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: 9764307
• 负责人: PAUL D. KAUFMAN
• 金额: $ 29.68万
• 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 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9764307
• 关键词: 10q; 3-Dimensional; Adopted; Affect; Biological; Biological Process; CRISPR/Cas technology; 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; stem cell differentiation; 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.