Senataxin and RNase H2 act redundantly to suppress genome instability during class switch recombination. Senataxin and RNase H2 act redundantly to suppress genome instability during class switch recombination

Class switch recombination generates antibody distinct isotypes critical to a robust adaptive immune system and defects are associated with auto-immune disorders and lymphomagenesis. Transcription is required during class switch to recruit the cytidine deaminase AID—an essential step for the formation of DNA doublestrand breaks—and strongly induces the formation of R loops within the immunoglobulin heavy chain locus. However, the impact of R loops on double-strand break formation and repair during class switch recombination remains unclear. Here we report that cells lacking two enzymes involved in R loop removal— Senataxin and RNase H2—exhibit increased R loop formation and genome instability at the immunoglobulin heavy chain locus without impacting class switch recombination efficiency, transcriptional activity, or AID recruitment. Senataxin and RNase H2-deficient cells also exhibit increased insertion mutations at switch junctions, a hallmark of alternative end joining. Importantly, these phenotypes were not observed in cells lacking Senataxin or RNase H2B alone. We propose that Senataxin acts redundantly with RNase H2 to mediate timely R loop removal, promoting efficient repair while suppressing AID-dependent genome instability and insertional mutagenesis. Overall design: Using LAMS-HTGTS and DRIP-seq to characterize translocation/mutation profile and R-loop formation at IgH region during class switch recombination in mouse B-cells which lack R-loop removal enzymes (setx-/-, rnaseh2b-/-, and setx-/- rnaseh2b-/-).

类别转换重组（Class switch recombination）可产生不同的抗体同种型，这对于构建强健的适应性免疫系统至关重要；该过程的缺陷与自身免疫性疾病及淋巴肿瘤发生密切相关。类别转换过程中需要转录以招募胞苷脱氨酶AID（cytidine deaminase AID）——这是DNA双链断裂（DNA double-strand breaks）形成的关键步骤——同时会强力诱导免疫球蛋白重链基因座（immunoglobulin heavy chain locus）内R环（R loop）的形成。然而，R环在类别转换重组过程中对DNA双链断裂形成与修复的影响仍不明确。
本研究发现，缺失两种参与R环清除的酶——Senataxin与核糖核酸酶H2（RNase H2）——的细胞，其免疫球蛋白重链基因座内的R环形成水平与基因组不稳定性均显著升高，但未对类别转换重组效率、转录活性或AID招募产生影响。Senataxin与RNase H2双缺陷细胞还在免疫球蛋白转换接头区域出现了更多插入突变，这是替代性末端连接（alternative end joining）的标志性特征。值得注意的是，仅缺失Senataxin或RNase H2B的细胞并未出现上述表型。
我们提出，Senataxin与RNase H2存在功能冗余，可介导R环的及时清除，在促进高效修复的同时抑制AID依赖的基因组不稳定性与插入突变。
整体实验设计：通过LAMS-HTGTS与DRIP-seq技术，对缺失R环清除酶（setx-/-、rnaseh2b-/-以及setx-/- rnaseh2b-/-）的小鼠B细胞在类别转换重组过程中，免疫球蛋白重链（IgH）区域的易位/突变谱与R环形成情况进行表征。