Nucleolar Genomics During Early Mammalian Development

 描述(申请人提供)：在所有真核生物中，大的核糖体RNA是从重复的核糖体DNA(RDNA)基因转录而来的。这些rDNA重复序列形成核仁，核仁是核糖体组装的位置，是专门的、非膜结合的亚核细胞器。此外，核仁是许多蛋白质穿梭的动态中枢。较少被研究的是核仁在组织哺乳动物基因组的三维结构中的作用。染色体的长距离相互作用是非常有意义的，因为它们可以调节哺乳动物细胞的发育时间或基因表达的多样性。从人体细胞系分离的核仁相关DNA的深度测序分析表明，称为核仁相关结构域(NADS)的特定基因座与核仁形成频繁的三维关联。NAD是动态的，当核仁改变时，通过抑制rDNA转录，重新分布到核外围或中心周围的异染色质焦点。到目前为止，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，也是因为在哺乳动物植入前胚胎发育过程中，着丝粒周围染色质和核仁周围区域之间的相互作用是特别动态的。因此，我们建议应该在一个能够分析哺乳动物发育过程的系统中探索由核仁维持的3D基因组关联的生物学重要性；也就是说，一个可以从四个维度探索这些相互作用的功能的系统。因此，我们首次提出绘制小鼠基因组中的核仁相关区域(NAD)，确定这些关联在胚胎干细胞(ESC)分化过程中是如何改变的，并开发工具来研究固定和活的单细胞中这些高阶染色体的相互作用。为了与NIH倡议的目标保持一致，我们打算生产数据库和工具，以了解通过NAD相互作用对哺乳动物基因组结构和功能的4D调节，作为哺乳动物发育生物学社区的全面基础。此外，我们将确定这些关联在分化为三个胚层时是如何改变的，以及它们与植入后外胚层中发生的全球基因组重组是如何相关的。除了这些群体测量，我们还将通过基于CRISPR的靶向生成工具，用于可视化与活的单细胞中的核仁相关的富含重复序列的DNA。通过这种方式，我们的项目将是第一个分析NAD介导的基因组组织在哺乳动物细胞分化过程中的动态。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领域的主要新工具。

 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.