Combined Cytological, Genomic, and Functional Mapping of Nuclear Genome Organization

 产品说明：几十年的显微镜研究表明，细胞核不是一个均匀的细胞器，而是由不同的隔室组成，如核仁、核斑点、核板层等结构。越来越多的证据表明，每个特定的基因组区域与这些隔室相关。这种基因组区室化与各种功能有关，但这些联系仍然知之甚少。有趣的是，板层相关结构域（LAD）共享特定的异染色质标记，定义具有不同遗传和表观遗传特性的染色质结构域。与其他核区室相关的基因组区域可以类似地定义不同类别的染色质结构域。一个主要的瓶颈，以更深入地了解核组织一直无法转换成全基因组的地图，显示哪些基因座与哪个车厢，以及如何染色体纤维之间的车厢之间的显微镜视图。此外，迫切需要更有效的方法来剖析大基因组区域靶向特定核区室的机制。最后，迫切需要高通量的方法来查询基因组区室化的功能相关性。 对于这个中心补助金，我们建议通过以下目标来满足这些需求：1。开发一种策略，将显微镜视图与全基因组图谱连接起来，再加上建模，揭示基因组区域相对于所有主要核区室的定位和动态。2.开发基因组的有效操作方法，以阐明将基因座靶向特定隔室的机制。3.开发测量、建模和验证核隔室功能相关性的方法。 这些方法的综合结果将揭示现在隐藏在纠缠的基因组，表观遗传和核组织特征之间的因果关系。这一提议的可行性包括广泛的核组织的结构和功能图，用于可视化内源性染色体位点的试剂，用于合成~ 100 kb DNA片段的强大管道，以及促进这些大片段重复、高保真地插入回基因组中的选定位点的细胞系。这些资源将为4D Nucleome Consortium的其他工作提供有力的补充。该中心提案的一个关键优势是其五名研究人员的经验和互补研究能力。他们将共同汇集他们的专业知识，对生物功能进行协调一致的调查， 核隔离的概念

 DESCRIPTION: Decades of microscopy have revealed that the nucleus is not a homogeneous organelle, but rather consists of distinct compartments such as nucleoli, nuclear speckles, the nuclear lamina, among other structures. Increasing evidence indicates that specific genomic regions each associate with these compartments. This genome compartmentalization has been linked to various functions, but these links are still poorly understood. Interestingly, Lamina Associated Domains (LADs) share specific heterochromatin marks, defining chromatin domains with distinct genetic and epigenetic properties. Genomic regions associating with other nuclear compartments may similarly define distinct classes of chromatin domains. One major bottleneck towards a deeper understanding of nuclear organization has been the inability to convert microscopy views of nuclear compartments into genome-wide maps that show which loci are associated with which compartment, and how the chromosomal fiber traverses between compartments. In addition, there is an urgent need for more efficient methods to dissect the mechanisms by which large genomic regions are targeted to specific nuclear compartments. Finally, there is an urgent need for high-throughput approaches that query the functional relevance of genome compartmentalization. For this Center grant, we propose to meet these needs through the following Aims: 1. Develop a strategy that connects microscopy views to genome-wide maps that, together with modeling, reveal the localization and dynamics of genomic regions relative to all major nuclear compartments. 2. Develop methods for efficient manipulation of the genome in order to elucidate mechanisms that target loci to specific compartments. 3. Develop methods to measure, model, and validate the functional relevance of nuclear compartments. The combined results of these approaches will reveal causal relationships now hidden among entangled genomic, epigenetic, and nuclear organization features. Deliverables of this proposal include a wide range of structural and functional maps of nuclear organization, reagents for visualizing endogenous chromosome loci, a powerful pipeline for synthesis of ~100kb DNA fragments, and cell lines facilitating repeated, high-fidelity insertio of these large fragments back into selected sites in the genome. These resources will provide a powerful complement to other 4D Nucleome Consortium efforts. A key strength of this Center proposal is the experience and complementary research capabilities of its five Investigators. Together they will pool their expertise for a concerted investigation into the biological functions of nuclear compartmentalization.