Although DNA N⁶-adenine methylation (6mA) is best known in prokaryotes, its presence in eukaryotes has recently generated great interest. Biochemical and genetic evidence supports that AMT1, an MT-A70 family methyltransferase (MTase), is crucial for 6mA deposition in unicellular eukaryotes. Nonetheless, the 6mA transmission mechanism remains to be elucidated. Taking advantage of single-molecule real-time circular consensus sequencing (SMRT CCS), here we provide definitive evidence for semiconservative transmission of 6mA in Tetrahymena thermophila. In wild-type (WT) cells, 6mA occurs at the self-complementary ApT dinucleotide, mostly in full methylation (full-6mApT); after DNA replication, hemi-methylation (hemi-6mApT) is transiently present on the parental strand, opposite to the daughter strand readily labeled by 5-bromo-2′-deoxyuridine (BrdU). In ΔAMT1 cells, 6mA predominantly occurs as hemi-6mApT. Hemi-to-full conversion in WT cells is fast, robust, and processive, whereas de novo methylation in ΔAMT1 cells is slow and sporadic. In Tetrahymena, regularly spaced 6mA clusters coincide with the linker DNA of nucleosomes arrayed in the gene body. Importantly, in vitro methylation of human chromatin by the reconstituted AMT1 complex recapitulates preferential targeting of hemi-6mApT sites in linker DNA, supporting AMT1's intrinsic and autonomous role in maintenance methylation. We conclude that 6mA is transmitted by a semiconservative mechanism: full-6mApT is split by DNA replication into hemi-6mApT, which is restored to full-6mApT by AMT1-dependent maintenance methylation. Our study dissects AMT1-dependent maintenance methylation and AMT1-independent de novo methylation, reveals a 6mA transmission pathway with a striking similarity to 5-methylcytosine (5mC) transmission at the CpG dinucleotide, and establishes 6mA as a bona fide eukaryotic epigenetic mark.

中文翻译：

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单细胞真核生物中 DNA N6-腺嘌呤甲基化的半保守传递


尽管 DNA N ⁶ -腺嘌呤甲基化 (6mA) 在原核生物中最为人所知，但它在真核生物中的存在最近引起了人们的极大兴趣。生化和遗传学证据表明，AMT1（一种 MT-A70 家族甲基转移酶 (MTase)）对于单细胞真核生物中的 6mA 沉积至关重要。尽管如此，6mA 传输机制仍有待阐明。利用单分子实时循环一致性测序 (SMRT CCS)，我们为嗜热四膜虫中 6mA 的半保守传输提供了明确的证据。在野生型 (WT) 细胞中，6mA 发生在自我互补的 ApT 二核苷酸处，大部分处于完全甲基化状态 (full-6mApT)； DNA 复制后，半甲基化 (hemi-6mApT) 短暂存在于亲本链上，与易于被 5-溴-2'-脱氧尿苷 (BrdU) 标记的子链相反。在 ΔAMT1 细胞中，6mA 主要以 hemi-6mApT 形式出现。 WT 细胞中的半向完全转化是快速、稳健且持续进行的，而 ΔAMT1 细胞中的从头甲基化是缓慢且零星的。在四膜虫中，规则间隔的 6mA 簇与排列在基因体中的核小体的连接 DNA 一致。重要的是，重组的 AMT1 复合物对人染色质的体外甲基化概括了接头 DNA 中 hemi-6mApT 位点的优先靶向，支持 AMT1 在维持甲基化中的内在和自主作用。我们得出结论，6mA 通过半保守机制传播：全 6mApT 通过 DNA 复制分裂成半 6mApT，半 6mApT 通过 AMT1 依赖性维持甲基化恢复为全 6mApT。 我们的研究剖析了 AMT1 依赖性维持甲基化和 AMT1 独立从头甲基化，揭示了 6mA 传递途径，与 CpG 二核苷酸上的 5-甲基胞嘧啶 (5mC) 传递惊人相似，并将 6mA 确立为真正的真核表观遗传标记。