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Methylation, a widely occurring natural modification serving diverse regulatory and structural functions, is carried out by a myriad of S -adenosyl-l-methionine (AdoMet)-dependent methyltransferases (MTases). The AdoMet cofactor is produced from l-methionine (Met) and ATP by a family of multimeric methionine adenosyltransferases (MAT). To advance mechanistic and functional studies, strategies for repurposing the MAT and MTase reactions to accept extended versions of the transferable group from the corresponding precursors have been exploited. Here, we used structure-guided engineering of mouse MAT2A to enable biocatalytic production of an extended AdoMet analogue, Ado-6-azide, from a synthetic methionine analogue, S -(6-azidohex-2-ynyl)-l-homocysteine (N 3 -Met). Three engineered MAT2A variants showed catalytic proficiency with the extended analogues and supported DNA derivatization in cascade reactions with M. Taq I and an engineered variant of mouse DNMT1 both in the absence and presence of competing Met. We then installed two of the engineered variants as MAT2A-DNMT1 cascades in mouse embryonic stem cells by using CRISPR-Cas genome editing. The resulting cell lines maintained normal viability and DNA methylation levels and showed Dnmt1-dependent DNA modification with extended azide tags upon exposure to N 3 -Met in the presence of physiological levels of Met. This for the first time demonstrates a genetically stable system for biosynthetic production of an extended AdoMet analogue, which enables mild metabolic labeling of a DNMT-specific methylome in live mammalian cells.

We present a method, named Mx-TOP, for profiling of three epigenetic regulatory layers-chromatin accessibility, general DNA modification, and DNA hydroxymethylation-from a single library. The approach is based on chemo-enzymatic covalent tagging of unmodified CG sites and hydroxymethylated cytosine (5hmC) along with GC sites in chromatin, which are then mapped using tag-selective base-resolution TOP-seq sequencing. Our in-depth validation of the approach revealed its sensitivity and informativity in evaluating chromatin accessibility and DNA modification interactions that drive transcriptional regulation. We employed the technology in a study of chromatin and DNA demethylation dynamics during in vitro neuronal differentiation. The study highlighted the involvement of gene body 5hmC in modulating an extensive decoupling between promoter accessibility and transcription. The importance of 5hmC in chromatin remodeling was further demonstrated by the observed resistance of the developmentally acquired open loci to the global 5hmC erasure in neuronal progenitors., Competing Interests: Declaration of interests S.K. and E.K. are inventors on patents related to TOP-seq analysis: EP2776575B1, US9347093B2, and US9988673B2., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Epigenetic phenomena play a central role in cell regulatory processes and are important factors for understanding complex human disease. One of the best understood epigenetic mechanisms is DNA methylation. In the mammalian genome, cytosines (C) in CpG dinucleotides were long known to undergo methylation at the 5-position of the pyrimidine ring (mC). Later it was found that mC can be oxidized to 5-hydroxymethylcytosine (hmC) or even further to 5-formylcytosine (fC) and to 5-carboxylcytosine (caC) by the action of 2-oxoglutarate-dependent dioxygenases of the TET family. These findings unveiled a long elusive mechanism of active DNA demethylation and bolstered a wave of studies in the area of epigenetic regulation in mammals. This review is dedicated to critical assessment of recent data on biochemical and chemical aspects of the formation and conversion of hmC in DNA, analytical techniques used for detection and mapping of this nucleobase in mammalian genomes as well as epigenetic roles of hmC in DNA replication, transcription, cell differentiation and human disease.