虽然DNAN6-腺嘌呤甲基化(6mA)在原核生物中最为人所知,它在真核生物中的存在最近引起了极大的兴趣。生化和遗传证据支持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传输机制还有待阐明。利用单分子实时循环一致测序(SMRTCCS),在这里,我们提供了在嗜热四膜虫中6mA在野生型(WT)细胞中的半保守传输的明确证据,6mA发生在自身互补的ApT二核苷酸,主要是完全甲基化(full-6mApT);DNA复制后,半甲基化(hemi-6mApT)瞬时存在于亲本链上,与容易被5-溴-2'-脱氧尿苷(BrdU)标记的子链相反。在ΔAMT1细胞中,6mA主要以半6mApT形式出现。WT细胞的半完全转化速度很快,健壮,和进程,而ΔAMT1细胞中的从头甲基化是缓慢和零星的。在四膜虫中,规则间隔的6mA簇与排列在基因体内的核小体的接头DNA一致。重要的是,通过重建的AMT1复合物对人染色质的体外甲基化概括了接头DNA中半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。我们得出的结论是,6mA是通过半保守机制传输的:全6mApT通过DNA复制分裂成半6mApT,通过AMT1依赖性维持甲基化恢复到全6mApT。我们的研究解剖了AMT1依赖性维持甲基化和AMT1非依赖性从头甲基化。揭示了6mA的传输途径,与CpG二核苷酸的5-甲基胞嘧啶(5mC)传输具有惊人的相似性,并建立6mA作为真正的真核表观遗传标记。
Although DNA N 6-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.