Transacting Genes Regulating Recombination Hotspot Activities

描述(申请人提供)：重组热点在人类和小鼠中的位置和它们经历遗传交叉的频率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；两者都是努力识别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种迄今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。这一调控系统中的遗传变异首先使其得以发现，现在提供了一种确定其组成部分及其相互作用的手段，这是理解其机制及其与人类遗传学、种群生物学和进化问题的相关性的关键一步。我们以这种方式确定的第一个调节蛋白PRDM9，通过改变染色质结构，使一系列小鼠热点能够重组，允许形成启动的双链断裂。另一些人同时认为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，这是理解重组调控的同等关注的问题。我们将通过：(A)应用一种新的、显著改进的定量检测系统，利用NextGen DNA测序来测量精子DNA样本中的热点活性；(B)使用该方法定位并克隆涉及的调控基因；(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；(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；最后，(D)测试它们是否控制重组的启动或导致互换与非互换基因转化的可选重组途径之间的决定。在单独的实验中，我们还将为识别在家分枝杆菌(B6)和肌肉分枝杆菌菌株PWD之间存在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，是否对重组的启动或重组的替代途径之间的选择施加控制，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将为解决它们的作用机制提供必要的信息。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。 公共卫生相关性：适当的基因重组对于包括人类在内的所有有性生殖有机体的成功繁殖至关重要。它确保了减数分裂时染色体的有序分离是进化过程的一个重要特征，并产生了使我们每个人都成为独特个体的遗传多样性。重组过程的任何失败都会导致不育。人类和老鼠重组的一个重要特征是将遗传交叉定位在染色体上的特定位置，称为热点。我们现在了解到有一个控制热点位置和活性的大分子调控系统，我们将在这个项目中研究这个系统，目的是了解它在允许适当重组方面的作用。

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. PUBLIC HEALTH RELEVANCE: Proper genetic recombination is essential for successful reproduction in all sexually reproducing organisms, including humans. It assures the orderly segregation of chromosomes at meiosis is an important feature of evolutionary processes and generates the genetic diversity that makes each of us a unique individual. Any failure of the recombination process results in sterility. An important feature of human and mouse recombination is the location of genetic crossovers at specialized sites along chromosomes called hotspots. We now understand that there is a macromolecular regulatory system controlling the location and activity of hotspots, and it is this system that we will be studying in this project with the intent of understanding its role in allowing proper recombination.