DNA methylation is stable after MMEJ and NHEJ double strand break repair. DNA methylation is stable after MMEJ and NHEJ double strand break repair

DNA double strand breaks (DSBs) are a major source of mutations. Both non-homologous-end-joining (NHEJ) and microhomology-mediated-end-joining (MMEJ) DSB repair pathways are error prone and produce deletions, which can lead to cancer. DSBs also lead to epigenetic changes, including demethylation, which is involved in carcinogenesis. Of specific interest is the MMEJ repair pathway, as it requires methylation restoration around the break, as a result of the resection and formation of single stranded (ssDNA) intermediates. While, methylation patterns after homologous recombination (HR) have been partially studied, the methylation status after MMEJ and NHEJ remains poorly reported, and can be relevant for cancer. To study methylation patterns around DSB after NHEJ and MMEJ repair, we used targeted bisulfite-sequencing (BS-seq) to quantify methylation of dozens of single cell clones after induction of DSB by CRISPR. Each single cell clone was classified according to the sequence signature to a specific repair mechanism: NHEJ or MMEJ. Comparison of single cell clones after DSB to control cells, without DSB, demonstrated correct restoration of the methylation levels. No difference in methylation patterns was noticed when comparing NHEJ to MMEJ. Methylation levels in gene body, highly methylated CpGs (n=61, 4000 base pairs around DSB) and in low methylation CpGs (n=19), remained stable after both MMEJ and NHEJ. Gene body methylation persisted even on the background of DNMT3A R882C mutation, the most prevalent preleukemic mutation, in which the de novo methylation machinery is compromised. An exception observed in a single CpG site (ASXL1 995) which demonstrated elevated methylation rate after DSB repair only in the presence of WT DNMT3A. In summary, DNA methylation restoration demonstrated high fidelity after DSB both in methylated and unmethylated gene body, even in cases where DNA resections and deletions occurred.

DNA双链断裂（DNA double strand breaks, DSBs）是突变的主要来源。非同源末端连接（non-homologous-end-joining, NHEJ）与微同源介导的末端连接（microhomology-mediated-end-joining, MMEJ）这两种DSB修复通路均具有致错性，会产生缺失突变，进而可能引发癌症。DSB还可诱导表观遗传变化，包括参与致癌过程的去甲基化。其中尤为值得关注的是MMEJ修复通路：由于其涉及核酸切除与单链（single stranded, ssDNA）中间体的形成，该通路需要在断裂位点周围进行甲基化修复。目前针对同源重组（homologous recombination, HR）后的甲基化模式已有部分研究，但NHEJ与MMEJ修复后的甲基化状态仍鲜有报道，且其与癌症发生的相关性不容忽视。为研究NHEJ与MMEJ修复后DSB位点周围的甲基化模式，本研究采用靶向亚硫酸氢盐测序（targeted bisulfite-sequencing, BS-seq），对CRISPR诱导DSB后的数十个单细胞克隆的甲基化水平进行定量检测。每个单细胞克隆均根据序列特征被归类至特定修复机制：NHEJ或MMEJ。将经DSB诱导的单细胞克隆与未发生DSB的对照组细胞进行比对，结果显示甲基化水平得到了正确恢复。对比NHEJ与MMEJ两组样本，未观察到甲基化模式存在显著差异。在基因本体区域、DSB周围4000碱基对范围内的高甲基化CpG位点（n=61）以及低甲基化CpG位点（n=19）中，无论是MMEJ还是NHEJ修复后，甲基化水平均保持稳定。即便在DNMT3A R882C突变（最常见的白血病前期突变，该突变会破坏从头甲基化机制）的背景下，基因本体区域的甲基化仍得以维持。仅在单个CpG位点（ASXL1 995）观察到例外情况：仅当存在野生型DNMT3A时，该位点在DSB修复后的甲基化水平才会升高。综上，无论在甲基化还是非甲基化的基因本体区域，即便发生了DNA切除与缺失突变，DSB修复后的DNA甲基化恢复均表现出较高的保真度。