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, 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 cellulo. The deposited covalent tags were exploited as clickhandles 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.

Automated summary

Enzymatic methylation of cytosine to 5-methylcytosine in DNA is a fundamental epigenetic mechanism involved in mammalian development and disease. In this study, the researchers engineered the Dnmt1 methyltransferase to catalyze the transfer of a synthetic cofactor analog onto DNA. By editing the Dnmt1 locus in mouse embryonic stem cells and using electroporation to introduce the cofactor, they were able to selectively tag Dnmt1-specific genomic targets. This new approach, called Dnmt-TOP-seq, allows for high-resolution tracking of Dnmt1 catalysis in mammalian cells and opens the door for selective studies of other methylation pathways in eukaryotic systems.