rahu is a mutant allele of Dnmt3c, encoding a DNA methyltransferase homolog required for meiosis and transposon repression in the mouse male germline [RNA-seq]

Transcriptional silencing by heritable cytosine-5 methylation is an ancient strategy to repress transposable elements. It was previously thought that mammals possess four DNA methyltransferase paralogs--Dnmt1, Dnmt3a, Dnmt3b and Dnmt3l--t establish and maintain cytosine-5 methylation. Here we identify a fifth paralog, Dnmt3c, that is essential for retrotransposon methylation and repression in the mouse male germline. From a phenotype-based forward genetics screen, we isolated a mutant mouse called 'rahu', which displays severe defects in double-strand-break repair and homologous chromosome synapsis during male meiosis, resulting in sterility. rahu is an allele of a transcription unit (Gm14490, renamed Dnmt3c) that was previously mis-annotated as a Dnmt3-family pseudogene. Dnmt3c encodes a cytosine methyltransferase homolog, and Dnmt3crahu mutants harbor a non-synonymous mutation of a conserved residue within one of its cytosine methyltransferase motifs, similar to a mutation in human DNMT3B observed in patients with immunodeficiency, centromeric instability and facial anomalies syndrome. The rahu mutation lies at a potential dimerization interface and near the potential DNA binding interface, suggesting that it compromises protein-protein and/or protein-DNA interactions required for normal DNMT3C function in vivo. Dnmt3crahu mutant males fail to establish normal methylation within LINE and LTR retrotransposon sequences in the germline and accumulate higher levels of transposon-derived transcripts and proteins, particularly from distinct L1 and ERVK retrotransposon families. Phylogenetic analysis indicates that Dnmt3c arose during rodent evolution by tandem duplication of Dnmt3b, after the divergence of the Dipodoidea and Muroidea superfamilies. These findings provide insight into the evolutionary dynamics and functional specialization of the transposon suppression machinery critical for mammalian sexual reproduction and epigenetic regulation. Six samples total: Dnmt3crahu/+ (three biological replicates) and Dnmt3crahu/rahu (three biological replicates) mice aged 14 days post partum可遗传的5-胞嘧啶甲基化介导的转录沉默是抑制转座因子的古老策略。此前学界认为哺乳动物拥有四类DNA甲基转移酶（DNA methyltransferase）旁系同源蛋白——Dnmt1、Dnmt3a、Dnmt3b与Dnmt3l——用于建立并维持5-胞嘧啶甲基化。本研究鉴定出第五类旁系同源蛋白Dnmt3c，其对小鼠雄性生殖系中的反转座子甲基化与抑制至关重要。我们基于表型的正向遗传学筛选，分离得到一株命名为“rahu”的突变小鼠，该小鼠在雄性减数分裂过程中出现双链断裂修复与同源染色体联会的严重缺陷，最终导致雄性不育。rahu是一个转录单元（Gm14490，后被重命名为Dnmt3c）的等位基因，该转录单元此前被错误注释为Dnmt3家族的假基因。Dnmt3c编码一类胞嘧啶甲基转移酶同源蛋白，而Dnmt3c^rahu突变体携带其一个胞嘧啶甲基转移酶基序内保守残基的非同义突变，这与免疫缺陷、着丝粒不稳定和面部异常综合征患者体内观察到的人类DNMT3B突变特征相似。rahu突变位点位于潜在的二聚化界面，且临近潜在的DNA结合界面，提示该突变会破坏体内维持DNMT3C正常功能所需的蛋白质-蛋白质及/或蛋白质-DNA相互作用。Dnmt3c^rahu突变雄性小鼠无法在生殖系的LINE与LTR反转座子序列内建立正常甲基化模式，并积累更高水平的转座子来源转录本与蛋白，尤其是来自特定L1与ERVK反转座子家族的转录本与蛋白。系统发育分析表明，Dnmt3c是在啮齿类动物进化过程中，由Dnmt3b通过串联复制产生，其分化时间晚于跳鼠总科（Dipodoidea）与鼠总科（Muroidea）超家族的分化事件。上述研究结果为阐明转座子抑制机制的进化动力学与功能特化提供了重要见解，而该机制对哺乳动物有性生殖与表观遗传调控至关重要。本研究共包含6个样本：产后14天的Dnmt3c^rahu/+（3个生物学重复）与Dnmt3c^rahu/rahu（3个生物学重复）小鼠。