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定义哺乳动物基因组中减数分裂重组区域的解剖和控制。各种模型的研究表明，重组发生在减数分裂i期间创造的不同区域热点。事实上，据估计，高等真核生物约有1-2%的基因组是重组的，而其余的基因组则处于“冷”状态。然而，对于定义重组热点的顺式作用特征，以及复杂哺乳动物基因组中反式作用因子如何控制减数分裂重组，所知甚少。为了定义小鼠重组热点的解剖结构和调控，在Aim 1中，我们将在小鼠中识别和表征新的重组热点。我们将重点研究小鼠19号染色体的重组热点，并通过直接精子和卵母细胞分型，以及分析这些位点的基因组结构和可塑性，来表征它们的重组率和交叉/非交叉（CO/NCO）谱。我们利用重组近交系小鼠作为交叉库的初步研究已经建立，我们可以快速确定真正的重组热点。重组热点的识别将允许我们在Aim 2中定义指导小鼠重组热点活动的顺式作用特征。我们在这里的重点是询问在重组热点核心的顺式作用序列的局部差异的影响。我们将研究核小体在热点的占用率，并评估它们在影响双链断裂起始位点中的作用。我们的初步数据，使用一种基于精细化FACS的方法来纯化各个减数分裂阶段，提供了对核小体在重组热点和周围重组惰性区域的占用的精确的直接见解。利用重组自交系的力量，我们提出评估重组热点活动的远程遗传控制取决于它们所面对的配对同源物之间的相互作用和它们固有的局部性质。总的来说，拟议的研究将为复杂哺乳动物基因组中减数分裂重组热点的解剖和控制提供基础知识，并且它们也可能为这些分子机器在诸如不孕症和癌症等疾病状态中如何出错提供非常重要的见解。

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.