PRDM9 and Control of Meiotic Recombination

项目A 尽管重组热点的位置和相对活性在确定模式时是至关重要的 代代相传，到目前为止，我们对这些 确定了参数。PRDM9的等位基因变异现在为我们提供了补救这一点的机会 缺乏症。该项目的长期目标是表征分子和遗传机制 规范热点位置和活动。为了实现这一点，我们将生成一系列KO和敲入小鼠 以确保唯一的遗传变量将是被测试的PRDM9等位基因的身份。使用此常量 遗传背景，我们将找出等位基因特异的热点，并研究等位基因偏好对 当同一减数分裂细胞中存在两个等位基因时，热点被激活。使用遗传变量 背景，我们还将确定PRDM9功能的遗传修饰物。 在目标1中，我们将：(A)确定一系列PRDM9等位基因特异的热点，其序列将有助于阐明 控制PRDM9等位基因特异性的规则；(B)确定是否存在常染色体和假性染色体 常染色体PRDM9非依赖热点，以及(C)以小鼠为载体的人类特征测试 PRDM9特异性。要深入研究人类PRDM9的生物学功能是不可能的 人类材料，作为由相同的PRDM9等位基因控制的热点，其活性从一个 个体到下一个，推测来自于遗传变异的多个其他热点调控因子(4)。测试 人类的等位基因也提供了一种有用的控制方法，它通过比较在基因突变的情况下重组 不同的ZNF域。在这样做的时候，正如导言中所概述的那样，我们认为重要的是对两者进行分析 表征重组过程的开始和遗传交叉的DSB 最终的、功能性的结果。这两项衡量标准提供了互补的见解。如果我们想 了解重组的功能后果，我们尽最大努力对 过程的开始和结束。 在目标2中，我们期望证实PRDM9等位基因激活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 热点本身，重要的是决定偏好是否通过PRDM9的相对数量来调节 存在的分子或它们与热点DNA序列的相对亲和力。这些实验有相当大的 对于理解在约束条件下运行的交叉自平衡现象的相关性 DSB的数量，以确保在每个减数分裂时有恒定的交换数量。 最后，在目标3中，我们将测试各种小鼠的遗传背景，以筛选基因的存在 PRDM9的修饰物的作用目的是确定它们的分子同一性。PRDM9不能在 分离，这里的目标是确定与PRDM9物理或功能相互作用的蛋白质 在本项目中产生的PRDM9等位基因的敲入菌株将用于B Petkov项目和 项目C Hibbs。 实现这些目标对于理解减数分裂的基本生物学和它的 对公共卫生的影响，因为减数分裂重组和配子发生失败是导致 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.