In mammals, meiotic recombination is initiated by the introduction of DNA double strand breaks (DSBs) into narrow segments of the genome, defined as hotspots, which is carried out by the SPO11/TOPOVIBL complex. A major player in the specification of hotspots is PRDM9, a histone methyltransferase that, following sequence-specific DNA binding, generates trimethylation on lysine 4 (H3K4me3) and lysine 36 (H3K36me3) of histone H3, thus defining the hotspots. PRDM9 activity is key to successful meiosis, since in its absence DSBs are redirected to functional sites and synapsis between homologous chromosomes fails. One protein factor recently implicated in guiding PRDM9 activity at hotspots is EWS, a member of the FET family of proteins that also includes TAF15 and FUS/TLS. Here, we demonstrate that FUS/TLS partially colocalizes with PRDM9 on the meiotic chromosome axes, marked by the synaptonemal complex component SYCP3, and physically interacts with PRDM9. Furthermore, we show that FUS/TLS also interacts with REC114, one of the axis-bound SPO11-auxiliary factors essential for DSB formation. This finding suggests that FUS/TLS is a component of the protein complex that promotes the initiation of meiotic recombination. Accordingly, we document that FUS/TLS coimmunoprecipitates with SPO11 in vitro and in vivo. The interaction occurs with both SPO11β and SPO11α splice isoforms, which are believed to play distinct functions in the formation of DSBs in autosomes and male sex chromosomes, respectively. Finally, using chromatin immunoprecipitation experiments, we show that FUS/TLS is localized at H3K4me3-marked hotspots in autosomes and in the pseudo-autosomal region, the site of genetic exchange between the XY chromosomes.

在哺乳动物中，减数分裂重组是通过将 DNA 双链断裂 (DSB) 引入基因组的窄片段（定义为热点）而启动的，这是由 SPO11/TOPOVIBL 复合体执行的。热点规范的主要参与者是 PRDM9，这是一种组蛋白甲基转移酶，在序列特异性 DNA 结合后，在组蛋白 H3 的赖氨酸 4 (H3K4me3) 和赖氨酸 36 (H3K36me3) 上产生三甲基化，从而定义热点。PRDM9 活性是减数分裂成功的关键，因为在它不存在的情况下，DSB 被重定向到功能位点，同源染色体之间的联会失败。最近涉及在热点处指导 PRDM9 活动的一个蛋白质因子是 EWS，它是 FET 蛋白质家族的成员，还包括 TAF15 和 FUS/TLS。这里，我们证明 FUS/TLS 在减数分裂染色体轴上与 PRDM9 部分共定位，由联会复合体成分 SYCP3 标记，并与 PRDM9 物理相互作用。此外，我们表明 FUS/TLS 还与 REC114 相互作用，REC114 是 DSB 形成所必需的轴绑定 SPO11 辅助因子之一。这一发现表明 FUS/TLS 是促进减数分裂重组启动的蛋白质复合物的组成部分。因此，我们记录了 FUS/TLS 在体外和体内与 SPO11 共免疫沉淀。这种相互作用发生在 SPO11β 和 SPO11α 剪接亚型上，据信它们分别在常染色体和男性性染色体中的 DSB 形成中发挥着不同的功能。最后，利用染色质免疫沉淀实验，