Transacting Genes Regulating Recombination Hotspot Activities

描述（由申请人提供）：人类和小鼠中重组热点的位置及其经历基因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；这两个问题都是确定对人类健康和疾病有重要影响的基因的关键问题。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它们的位置和相对活性。这种调控系统的遗传变异首先使其得以发现，现在提供了一种识别其组成部分及其相互作用的手段，这是理解其机制以及与人类遗传学、种群生物学和进化问题相关的重要一步。我们以这种方式确定的第一个调节蛋白，PRDM9，通过修改染色质结构，允许形成初始双链断裂，从而在小鼠热点家族中实现重组。另一些人同时发现PRDM9是人类重组热点的调节因子。在这个项目中，我们将确定在特定热点控制重组抑制的互补调控系统的组成部分，这对理解重组调控同样重要。我们将通过以下方法鉴定小鼠Chr 1上5个热点抑制基因的编码，这些热点的活性是由m.m.驯化菌株C57BL/6J （B6）和m.m. castaneus菌株CAST/EiJ （CAST）之间不同的基因调控的：(a)采用一种新的、显著改进的定量分析系统，使用NextGen DNA测序测量精子DNA样本中的热点活性；(b)利用该试验绘制和克隆相关调控基因；(c)测试它们之间的相互作用以及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，(d)测试它们是否控制重组的启动，或者在导致交叉与非交叉基因转换的可选重组途径之间的决定。在单独的实验中，我们还将为确定家养m.m. （B6）和肌肉m.m.菌株PWD之间的等位基因差异的其他调节因子奠定基础。我们希望了解每个热点是否有自己独特的调控系统或是否有共同的调控元件，这些分子是什么，是否对重组的启动施加控制或在重组的替代途径之间进行选择，以及这些基因相互作用的方式。这些数据以及这些基因的分子特性将为解决它们的作用机制提供必要的信息。这一结果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): 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.