Aims An intrinsic feature of gene transcription is the formation of DNA superhelices near the transcription bubble, which are resolved upon induction of transient double-stranded breaks (DSBs) by topoisomerases. Unrepaired DSBs are pathogenic as they lead to cell cycle arrest, senescence, inflammation, and organ dysfunction. We posit that DSBs would be more prevalent at the genomic sites that are associated with gene expression. The objectives were to identify and characterize genome-wide DSBs at the nucleotide resolution and determine the association of DSBs with transcription in cardiac myocytes. Methods and results We identified the genome-wide DSBs in ∼1 million cardiac myocytes per heart in three wild-type and three myocyte-specific LMNA-deficient (Myh6-Cre:LmnaF/F) mice by END-Sequencing. The prevalence of DSBs was 0.8% and 2.2% in the wild-type and Myh6-Cre:LmnaF/F myocytes, respectively. The END-Seq signals were enriched for 8 and 6764 DSBs in the wild-type and Myh6-Cre:LmnaF/F myocytes, respectively (q < 0.05). The DSBs were preferentially localized to the gene regions, transcription initiation sites, cardiac transcription factor motifs, and the G quadruplex forming structures. Because LMNA regulates transcription through the lamin-associated domains (LADs), we defined the LADs in cardiac myocytes by a Cleavage Under Targets & Release Using Nuclease (CUT&RUN) assay (N = 5). On average there were 818 LADs per myocyte. Constitutive LADs (cLADs), defined as LADs that were shared by at least three genomes (N = 2572), comprised about a third of the mouse cardiac myocyte genomes. Transcript levels of the protein-coding genes located at the cLADs (N = 3975) were ∼16-fold lower than those at the non-LAD regions (N = ∼17 778). The prevalence of DSBs was higher in the non-LAD as compared to the cLAD regions. Likewise, DSBs were more common in the loss-of-LAD regions, defined as the genomic regions in the Myh6-Cre:LmnaF/F that were juxtaposed to the LAD regions in the wild-type myocytes. Conclusion To our knowledge, this is the first identification of the DSBs, at the nucleotide resolution in the cardiovascular system. The prevalence of DSBs was higher in the genomic regions associated with transcription. Because transcription is pervasive, DSBs are expected to be common and pathogenic in various states and aging.

中文翻译：

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DNA 双链断裂是衰老的标志，以核苷酸分辨率定义，在 LMNA 心肌病中，DNA 双链断裂增加并与心肌细胞的转录相关

目的 基因转录的一个内在特征是在转录泡附近形成 DNA 超螺旋，这些超螺旋在拓扑异构酶诱导瞬时双链断裂 (DSB) 时被分解。未修复的 DSB 具有致病性，因为它们会导致细胞周期停滞、衰老、炎症和器官功能障碍。我们假设 DSB 在与基因表达相关的基因组位点上更为普遍。目的是在核苷酸分辨率下识别和表征全基因组 DSB，并确定 DSB 与心肌细胞转录的关联。方法和结果 我们通过 END 测序鉴定了三只野生型和三只肌细胞特异性 LMNA 缺陷 (Myh6-Cre:LmnaF/F) 小鼠每颗心脏约 100 万个心肌细胞中的全基因组 DSB。 DSB 在野生型和 Myh6-Cre:LmnaF/F 肌细胞中的患病率分别为 0.8% 和 2.2%。野生型和 Myh6-Cre:LmnaF/F 肌细胞中的 END-Seq 信号分别富集了 8 个和 6764 个 DSB（q < 0.05）。 DSB 优先定位于基因区域、转录起始位点、心脏转录因子基序和 G 四链体形成结构。由于 LMNA 通过核纤层蛋白相关结构域 (LAD) 调节转录，因此我们通过目标下切割和使用核酸酶释放 (CUT&RUN) 测定 (N = 5) 定义了心肌细胞中的 LAD。每个肌细胞平均有 818 个 LAD。组成型 LAD (cLAD) 定义为至少三个基因组共享的 LAD (N = 2572)，约占小鼠心肌细胞基因组的三分之一。位于 cLAD 的蛋白质编码基因的转录水平 (N = 3975) 比非 LAD 区域的转录水平低 ∼16 倍 (N = ∼17 778)。与 cLAD 区域相比，非 LAD 区域的 DSB 患病率更高。同样，DSB 在 LAD 缺失区域中更为常见，LAD 缺失区域定义为 Myh6-Cre:LmnaF/F 中与野生型肌细胞中的 LAD 区域并列的基因组区域。结论 据我们所知，这是在心血管系统中首次以核苷酸分辨率鉴定 DSB。 DSB 在与转录相关的基因组区域中的发生率较高。由于转录是普遍存在的，因此 DSB 预计在各种状态和衰老过程中都很常见并具有致病性。