PRDM9 and Control of Meiotic Recombination

项目A 虽然重组热点的位置和相对活动在决定模式方面至关重要， 从一代到下一代的遗传，到目前为止，我们只有一个基本的了解，这些如何 参数已确定。PRDM 9中的等位基因变异现在为我们提供了纠正这一点的机会 缺陷该项目的长期目标是描述分子和遗传机制， 调节热点位置和活动。为了实现这一点，我们将产生一系列KO和敲入小鼠 以确保唯一的遗传变量将是被测试的PRDM 9等位基因的身份。利用这个常数 遗传背景，我们将确定等位基因特异性热点，并研究等位基因偏好的作用， 当两个等位基因存在于同一减数分裂细胞中时热点激活。利用基因变异 在本发明的背景下，我们还将鉴定PRDM 9功能的遗传修饰剂。 在目标1中，我们将：（a）鉴定一系列PRDM 9等位基因特异性热点，其序列将有助于阐明 控制PRDM 9等位基因特异性的规则;（B）确定是否存在常染色体以及假- 常染色体PRDM 9非依赖性热点，和（c）使用小鼠作为表征人PRDM 9非依赖性热点的媒介物的测试。 PRDM 9特异性。不可能使用生物学方法深入表征人PRDM 9的生物学功能。 人类材料，因为由相同PRDM 9等位基因控制的热点在活性上从一个PRDM 9等位基因变化超过十五倍。 个体到下一个，可能来自多个其他热点调节因子的遗传变异（4）。测试 人等位基因还提供了一种有用的对照， 不同的ZNF结构域。在这样做的时候，正如引言中所概述的，我们认为重要的是要分析这两个方面， DSB是重组过程开始的特征， 最终的功能性成果。这两项测量提供了互补的见解。如果我们想 为了理解重组的功能后果，我们尽最大努力提供对重组的描述。 过程的开始和结束。 在目的2中，我们期望证实PRDM 9等位基因激活的能力中存在等位基因偏好。 重组，然后测试对这种效应的敏感性是否取决于重组的相对强度。 热点本身，重要的是决定偏好是否由PRDM 9的相对数量介导。 分子存在或其对热点DNA序列的相对亲和力。这些实验具有相当大的 相关性的理解交叉稳态的现象，在限制内运作的 DSB的数量，以确保在每次减数分裂的交换数量恒定。 最后，在目标3中，我们将测试各种小鼠遗传背景，以筛选遗传背景的存在。 PRDM 9的修饰物起作用的目的是确定它们的分子身份。PRDM 9不能在 分离，这里的目标是鉴定与PRDM 9物理或功能相互作用的蛋白质 本项目中产生的PRDM 9等位基因敲入菌株将用于项目B Petkov， C计划希布斯 实现这些目标对于理解减数分裂的基础生物学和其生物学特性都具有重要意义。 公共卫生的影响，因为减数分裂重组和配子发生的失败是主要的贡献者， 人类的生育能力和胚胎致死率。

PROJECT A Although the locations and relative activities of recombination hotspots are crucial in determining patterns of inheritance from one generation to the next, as yet we have only a rudimentary understanding of how these parameters are determined. Allelic variation in PRDM9 now provides us the opportunity to remedy this deficiency. The long-term goal of this project is to characterize the molecular and genetic mechanisms that regulate hotspot location and activity. To accomplish this, we will generate a series of KO and knock-in mice to assure that the only genetic variable will be the identity of PRDM9 allele being tested. Using this constant genetic background, we will identify allele-specific hotspots and examine the role of allelic preference for hotspot activation when two alleles are present in the same meiotic cells. Using genetically variable backgrounds, we will also identify genetic modifiers of PRDM9 function. In Aim 1 we will: (a) identify a series of PRDM9 allele-specific hotspots whose sequences will help clarify the rules governing PRDM9 allelic specificity; (b) determine if there are autosomal as well as pseudo- autosomal PRDM9-independent hotspots, and (c) test using mice as a vehicle for characterizing human PRDM9 specificity. It is not possible to characterize the biological functions of human PRDM9 in depth using human material, as hotspots controlled by the same PRDM9 allele vary over fifteen fold in activity from one individual to the next, presumably from genetic variation at multiple other hotspot regulatory factors (4). Testing the human allele also provides a useful control by comparing recombination in the presence of a drastically different ZNF domain. In doing so, as outlined in the Introduction, we believe that it is important to assay both the DSBs that characterize the beginnings of the recombination process and the genetic crossovers that are the final, functional outcome. The two measurements provide complementary insights. If we want to understand the functional consequences of recombination, we do best to provide descriptions of both the beginning and end of the process. In Aim 2 we expect to confirm the existence of allelic preference in the ability of PRDM9 alleles to activate recombination, and then test whether susceptibility to this effect depends on the relative strength of the hotspots themselves, and importantly determine if preference is mediated by the relative numbers of PRDM9 molecules present or their relative affinity for hotspot DNA sequences. These experiments have considerable relevance for understanding the phenomenon of crossover homeostasis that operates within constraints on the numbers of DSBs to assure a constant number of crossovers at each meiosis. Finally, in Aim 3 we will test a variety of mouse genetic backgrounds to screen for the existence of genetic modifiers of PRDM9 function with the goal of determining their molecular identity. PRDM9 cannot act in isolation, and the goal here is to identify proteins that physically or functionally interact with PRDM9 The knock-in strains of PRDM9 alleles generated in this Project will be used in Project B Petkov and Project C Hibbs. Carrying out these goals has significance both for understanding the basic biology of meiosis and for its public health implications, as failures of meiotic recombination and gametogenesis are major contributors to human in fertility and embryonic lethality.