Functional analysis of programmed genome rearrangement

描述(由申请人提供)： 这一建议源于我们最近的发现，即七鳃鳗(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组。在早期胚胎体细胞谱系的建立过程中，程序性基因组重排(PGR)导致~20%的七鳃鳗基因组高度可重复地消除。最近的工作揭示：1)数百个编码基因的片段被消除，2)人类丢失基因的同源物通常具有“多潜能”功能，3)对“下一代”序列数据集的计算分析确定了介导缺失事件的预测重组位点，并已得到实验验证，表明PGR部分通过重组介导，4)在早期胚胎中表达的蛋白质显示序列特异性结合到有效的重组位点。随着广泛的七鳃鳗序列资源以及基因敲除和转录替换方法的出现，七鳃鳗将继续为脊椎动物基因组的重排生物学和体细胞与生殖系细胞命运的遗传学提供独特的见解。因此，这项研究与NIGMS在遗传学和发育生物学方面的研究计划广泛相关，特别是在了解基因组、染色体和表观遗传学、脊椎动物发育遗传学、干细胞生物学、DNA重组/修复的遗传机制和肿瘤发生方面。这项建议的具体目的将寻求在遗传水平上进一步描述七鳃鳗PGR的特征，以推断其分子机制，并利用这些信息指导对基因组重排的生物学原因和后果的功能研究。具体目标1将使我们能够通过开发七鳗鱼基因组的减数分裂图谱并将其与现有的体细胞和生殖系基因组组合相结合，进一步完善我们的PGR功能模型。对这一图谱的分析将对缺失的规模、分布、生物学功能和可能的机制提供关键的线索。特定目的2剖析通过PGR删除的基因的生物学功能。在这一目标下进行的实验将使用吗啉介导的基因敲除和转录替换来阐明体细胞缺失基因及其人类同源物的功能。特异靶3识别介导PGR的基因，并表征其分子功能。在这一目标下进行的实验将使用吗啉介导的敲除来阐明在PGR过程中结合重组位点的基因的功能，然后使用转录替换来测试人类同源物是否可以部分或完全取代这些功能。这将为已鉴定的人类同源物参与体细胞重组的能力提供直接的测试。关键是，这些方法不依赖于关于人类基因功能的预先存在的知识，因此在发现新的基因功能方面具有巨大的潜力。这些研究有望为脊椎动物基因组生物学提供独特的见解，并产生在PGR之外可能无法获得的信息。

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.