Semiconservative transmission of DNA N6-adenine methylation in a unicellular eukaryote
- Yalan Sheng1,2,6,7,
- Yuanyuan Wang1,2,6,
- Wentao Yang3,6,
- Xue Qing Wang3,
- Jiuwei Lu4,
- Bo Pan1,2,
- Bei Nan1,2,
- Yongqiang Liu1,2,
- Fei Ye1,2,
- Chun Li5,
- Jikui Song4,
- Yali Dou3,
- Shan Gao1,2 and
- Yifan Liu3
- 1MOE Key Laboratory of Evolution and Marine Biodiversity and Institute of Evolution and Marine Biodiversity, Ocean University of China, Qingdao 266003, China;
- 2Laboratory for Marine Biology and Biotechnology, Qingdao Marine Science and Technology Center, Qingdao 266237, China;
- 3Department of Biochemistry and Molecular Medicine, Keck School of Medicine, University of Southern California, Los Angeles, California 90033, USA;
- 4Department of Biochemistry, University of California Riverside, Riverside, California 92521, USA;
- 5Division of Biostatistics, Department of Preventive Medicine, University of Southern California, Los Angeles, California 90033, USA
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↵6 These authors contributed equally to this work.
Abstract
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.
Footnotes
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[Supplemental material is available for this article.]
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Article published online before print. Article, supplemental material, and publication date are at https://www.genome.org/cgi/doi/10.1101/gr.277843.123.
- Received March 3, 2023.
- Accepted April 15, 2024.
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