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（由适用提供）：在所有真核生物中，大型核糖体RNA都是从重复的核糖体DNA（rDNA）基因转录的。这些rDNA重复序列形成核仁，它们是核糖体组装部位的专业，非膜结合的亚核细胞器。此外，核橄榄是动态枢纽，众多蛋白质穿梭。核能在组织哺乳动物基因组的三维结构中的作用较少。远程染色体相互作用引起了极大的关注，因为它们可以调节哺乳动物细胞中基因表达的发育时机或变异。与从人体细胞系中分离的核核相关的DNA的深层测序分析表明，特定的局部称为核相关结构域（NAD），经常形成与核的三维关联。 NAD是动态的，通过抑制rDNA转录，将其重新分布在核周围或周围的异染色质焦点。迄今为止已经研究了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。早期发展是研究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，还因为哺乳动物植入前胚胎胚胎胚胎发育期间，周围粒层粒染色质和核酸区域之间的相互作用尤其动态。因此，我们建议在允许分析哺乳动物发育过程的系统中探索核维持的3D基因组关联的生物学重要性；也就是说，可以在四个维度中探索这些相互作用的功能的系统。因此，我们首次提出了小鼠基因组中与核仁相关结构域（NAD）的映射，确定在胚胎干细胞（ESC）分化过程中如何改变这些关联，并开发用于研究这些高阶染色体相互作用的工具。为了符合NIH倡议的目标，我们打算为通过NAD互动来理解哺乳动物基因组结构和功能的4D调节的数据库和工具，作为哺乳动物发育生物学社区的全面基础。此外，我们将在分化为三个细菌层中的每个层时如何改变这些关联，以及它们与在植入后植入层植入植物中发生的全球基因组重组的相关性。除了这些种群测量值外，我们还将生成工具，以通过基于CRISPR基于CRISPR的靶向实现活体细胞中与核仁相关的重复富集DNA。以这种方式，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力学的人。总而言之，该项目创建的综合数据库将构成哺乳动物发育生物学和基因组学群落的主要新工具。