Nucleolar Genomics During Early Mammalian Development

 描述（由申请人提供）：在所有真核生物中，大核糖体RNA由重复核糖体DNA（rDNA）基因转录。这些rDNA重复序列形成核仁，核仁是专门的、非膜结合的亚核细胞器，是核糖体组装的位点。此外，核仁是许多蛋白质穿梭的动态枢纽。 较少研究的是核仁在组织哺乳动物基因组三维结构中的作用。染色体的长程相互作用是哺乳动物细胞中重要的研究对象，因为它们可以调控细胞的发育时间或基因表达的多样性。对与来自人类体细胞系的分离的核仁相关的DNA的深度测序分析已经表明，被称为核仁相关结构域（NAD）的特定基因座与核仁形成频繁的三维关联。NAD是动态的，通过抑制rDNA转录，在核仁改变后重新分布到核周边或着丝粒旁异染色质灶。 迄今为止研究NAD的人类细胞系不适合回答关于NAD在哺乳动物发育中的作用的广泛问题。早期发育是研究NAD生物学功能的关键时期，这不仅是因为发生的基本生物学事件，而且还因为在哺乳动物植入前胚胎发育期间，近着丝粒染色质和近核仁区域之间的相互作用特别动态。因此，我们建议，由核仁维持的3D基因组协会的生物学重要性，应探讨在一个系统，允许分析哺乳动物的发育过程，也就是说，在该系统中，这些相互作用的功能可以在四个维度进行探索。因此，我们首次提出在小鼠基因组中绘制核仁相关结构域（NAD），确定这些关联在胚胎干细胞（ESC）分化过程中如何改变，并开发用于研究固定和活单细胞中这些高阶染色体相互作用的工具。为了与NIH计划的目标保持一致，我们打算生产数据库和工具，以了解哺乳动物基因组结构和功能通过NAD相互作用的4D调节，作为哺乳动物发育生物学社区的全面基础。此外，我们将确定这些协会是如何改变分化成三个胚层的每一个，以及它们是如何与全球基因组重组发生在植入后外胚层。除了这些群体测量之外，我们还将通过基于CRISPR的靶向生成用于可视化活单细胞中与核仁相关的富含重复序列的DNA的工具。通过这种方式，我们的项目将是第一个分析哺乳动物细胞分化过程中NAD介导的基因组组织动态的项目。总之，该项目创建的综合数据库将成为哺乳动物发育生物学和基因组学界的一个重要新工具。

 DESCRIPTION (provided by applicant): In all eukaryotes, the large ribosomal RNAs are transcribed from repeated ribosomal DNA (rDNA) genes. These rDNA repeats form nucleoli, which are specialized, non-membrane-bound sub-nuclear organelles that are the sites of ribosome assembly. Additionally, nucleoli are dynamic hubs through which numerous proteins shuttle. Less well investigated is the role of nucleoli in organizing the three dimensional structure of mammalian genomes. Long-range chromosome interactions are of great interest because they can regulate the developmental timing or the variegation of gene expression in mammalian cells. Deep sequencing analyses of DNA associated with isolated nucleoli from human somatic cell lines have shown that specific loci, termed nucleolar-associated domains (NADs), form frequent three-dimensional associations with nucleoli. NADs are dynamic, being redistributed to the nuclear periphery or to pericentric heterochromatin foci upon nucleolar alteration via inhibition of rDNA transcription. The human cell lines in which NADs have been studied to date are not suited for answering broad questions about the role of NADs in mammalian development. Early development is a critical period to study NAD biological function, not just because of the fundamental biological events that occur, but also because interactions between pericentromeric chromatin and perinucleolar regions are particularly dynamic during mammalian preimplantation embryonic development. We therefore propose that the biological importance of the 3D genome associations maintained by nucleoli should be explored in a system that allows analysis of mammalian developmental processes; that is, a system in which the functionality of these interactions can be explored in four dimensions. Therefore, we propose for the first time to map of the nucleolar-associated domains (NADs) in the mouse genome, determine how these associations are altered during embryonic stem cell (ESC) differentiation, and develop tools for study of these higher-order chromosome interaction in fixed and live single cells. In keeping with the goals of the NIH Initiative, we intend to produe databases and tools for understanding the 4D regulation of mammalian genome structure and function via NAD interactions, as a comprehensive foundation for the mammalian developmental biology community. Furthermore, we will determine how these associations are altered upon differentiation into each of the three germ layers, and how they are correlated with the global genome reorganization that occurs in post-implantation epiblasts. In addition to these population measurements, we will generate tools for the visualization of the repeat-rich DNAs associated with nucleoli in live, single cells via CRISPR-based targeting. In this manner, our project will be the first to analyze the dynamics of NAD-mediated genome organization during mammalian cell differentiation. In sum, the comprehensive database created by this project will constitute a major new tool for the mammalian developmental biology and genomics communities.