5-Methylcytosine (5mC) is one of the most critical epigenetic modifications in mammals that formed by DNA methyltransferase (DNMT) through adding methyl groups to the carbon-5′position of cytosine. Being regarded as the fifth base in DNA, 5mC plays a pivotal role in regulating gene expression, embryonic development, X chromosome inactivation, and genomic imprinting. The comprehensive mapping of DNA methylation in genomes is crucial for understanding their dynamic changes and biological significance.
Previously, researchers have shown that the Naegleria TET-like proteins (nTET) identified in the heterolobosean amoeboflagellate Naegleria gruberi exhibit robust catalytic activity on 5mC in a broader sequence context compared with its mammalian homologs.
"In the current study, the wtA3A deaminase was engineered and we obtained an A3A mutant (A3Am) that efficiently deaminates C and 5mC, but not 5hmC, 5fC and 5caC in various DNA contexts." Yuan said. Leveraging the unique properties of nTET and A3Am, they established a novel sequencing technology termed Naegleria TET-assisted deaminase sequencing (NTD-seq). The NTD-seq method utilizes a nTET to oxidize 5mC, generating 5-methylcytosine oxidation products (5moC). Treatment with A3Am results in the conversion of cytosine to uracil, while 5moC remains unchanged.
Consequently, NTD-seq enables the direct deamination of cytosine to uracil by A3Am, which is sequenced as thymine, whereas 5mC, once oxidized to 5moC by nTET, resists deamination and is sequenced as cytosine. Therefore, the cytosines that persist in the sequencing data represent the original 5mC sites.
The team further applied NTD-seq to HEK293T cells, generating a base-resolution map of 5mC that exhibits strong concordance with maps generated by conventional BS-seq. NTD-seq emerges as a powerful, bisulfite-free approach for the single-base resolution mapping of 5mC stoichiometry in genomic DNA.
