The Anatomy and Regulation of Mouse Recombination Hotspots.

描述（由申请人提供）：拟议研究的长期目标是确定哺乳动物基因组中减数分裂重组区域的解剖学和控制。在各种模型中的研究表明，重组发生在减数分裂I期间创造的热点的不同区域。事实上，据估计，高等真核生物的基因组中约1-2%是重组基因组，而其余的基因组则处于“冷”状态。然而，很少有人知道关于定义重组热点的顺式作用特征，也没有反式作用因子如何控制复杂的哺乳动物基因组中的减数分裂重组。为了定义小鼠重组热点的解剖学和调控，在目标1中，我们将鉴定和表征小鼠中的新重组热点。我们将集中精力定义小鼠19号染色体的重组热点，并通过直接的精子和卵母细胞分型，并通过分析野生小鼠种群中这些位点的基因组结构和可塑性来表征其速率和交叉/非交叉（CO/NCO）特征。我们使用重组近交系小鼠作为交叉文库的初步研究已经确定，我们可以快速鉴定真正的重组热点。重组热点的鉴定将允许我们在目的2中定义指导小鼠中重组热点活性的顺式作用特征。我们在这里的重点是询问在重组热点的核心顺式作用序列的局部差异的影响。我们将研究核小体占据热点，并评估其在影响双链断裂（DSB）起始位点的作用。我们的初步数据，使用一个完善的基于流式细胞仪的方法来纯化的各个减数分裂阶段，提供了精致的直接洞察重组热点和周围的重组惰性区域的核小体占用。利用重组近交系的力量，我们建议根据它们所面临的配对同源物和它们内在的局部性质之间的相互作用来评估重组热点活性的远程遗传控制。总的来说，拟议的研究将提供关于复杂哺乳动物基因组中减数分裂重组热点的解剖和控制的基础知识，它们也可能提供关于这些分子机器如何在不孕症和癌症等疾病状态中出错的非常重要的见解。 公共卫生相关性：这项修订后的R 01拨款申请的拟议研究与我们对基因组周转和进化的理解直接相关，我们试图定义重组热点的解剖和控制，这些热点是染色体在每一代之间重新洗牌的首选位点。令人惊讶的是，这些热点仅代表整个基因组的一小部分（1%至2%），迄今为止，这些位点的大多数分析仅限于酵母，酵母的基因组比高等真核生物的基因组小得多，也简单得多。拟议的研究将定义小鼠基因组中新重组热点的解剖学和活性，并将评估已知在重组中起关键作用的因素对它们的控制。这些研究将为理解复杂哺乳动物基因组中重组是如何控制的奠定基础。

DESCRIPTION (provided by applicant): The long-term goal of the proposed research is to define the anatomy and control of meiotic recombinogenic regions in mammalian genomes. Studies in a variety of models have demonstrated that recombination occurs in distinct regions coined hotspots during meiosis I. Indeed, it has been estimated that ~1-2% of the genomes of higher eukaryotes are recombinogenic, while the remainder of the genome is left in the "cold". However, very little is known regarding the cis-acting features that define recombination hotspots, nor how trans-acting factors control meiotic recombination in complex mammalian genomes. To define the anatomy and regulation of mouse recombination hotspots, in Aim 1 we will identify and characterize novel recombination hotspots in the mouse. We will focus our efforts on defining the recombination hotspots of mouse chromosome 19, and will characterize their rates and crossover/non-crossover (CO/NCO) profiles by direct sperm and oocyte typing, and by analyzing the genomic structure and plasticity of these loci in wild-house mouse populations. Our Preliminary Studies using recombinant inbred mice as a crossover library have established that we can rapidly identify bona fide recombination hotspots. The identification of recombination hotspots will allow us in Aim 2 to define the cis-acting features that direct recombination hotspot activity in the mouse. Our focus here is to interrogate the influence of local differences of cis-acting sequences at the cores of recombination hotspots. We will study nucleosome occupancy at hotspots and assess their role in influencing double strand break (DSB) initiation sites. Our preliminary data, using a refined FACS based method to purify the various meiotic stages, have provided exquisite direct insight into the nucleosome occupancy at recombination hotspots and the surrounding recombinogenic inert regions. Using the power of recombinant inbred strains we propose to assess the long-range genetic control of recombination hotspots activity depending on the interplay between the paired homolog they are faced to and their intrinsic local nature. Collectively, the proposed studies will provide fundamental knowledge regarding the anatomy and control of meiotic recombination hotspots in complex mammalian genomes, and they may also provide very important insights into how these molecular machines go awry in disease states such as infertility and cancer. PUBLIC HEALTH RELEVANCE: The proposed research of this revised R01 grant application is directly relevant to our understanding of genome turnover and evolution, where we seek to define the anatomy and control of recombination hotspots, which are preferred sites where chromosomes reshuffle between each generation. Surprisingly, these hotspots only represent a small fraction (1% to 2%) of the whole genome, and to date most analyses of these sites have been limited to the yeast, which have a much smaller and simpler genome than those of higher eukaryotes. The proposed studies will define the anatomy and activity of new recombination hotspots in the mouse genome, and will assess their control by factors known to play critical roles in recombination. These studies will lay the foundation for understanding how recombination is controlled in complex mammalian genomes.