Transacting Genes Regulating Recombination Hotspot Activities

描述（由申请人提供）：人和小鼠中重组热点的位置及其经历遗传交叉的频率决定了连锁不平衡的模式和紧密连锁基因共同遗传的可能性;两者都是鉴定对人类健康和疾病重要的基因的关键问题。我们的实验揭示了一个迄今为止未知的大分子调控系统的存在，该系统通过激活、抑制和调节哺乳动物重组热点的活性来控制其位置和相对活性。这个调控系统中的遗传变异首先使其得以发现，现在提供了一种鉴定其组分及其相互作用的方法，这是理解其机制的重要一步，也是其与人类遗传学、群体生物学和进化问题的相关性。我们以这种方式鉴定的第一个调节蛋白PRDM 9通过修饰染色质结构，允许形成起始双链断裂，使小鼠热点家族发生重组。其他人同时将PRDM 9鉴定为人类重组热点的调节剂。 在这个项目中，我们将确定控制抑制重组在特定的热点，在理解重组的调节同样值得关注的互补调控系统的组件。我们将鉴定编码小鼠Chr 1上五个热点抑制因子的基因，这些抑制因子的活性受M. M. C57 BL/6 J（B6）和M. M. castaneus strain CAST/EiJ（CAST），通过：（a）应用新的、显著改进的定量测定系统，该系统使用NextGen DNA测序测量精子DNA样品中的热点活性;（B）使用该测定来定位和克隆所涉及的调控基因;（c）测试它们彼此之间的相互作用以及它们是否以剂量依赖性方式起作用，表明它们是催化作用还是化学计量作用;最后，（d）测试它们是否控制重组的起始或在导致交换与非交换基因转化的备选重组途径之间的决定。在单独的实验中，我们还将为鉴定M. M. Aceticus（B6）和M. M.肌细胞株PWD。 我们希望了解每个热点是否有自己独特的调控系统，或者是否有共同的调控元件，这些分子是什么，是否控制重组的启动或重组的替代途径之间的选择，以及这些基因相互作用的方式。这些数据与这些基因的分子特性一起将提供解决其作用机制所必需的信息。 这些结果将大大增强我们对最基本的生物过程之一--基因重组的理解。

DESCRIPTION (provided by applicant): The locations of recombination hotspots in humans and mice and their frequencies of undergoing genetic crossing over determine patterns of linkage disequilibrium and the possibilities for closely linked genes to be co-inherited; both are critical issues in efforts to identify genes important in human health and disease. Our experiments have revealed the existence of a hitherto unknown, macromolecular regulatory system that controls the location and relative activity of mammalian recombination hotspots by activating, suppressing and modulating their activity. Genetic variation in this regulatory system first enabled its discovery and now provides a means of identifying its components and their interactions, an essential step in understanding its mechanisms, as well as its relevance to issues of human genetics, population biology and evolution. The first regulatory protein we identified in this way, PRDM9, enables recombination at a family of mouse hotspots by modifying chromatin structure, allowing formation of the initiating double strand break. Others have simultaneously identified PRDM9 as a regulator of human recombination hotspots. In this project we will identify components of the complementary regulatory system controlling suppression of recombination at specific hotspots, a matter of equal concern in understanding the regulation of recombination. We will identify the genes encoding suppressors of five hotspots on mouse Chr 1 whose activities are regulated by genes that differ between the M. m. domesticus strain C57BL/6J (B6) and the M. m. castaneus strain CAST/EiJ (CAST) by: (a) applying a new, considerably improved, quantitative assay system that measures hotspot activities in sperm DNA samples using NextGen DNA sequencing; (b) using this assay to map and clone the regulatory genes involved; (c) testing their interactions with each other and whether they act in a dose dependent manner, indicating whether they act catalytically or stoichimetrically; and finally, (d) testing whether they control the initiation of recombination or the decision between the alternative recombination pathways leading to crossing over v. non-crossover gene conversions. In separate experiments we will also lay the groundwork for identifying additional regulatory factors with allelic differences between M. m. domesticus (B6) and the M. m. musculus strain PWD. We expect to learn whether each hotspot has its own unique regulatory system or whether there are shared regulatory elements, what these molecules are, 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 provide information essential to resolving their mechanism of action. The results will considerably enhance our understanding of one of the most basic of biological processes, genetic recombination.