激发和耗尽的发射体控制，实现了定量、随机和单分子成像。这导致了核染色质的1-5纳米各向同性结构分辨率，并能够 以辨别DNA元件，如增强子、抑制子或可被定位和追踪的基因位点。第二种方法，称为3D-SMRT显微镜(三维单分子实时显微镜，提交手稿)，是最近描述的MFM(多焦点显微镜)的扩展。它在活细胞中以毫秒到小时的时间尺度提供实时、同步、多色、30-80纳米分辨率的跟踪。通过实施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上的运动。为了能够分析数百个处于不同激活状态的细胞，我们还描述了我们针对寿司和3D-SMRT的简化图像处理工作流程，允许自动分析数百个单个细胞和许多激活状态的多个位置。我们将把显微镜放置在UMMS的核心设施中，使4D核组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。我们把重点放在Fos基因座上，因为这个“即刻-早期反应”基因在静止状态下c-Fos的mRNA和蛋白表达很低或根本没有表达。然而，一旦激活，就会迅速诱导c-Fos，这种诱导只持续几个小时。热图描绘了FOS基因周围的染色体内相互作用矩阵，表明该基因在该位点上存在广泛的环状结构。通过标记活细胞中的DNA，我们可以解决该基因座及其周围增强子周围的染色质环状变化，确定潜在的基因座向核周和核孔的位置移动，并将其与静止和激活时的mRNA表达和输出相关联。通过与4D基因组联盟的成员合作，我们设想该项目的最后阶段将包括与生化、结构和全基因组图谱衍生数据的直接关联，从而揭示基因组信息如何指定静止、激活、细胞发育期间和疾病状态下基因表达的空间和时间表达的正确执行。

 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.