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

 描述(申请人提供)：为了了解人类基因组的功能，仅知道基因组序列是不够的。我们还需要知道基因组中所有DNA分子(染色体)在细胞核内的物理3D路径。对于基本的基因组活动，例如转录、复制及其传递给 在下一代细胞分裂中，进一步了解3D架构如何动态变化是必要的。到目前为止，传统光学显微镜在单细胞水平上的洞察仍然停留在整个染色体区域的表面水平，因为拓扑相关结构域(TADS)及其连接纤维的关键基因组结构元素位于光的衍射极限以下。此外，我们对动态知识知之甚少，因为在活细胞中进行序列特异性标记一直很困难，而且染色质动力学对光特别敏感。两项技术突破，即超分辨显微镜(SRM)的出现和新的计算数据分析算法的结合，以及通过基于基因组编辑的工具对活细胞中任何感兴趣的DNA序列进行荧光标记的能力，现在使解决这一阻碍我们进步的根本障碍成为可能。在这里，我们建议开发3D和4D SRM技术，使我们能够确定稳定染色质结构域的3D结构，解决这些结构域如何在3D中相互连接和组织形成染色体，并在细胞分裂过程中实时监测结构动态的DNA序列。我们将提供(1)现实的 计算机模拟细胞核中的所有染色质纤维，重建算法可以将SRM产生的一系列探针位置转换为3D染色质路径；(2)新的标记策略，使用与SRM兼容的探针以~10种可分辨的颜色标记整个基因组，并以两种颜色标记活细胞中的多个特定位点；以及(3)多色超临界角度和倒置晶格光片显微镜，分别以高通量样本和活细胞处理的~20 nm和~30 nm分辨率进行标记。这些计算、实验和成像技术的集成将产生一个集成的工作流程，用于3D和4D、强大和完全自动化的基因组结构分析。然后，我们将使用这一工作流程在三个结构水平上分析基因组：单个TADS、TAD簇和整个染色体，并通过活细胞3D超分辨率显微镜跟踪整个染色体在细胞周期中的结构变化。由此产生的数据将是我们科学知识的突破--第一张人类单个细胞细胞核中线性基因组序列的3D路径图，以及这种3D组织如何在细胞周期中变化的第一个表征。确定单个细胞基因组结构的可靠成像技术将对基因组生物学的所有领域以及 细胞周期和有丝分裂研究，并为临床应用提供了许多令人兴奋的可能性--更好地理解和诊断与基因组不稳定相关的疾病，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.