Reconstructing the dynamic 3D architecture of the human genome by superresolution microscopy and DNA sequence modelling.

 描述（由申请人提供）：为了理解人类基因组的功能，仅知道基因组序列是不够的。我们还需要知道细胞核内基因组的所有DNA分子（染色体）的物理3D路径。对于基本的基因组活动，例如转录、复制及其向细胞的传递， 在下一代细胞分裂中，更有必要了解3D结构如何动态变化。从传统的光学显微镜在单细胞水平上的见解迄今为止仍然停留在整个染色体领域的表面水平，因为拓扑相关域（TADs）的关键基因组结构元素及其连接纤维低于光的衍射极限。此外，我们有很少的动态知识，因为在活细胞中的序列特异性标记一直是困难的，染色质动力学是特别光敏。两项技术突破，即具有几个核小体的分辨率的超分辨率显微镜（SRM）的出现与新的计算数据分析算法相结合，以及通过基于基因组编辑的工具在活细胞中荧光标记任何感兴趣的DNA序列的能力，现在使得有可能解决我们进步的这一根本障碍。在这里，我们建议开发3D和4D SRM技术，使我们能够确定稳定的染色质结构域的3D结构，解决这些结构域是如何相互连接和组织在3D中形成染色体，并监测结构动态DNA序列在细胞分裂过程中的真实的时间。我们将提供（1）现实的 细胞核中所有染色质纤维的计算机模拟，可以将一系列SRM生成的探针位置转换成3D染色质路径的重建算法：（2）用SRM兼容探针以约10种可辨别颜色以10 kb分辨率标记整个基因组的新标记策略，以及以两种颜色标记活细胞中多个特异性基因座的方法;和（3）多色超临界角和倒置晶格光片显微镜，分辨率分别为~20 nm和~30 nm，具有高通量样品和活细胞处理。这些计算、实验和成像技术的整合将为3D和4D、鲁棒和全自动的基因组结构分析提供集成的工作流程。然后，我们将使用此工作流程在三个结构水平上分析基因组：单个TADs，TADs簇和整个染色体，并通过活细胞3D超分辨率显微镜跟踪细胞周期期间整个染色体的结构变化。由此产生的数据将是我们科学知识的一个突破-单个人类细胞核中线性基因组序列的3D路径的第一张地图，以及这种3D组织在整个细胞周期中如何变化的第一个表征。用于确定单细胞基因组结构的可靠成像技术对于基因组生物学的所有领域以及对于 细胞周期和有丝分裂的研究，并提供了许多令人兴奋的可能性，为临床应用，以更好地了解和诊断疾病与基因组不稳定性，如癌症。

 DESCRIPTION (provided by applicant): In order to understand the function of the human genome, knowing the genome sequence alone is not sufficient. We also need to know the physical 3D path of all DNA molecules (chromosomes) of the genome within the nucleus of a cell. For essential genome activities, e.g. transcription, replication, and its transmission to the next generation in cell division, it is furthermore necessary to understand how the 3D architecture dynamically changes. Insights at the single cell level from conventional light microscopy have so far remained at the superficial level of whole chromosome territories since the critical genome structure elements of topologically associated domains (TADs) and their connecting fibers lie below the diffraction limit of light. Furthermore, we have very little dynami knowledge since sequence-specific labeling in live cells has been difficult, and chromatin dynamics are particularly light sensitive. Two technological breakthroughs, i.e. the advent of super-resolution microscopy (SRM) with a resolution of a few nucleosomes combined with novel computational data analysis algorithms, and the ability to label any DNA sequence of interest fluorescently in living cells by genome editing- based tools, now make it possible to address this fundamental barrier to our progress. Here, we propose to develop 3D and 4D SRM technologies to enable us to determine the 3D structure of stable chromatin domains, resolve how such domains are interconnected and organized in 3D to form a chromosome, and monitor the structurally dynamic DNA sequences in real time during cell division. We will provide (1) realistic computer simulations of all chromatin fibers in the nucleus, reconstruction algorithms which can transform a series of SRM- generated probe positions into the 3D chromatin path; (2) new labeling strategies to label the entire genome with 10 kb resolution with SRM-compatible probes in ~10 discernible colors, and a method to label multiple specific loci in living cells in two colos; and (3) multi-color supercritical angle and inverted lattice light-sheet microscopy with ~20 nm and ~30 nm resolution, respectively, with high-throughput sample and live cell handling. Integration of these computational, experimental, and imaging technologies will result in an integrated workflow for 3 and 4D, robust, and fully automated genome structure analyses. We will then use this workflow to analyze the genome on three structural levels: individual TADs, TAD clusters, and whole chromosomes, and follow structural changes of whole chromosomes during the cell cycle by live cell 3D super-resolution microscopy. The resulting data will be a breakthrough for our scientific knowledge - the first map of the 3D path of the linear genome sequence in the nucleus of a single human cell and the first characterization of how this 3D organization changes throughout the cell cycle. Reliable imaging technology to determine the genome structure of single cells will be invaluable for all fields of genome biology as well as for cell cycle and mitosis research, and offers many exciting possibilities for clinical applications t better understand and diagnose diseases associated with genome instability such as cancer.