Selective chemical tracking of Dnmt1 catalytic activity in live cells

Enzymatic methylation of cytosine to 5-methylcytosine in DNA is a fundamental epigenetic mechanism involved in mammalian development and disease. DNA methylation is brought about by collective action of three AdoMet-dependent DNA methyltransferases (DNMTs), whose catalytic interactions and temporal interplay are poorly understood. We used structure-guided engineering of the Dnmt1 methyltransferase to enable catalytic transfer of azide tags onto DNA from a synthetic cofactor analog, Ado-6-azide, in vitro . We then CRISPR-edited the Dnmt1 locus in mouse embryonic stem cells to install the engineered codon, which, following pulse- internalization of the Ado-6-azide cofactor by electroporation, permitted selective azide-tagging of Dnmt1-specific genomic targets in cellula . The deposited covalent tags were exploited as ‘click’ handles for reading adjoining sequences and precise genomic mapping of the methylation sites. The proposed approach, Dnmt-TOP-seq, enables high-resolution temporal tracking of the Dnmt1 catalysis in mammalian cells paving the way to selective studies of other methylation pathways in eukaryotic systems. Genome-wide tracking of DNA methyltransferase Dnmt1 action in mouse embryonic stem cells. Mouse Dnmt1-directed DNA modifications were analyzed in E14TG2a cells bearing the homozygous Dnmt1N1580A knock-in substitution following the delivery of Ado-6-azide by electroporation. Wild-type Dnmt1 cells were used as a control. All experiments were carried out in triplicates and included two technical replicates for each input.