Nucleolar Genomics During Early Mammalian Development

哺乳动物早期发育过程中的核仁基因组学

• 批准号: 9326974
• 负责人: PAUL D. KAUFMAN
• 金额: $ 39.5万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-01 至 2020-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9326974
• 关键词: 10q; Adopted; Affect; Biological; Biological Process; Cell Differentiation process; Cell Line; Cell Nucleolus; Cells; Chimeric Proteins; Chromatin; Chromosome Structures; Chromosomes; Clustered Regularly Interspaced Short Palindromic Repeats; Color; Communities; Computing Methodologies; Cytology; D4Z4; DNA; DNA Sequence; DNA analysis; Data; Data Set; Databases; Detection; Development; Developmental Biology; Developmental Process; Dimensions; Embryo; Embryonic Development; Epiblast; Eukaryota; Event; Fibroblasts; Foundations; Four-dimensional; Gene Expression; Genes; Genetic Transcription; Genome; Genomics; Germ Layers; Goals; Heterochromatin; Human; Human Cell Line; Karyotype; Laboratories; Mammalian Cell; Maps; Measurement; Measures; Mediating; Methods; Mus; Nuclear; Nuclear Lamina; Nucleolus Organizer Region; Organelles; Organism; Population; Proteins; Regulation; Repetitive Sequence; Resolution; Ribosomal DNA; Ribosomal RNA; Ribosomes; Role; Side; Site; Somatic Cell; Structure; Sum; System; Testing; Time; United States National Institutes of Health; Visualization software; arm; base; chromosome conformation capture; critical period; crosslink; deep sequencing; embryonic stem cell; experimental study; genome-wide; implantation; interest; live cell imaging; mammalian embryology; mammalian genome; mouse genome; preimplantation; public health relevance; rRNA Genes; telomere; three dimensional structure; tool

 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.

 描述（由申请人提供）：在所有真核生物中，大核糖体 RNA 都是由重复的核糖体 DNA (rDNA) 基因转录而成，这些 rDNA 重复形成核仁，核仁是特殊的非膜结合的亚核细胞器，是核糖体的位点。此外，核仁是许多蛋白质穿梭的动态中心，但核仁在组织三维空间中的作用却很少被研究。哺乳动物基因组的结构引起了人们极大的兴趣，因为它们可以调节哺乳动物细胞中的发育时间或基因表达的变化，对与人类体细胞系分离的核仁相关的 DNA 进行的深度测序分析表明，特定位点称为核仁相关结构域 (NAD)，与核仁形成频繁的三维关联，NAD 是动态的，重新分布到核外围或中心周异染色质灶。迄今为止，研究 NAD 的人类细胞系不适合回答有关 NAD 在哺乳动物发育中的作用的广泛问题，而不是研究 NAD 生物学功能的关键时期。不仅因为发生的基本生物学事件，而且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特别动态。由核仁维持的 3D 基因组关联应该在一个允许分析哺乳动物发育过程的系统中进行探索；也就是说，一个可以在四个维度上探索这些相互作用的功能的系统。小鼠基因组中的核仁相关结构域（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。的目标NIH 计划，我们打算开发数据库和工具，通过 NAD 相互作用来了解哺乳动物基因组结构和功能的 4D 调控，作为哺乳动物发育生物学界的综合基础。此外，我们将确定这些关联如何分化为每种关联。三个胚层，以及它们如何与植入后外胚层中发生的全局基因组重组相关。除了这些群体测量之外，我们还将生成用于可视化与活体核仁相关的富含重复 DNA 的工具。通过这种方式，我们的项目将成为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构成一个重要的新工具。哺乳动物发育生物学和基因组学界。