Genomic Organization of Recombination Hot Spots

描述(申请人提供)：我们的实验揭示了一种迄今未知的、反式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。这一调控系统的遗传变异使其得以发现，现在提供了一种确定其组成部分及其相互作用的手段，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的相关性的关键的第一步。在人类和小鼠(我们主要的哺乳动物研究模式生物)中，热点的位置和它们的相对活动决定了连锁不平衡的模式和紧密相连的基因共同遗传的可能性，这是努力识别对人类健康和疾病重要的基因的重要问题。我们鉴定的第一个调控基因Prdm9通过激活那里的重组来确定热点位置；它没有定量修饰物，它的作用与基因剂量无关。在这里，我们将研究控制热点活动的数量水平的互补调控系统，这是阐明这一新的调控系统的结构及其影响遗传模式的能力同样值得关注的问题。我们将研究三个热点的定量调控，它们的活性受C57BL/6J和CAST/EIJ小鼠品系之间的不同基因调控；两个热点的活性因CAST等位基因的存在而降低，一个热点的活性增强。为此，我们将应用一种新的、显著改进的、依赖于高通量DNA测序的精子样本热点活动的定量分析系统；绘制和克隆相关的调控基因；测试它们之间的相互作用；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作用；最后，测试它们是否控制重组的启动速度或导致交叉或非交叉基因转换形成的替代重组途径之间的决定。我们希望了解每个热点是否有自己独特的调控系统或有共同的调控元件，上调和下调是否由相同的基因影响，是否对重组的启动或在不同重组途径之间的选择进行控制，以及这些基因相互作用的方式。这些数据和这些基因的分子同一性可能会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.