Functional analysis of programmed genome rearrangement

程序化基因组重排的功能分析

• 批准号: 8911843
• 负责人: Jeramiah James Smith
• 金额: $ 28.64万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-01 至 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8911843
• 关键词: Binding; Biological; Biological Assay; Biological Process; Biology; Candidate Disease Gene; Cell Death; Cell Lineage; Cell Proliferation; Cells; Chromosomes; Code; Computer Analysis; Control Animal; DNA; Data; Data Set; Development; Developmental Biology; Disease; Embryo; Embryonic Development; Epigenetic Process; Event; Fertility; Fertilization in Vitro; Gene Deletion; Genes; Genetic; Genetic Polymorphism; Genetic Recombination; Genome; Genome Stability; Genomics; Health; Homologous Gene; Human; Human Genome; Individual; Knowledge; Lampreys; Light; Maintenance; Maps; Mass Spectrum Analysis; Measures; Mediating; Meiosis; Methods; Modeling; Molecular; National Institute of General Medical Sciences; Outcome; Pattern; Petromyzon marinus; Play; Population; Property; Protein Binding; Proteins; Regulation; Research; Resistance; Resolution; Resources; Role; Site; Somatic Cell; Specificity; Staging; Study models; Testing; Transcript; Vertebrates; Work; analog; base; comparative; design; developmental genetics; gene function; genome sequencing; genome-wide; germline stem cells; insight; next generation sequencing; novel; pleiotropism; pluripotency; programs; promoter; recombinational repair; research study; scaffold; stem; stem cell biology; tumorigenesis; vertebrate genome

DESCRIPTION (provided by applicant): This proposal stems from our recent discovery that the sea lamprey (unlike other vertebrates) undergoes large- scale genome rearrangements during the normal course of its embryonic development. Programmed genome rearrangement (PGR) results in the highly reproducible elimination of ~20% of the lamprey's genome during the establishment of somatic cell lineages in the early embryo. Recent work has revealed that: 1) hundreds of gene-coding fragments are eliminated, 2) human homologs of lost genes generally possess "pluripotency" functions, 3) computational analysis of "next-gen" sequence datasets identify predicted recombination sites that mediate deletion events, which have been validated experimentally, indicating that PGR is mediated in part by recombination and 4) proteins expressed in the early embryo show sequence-specific binding to validated recombination sites. With the advent of extensive sequence resources for lamprey and methods for gene knockdown and transcript replacement, lamprey will continue to provide unique insights into the rearrangement biology of vertebrate genomes and the genetics of somatic vs. germline cell fate. As such, this research is broadly relevant to NIGMS research programs in genetics and developmental biology, particularly towards understanding the biology of genomes, chromosomes and epigenetics, vertebrate developmental genetics, stem cell biology, genetic mechanisms of DNA recombination/repair, and oncogenesis. The specific aims of this proposal will seek to further characterize lamprey PGR at the genetic level in order to deduce its molecular mechanisms and use this information to guide functional studies of the biological causes and consequences of genome rearrangement. Specific Aim 1 will allow us to further refine our functional models of PGR by developing a meiotic map for the lamprey genome and integrating this with existing somatic and germline genome assemblies. Analyses of this map will shed critical light on the scale, distribution, biological function, and likely mechanisms of deletion. Specific Aim 2 dissects the biological function of genes that are deleted through PGR. Experiments performed under this aim will use morpholino mediated gene knockdowns and transcript replacement to elucidate the function of somatically deleted genes and their human homologs. Specific Aim 3 identifies genes that mediate PGR and characterizes their molecular function. Experiments performed under this aim will use morpholino-mediated knockdown to elucidate the function of genes that bind recombination sites during PGR then use transcript replacement to test whether human homologs can partly or completely replace these functions. This will provide a direct assay for the capacity of identified human homologs to participate in somatic recombination. Critically, these approaches do not rely on pre-existing knowledge regarding the function of human genes and therefore hold great potential for the discovery of new gene functions. These studies are expected to provide unique insight into vertebrate genome biology and yield information that might not be achievable outside of the context of PGR.

描述（由申请人提供）： 这项提议源于我们最近发现的，海七lamp虫（与其他脊椎动物不同）在其胚胎发育的正常过程中经历了大规模的基因组重排。程序基因组重排（PGR）导致在早期胚胎中建立体细胞谱系期间，高度可重复地消除了约20％的lamprey基因组。 Recent work has revealed that: 1) hundreds of gene-coding fragments are eliminated, 2) human homologs of lost genes generally possess "pluripotency" functions, 3) computational analysis of "next-gen" sequence datasets identify predicted recombination sites that mediate deletion events, which have been validated experimentally, indicating that PGR is mediated in part by recombination and 4) proteins expressed in the early embryo show序列特异性结合与经过验证的重组位点。随着七lamp虫的广泛序列资源的出现以及用于基因敲低和替代的基因敲低和替代方法的方法，Lamprey将继续为脊椎动物基因组的重排生物学以及躯体与生殖细胞命运的遗传学提供独特的见解。因此，这项研究与遗传学和发育生物学的NIGMS研究计划广泛相关，特别是旨在了解基因组，染色体和表观遗传学的生物学，脊椎动物发育遗传学，干细胞生物学，DNA重组/修复的遗传学机制。该提案的具体目的将旨在进一步表征遗传水平上的七语PGR，以推断其分子机制，并使用此信息来指导基因组重排的生物学原因和后果的功能研究。特定的目标1将使我们能够通过为七lamp子基因组开发减数分裂图，并将其与现有的体细胞和种系基因组组合相结合，从而进一步完善我们的PGR功能模型。该地图的分析将使批判性地阐明量表，分布，生物学功能以及可能的缺失机制。特定目标2剖析了通过PGR删除的基因的生物学功能。在此目的下进行的实验将使用莫德诺里诺介导的基因敲低和替换成绩单来阐明体形删除的基因及其人类同源物的功能。特定目标3识别介导PGR并表征其分子功能的基因。在此目标下进行的实验将使用morpholino介导的敲低来阐明在PGR期间结合重组位点的基因的功能，然后使用转录本替换来测试人类同源物是否可以部分或完全替代这些功能。这将为确定的人类同源物参与躯体重组的能力提供直接测定。至关重要的是，这些方法不依赖于有关人类基因功能的知识，因此在发现新基因功能方面具有巨大潜力。预计这些研究将提供对脊椎动物基因组生物学的独特见解，并产生在PGR背景外无法实现的信息。