Rapid genome-wide mapping of mammalian meiotic recombination hotspots.

描述(由申请人提供)：减数分裂过程中发生的重组对遗传多样性、物种适合性和进化至关重要。在这里，染色体的洗牌不是随机发生的，而是在真核基因组中不同的“热点”发生的。重组热点的解剖和控制还知之甚少，特别是在高等真核生物中，因为很难绘制这些位点的图谱，所以很少被详细描述。从历史上看，该领域一直依赖耗时且昂贵的人类种群研究(HapMap项目)或在小鼠身上进行广泛的重组近交系定位。在R21的这一应用中，我们寻求开发快速、稳健和成本效益高的方法来生成高等真核生物重组热点的全基因组图谱。对酵母等低等真核生物的研究表明，在Spo11启动DSB之后，会发生一系列精确的事件。利用这一坚实的基础，加上我们在FACS结合染色质免疫沉淀(CHIP)纯化减数分裂细胞方面的独特专业知识，我们的目标是通过靶向与Dmc1蛋白结合的DNA在DSB形成后立即引导链侵袭，在小鼠中产生DSB“重组组”图谱。真正的热点图谱将通过重组近交系的交叉分析以及其他染色体上的其他组来验证一些新的热点。总的来说，拟议的研究将验证重组热点的全基因组图谱的创新方法，然后该领域可以应用这些方法来询问它们的解剖和控制。此外，我们相信本文开发的方法可以快速应用于跨菌株和物种(包括人类)的重组热点的定位，这一知识将为理解减数分裂重组在指导遗传多样性和基因组进化中的作用奠定基础。 公共卫生相关性：减数分裂过程中染色体的重新洗牌确保了每一代有性繁殖配子的遗传多样性。这种重新洗牌不是随机的，而是发生在被称为重组热点的非常特定的区域，这些热点只占整个基因组的一小部分(1-2%)。高等真核生物中重组热点的解剖和控制知之甚少，这反映了识别热点的困难，到目前为止，识别热点一直依赖于大规模的人类群体研究(HapMap项目)或在小鼠中进行广泛的育种和低分辨率的图谱研究。这项拟议的研究旨在通过获得与DMC1蛋白结合的DNA来快速生成高分辨率的全基因组重组热点图谱，DMC1蛋白在重组的初始步骤中引导链入侵。R21中提出的研究将提供关于减数分裂重组在引导遗传多样性和推动进化中所起作用的基础知识。

DESCRIPTION (provided by applicant): Recombination that occurs during meiosis is essential for genetic diversity, species fitness, and evolution. Here the shuffling of chromosomes does not occur at random but rather at distinct "hotspots" in eukaryotic genomes. The anatomy and control of recombination hotspots is poorly understood, especially in higher eukaryotes, as very few have been characterized in detail due to difficulties in mapping these sites. Historically the field has relied on time-consuming and expensive human population studies (the HapMap project) or extensive recombinant inbred mapping in mice. In this R21 application we seek to develop rapid, robust and cost effective methods to generate genome-wide maps of recombination hotspots in higher eukaryotes. Studies in lower eukaryotes such as yeast have revealed that following Spo11 initiated DSB, a precise cascade of events occur. Using this strong foundation together with our unique expertise in purifying meiotic cells by FACS combined with chromatin immuno-precipitation (ChIP), we are aiming to generate DSB "recombinome" atlases in mice by targeting DNA bound to the Dmc1 protein, which directs strand invasion immediately following DSB formation. The mapping of bona fide hotspots will be validated for some of the new hotspots identified by crossover analyses of recombinant inbred strains as well as by other groups on other chromosomes. Collectively, the proposed studies will validate innovative approaches for genome-wide mapping of recombination hotspots, which then can be applied by the field to interrogate their anatomy and control. Further, we believe the methods developed herein can be rapidly applied to map recombination hotspots across strains and species, including humans, and that this knowledge will lay the foundation for understanding the roles of meiotic recombination in directing genetic diversity and genome evolution. PUBLIC HEALTH RELEVANCE: The reshuffling of chromosomes during meiosis ensures genetic diversity of gametes for sexual reproduction at each generation. This reshuffling is not random but rather occurs at very specific regions coined recombination hotspots, which only represent a small fraction (1-2%) of the whole genome. The anatomy and control of recombination hotspots in higher eukaryotes is poorly understood, and this reflects difficulties in identifying hotspots, which to date has relied on large human population studies (the HapMap project) or extensive breeding and low-resolution mapping studies in the mouse. The proposed research seeks funding to rapidly generate high-resolution genome-wide atlases of recombination hotspots by obtaining DNA bound to the DMC1 protein which directs strand invasion during the initial steps of recombination. The research proposed in this R21 will provide fundamental knowledge regarding the roles of meiotic recombination in directing genetic diversity and in driving evolution.