RT Journal
A1 Sheng, Yalan
A1 Wang, Yuanyuan
A1 Yang, Wentao
A1 Wang, Xue Qing
A1 Lu, Jiuwei
A1 Pan, Bo
A1 Nan, Bei
A1 Liu, Yongqiang
A1 Ye, Fei
A1 Li, Chun
A1 Song, Jikui
A1 Dou, Yali
A1 Gao, Shan
A1 Liu, Yifan
T1 Semiconservative transmission of DNA N6-adenine methylation in a unicellular eukaryote
JF Genome Research 
JO Genome Research 
YR 2024 
FD May 01 
VO 34 
IS 5 
SP 740 
OP 756 
DO 10.1101/gr.277843.123 
UL http://genome.cshlp.org/content/34/5/740.abstract 
AB 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.