F1 hybrids between mouse inbred strains PWD and C57BL/6 represent the most thoroughly genetically defined model of hybrid sterility in vertebrates. Hybrid male sterility can be fully reconstituted from three components of this model, the Prdm9 gene, intersubspecific homeology of Mus musculus musculus and Mus musculus domesticus autosomes, and the X-linked Hstx2 locus. Hstx2 modulates the extent of Prdm9-dependent meiotic arrest and harbors two additional factors responsible for intersubspecific introgression-induced oligospermia (Hstx1) and meiotic recombination rate (Meir1). To facilitate positional cloning and to overcome the recombination suppression within the 4.3 Mb encompassing the Hstx2 locus, we designed Hstx2-CRISPR and SPO11/Cas9 transgenes aimed to induce DNA double-strand breaks specifically within the Hstx2 locus. The resulting recombinant reduced the Hstx2 locus to 2.70 Mb (chromosome X: 66.51-69.21 Mb). The newly defined Hstx2 locus still operates as the major X-linked factor of the F1 hybrid sterility, and controls meiotic chromosome synapsis and meiotic recombination rate. Despite extensive further crosses, the 2.70 Mb Hstx2 interval behaved as a recombination cold spot with reduced PRDM9-mediated H3K4me3 hotspots and absence of DMC1-defined DNA double-strand-break hotspots. To search for structural anomalies as a possible cause of recombination suppression, we used optical mapping and observed high incidence of subspecies-specific structural variants along the X chromosome, with a striking copy number polymorphism of the microRNA Mir465 cluster. This observation together with the absence of a strong sterility phenotype in Fmr1 neighbor (Fmr1nb) null mutants support the role of microRNA as a likely candidate for Hstx2.

中文翻译：

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小鼠 Prdm9 依赖性杂交雄性不育的 Hstx2 修饰剂的基因组结构。


小鼠近交系 PWD 和 C57BL/6 之间的 F1 杂交代表了脊椎动物杂交不育的最彻底的遗传定义模型。杂种雄性不育可以由该模型的三个组成部分完全重建，即 Prdm9 基因、小家鼠和小家鼠常染色体的亚种间同源性以及 X 连锁的 Hstx2 基因座。 Hstx2 调节 Prdm9 依赖性减数分裂停滞的程度，并包含两个额外的因子，负责亚种间渗入诱导的少精子症 (Hstx1) 和减数分裂重组率 (Meir1)。为了促进定位克隆并克服包含 Hstx2 基因座的 4.3 Mb 内的重组抑制，我们设计了 Hstx2-CRISPR 和 SPO11/Cas9 转基因，旨在特异性地在 Hstx2 基因座内诱导 DNA 双链断裂。所得重组体将 Hstx2 位点减少至 2.70 Mb（X 染色体：66.51-69.21 Mb）。新定义的Hstx2基因座仍然作为F1杂种不育的主要X连锁因子发挥作用，并控制减数分裂染色体突触和减数分裂重组率。尽管进行了广泛的进一步杂交，2.70 Mb Hstx2 间隔仍表现为重组冷点，PRDM9 介导的 H3K4me3 热点减少，并且不存在 DMC1 定义的 DNA 双链断裂热点。为了寻找结构异常作为重组抑制的可能原因，我们使用光学图谱并观察到 ​​X 染色体上亚种特异性结构变异的高发生率，其中 microRNA Mir465 簇具有惊人的拷贝数多态性。这一观察结果加上 Fmr1 邻居 (Fmr1nb) 无效突变体中不存在强不育表型，支持 microRNA 作为 Hstx2 的可能候选者的作用。