Semi-conservative transmission of DNA N6-adenine methylation in a unicellular eukaryote

Abstract

AbstractWhile DNA N6-adenine methylation (6mA) is best known in prokaryotes, its presence in eukaryotes has generated great interest recently. Biochemical and genetic evidence supports that AMT1, a MT-A70 family methyltransferase (MTase), is crucial for 6mA deposition in unicellular eukaryotes. Nonetheless, 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 semi-conservative transmission of 6mA, showcased in the unicellular eukaryoteTetrahymena thermophila. In wildtype (WT) cells, 6mA occurs at the self-complementary ApT dinucleotide, mostly in full methylation (full-6mApT); hemi-methylation (hemi-6mApT) is transiently present on the parental strand of newly replicated DNA. In ΔAMT1cells, 6mA predominantly occurs as hemi-6mApT. Hemi-to-full conversion in WT cells is fast, robust, and likely processive, whilede novo6mA deposition in ΔAMT1cells is slow and sporadic. InTetrahymena, regularly spaced 6mA clusters coincide with linker DNA of the canonical nucleosome arrays in the gene body. Importantly,in vitromethylation of human chromatin by 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 semi-conservative 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-independentde novomethylation, reveals a molecular pathway for 6mA transmission with striking similarity to 5-methyl cytosine (5mC) transmission at the CpG dinucleotide, and establishes 6mA as abona fideeukaryotic epigenetic mark.