Genomic Organization of Recombination Hot Spots

描述（由申请人提供）：我们的实验揭示了一个迄今为止未知的、交互作用的大分子调控系统的存在，该系统通过激活、抑制和调节特定热点的活性来控制哺乳动物重组热点的位置和相对活性。这种调控系统的遗传变异使其得以发现，现在提供了一种识别其组成部分及其相互作用的方法，这是理解其与人类遗传学问题以及种群生物学和进化问题相关的重要的第一步。热点的位置及其在人类和小鼠（我们主要的哺乳动物研究模式生物）中的相对活动决定了连锁不平衡的模式和密切相关基因共同遗传的可能性，这是确定对人类健康和疾病重要基因的努力中的重要问题。我们发现的第一个调控基因Prdm9通过激活热点位置的重组来确定热点位置；它没有数量修饰因子，其作用与基因剂量无关。在这里，我们将研究控制热点活动数量水平的互补调控系统，这是阐明这种新型调控系统的结构及其影响遗传模式的能力的同等重要的问题。我们将研究C57BL/6J和CAST/EiJ小鼠品系不同基因调控的三个热点的定量调控；其中两种基因的活性因CAST等位基因的存在而降低，另一种基因的活性则增强。我们将通过应用一种新的、显著改进的、依赖于高通量DNA测序的精子样本热点活动定量分析系统来实现这一目标；绘制和克隆相关调控基因；测试他们之间的互动；确定它们是否以剂量依赖的方式起作用，表明它们是催化作用还是化学计量作用，最后，测试它们是否控制重组启动的速率，或在导致交叉或形成非交叉基因转换的可选重组途径之间做出决定。我们希望了解每个热点是否有自己独特的调控系统，还是有共同的调控元件，上下调控是否受到相同基因的影响，是否对重组的启动或重组替代途径的选择施加控制，以及这些基因之间的相互作用方式。这些数据连同这些基因的分子特性将可能为它们的作用机制提供重要线索。这些问题的答案将大大提高我们对最基本的生物过程之一——基因重组的理解。

DESCRIPTION (provided by applicant): Our experiments have revealed the existence of a hitherto unknown, trans-acting, macromolecular regulatory system controlling the location and relative activity of mammalian recombination hotspots by activating, suppressing and modulating the activity of specific hotspots. Genetic variation of this regulatory system enabled its discovery and now provides a means of identifying its components and their interactions, an essential first step in understanding its relevance to issues of human genetics as well as questions of population biology and evolution. The locations of hotspots and their relative activity in humans and mice (our principal mammalian research model organism) determine patterns of linkage disequilibrium and the possibilities for closely linked genes to be coinherited, important issues in efforts to identify genes important in human health and disease. The first regulatory gene we identified, Prdm9, determines hotspot locations by activating recombination there; it has no quantitative modifiers, and its role is independent of gene dosage. Here we will investigate the complementary regulatory system controlling quantitative levels of hotspot activity, a matter of equal concern in elucidating the structure of this novel regulatory system and its ability to influence inheritance patterns. We will study the quantitative regulation of three hotspots whose activities are regulated by genes that differ between the mouse strains C57BL/6J and CAST/EiJ; two have their activity reduced by the presence of CAST alleles, and one has its activity enhanced. We will do so by applying a new, considerably improved, quantitative assay system for hotspot activities in sperm samples that relies on high throughput DNA sequencing; mapping and cloning the regulatory genes involved; testing their interactions with each other; determining whether they act in a dose dependent manner, indicating whether they act catalytically or stoichimetrically, and finally, testing whether they control the rate of initiation of recombination or the decision between the alternative recombination pathways leading to crossing over or the formation of non-crossover gene conversions. We expect to learn whether each hotspot has its own unique regulatory system or there are shared regulatory elements, whether up and down regulation are effected by the same genes, whether controls are exerted on the initiation of recombination or the choice between alternate pathways of recombination, and the manner in which any of these genes interact with each other. These data together with the molecular identity of these genes will likely provide important clues as to their mechanism of action. The answers to these questions will considerably enhance our understanding of one of the most basic biological processes, genetic recombination. PUBLIC HEALTH RELEVANCE: Genetic recombination is the biological process that enables us to screen human populations for genes determining human health and disease. The proposed experiments will enhance our ability to carry out such studies by increasing our understanding of how recombination is regulated. The findings are applicable to every human condition that is genetically influenced, including all of our major diseases.