Abstract: Targeted enzymatic modification of DNA, RNA and proteins by addition of a methyl group is the prevalent epigenetic regulatory mechanism in higher eukaryotes. Methylation has been linked to a variety of biological processes including regulation of gene expression and cell identity. In DNA, three methyltransferases - DNMT1, DNMT3A, and DNMT3B - facilitate the transfer of a methyl group to the fifth carbon of a cytosine residue in CpG dinucleotides generating 5-methylcytosine. Aberrant genomic methylation profiles are associated with and serve as diagnostic markers for numerous human diseases. DNA methyltransferases are attractive therapeutic targets for drug design, however, the interplay and exact roles of the three DNMT enzymes in different biological contexts remains understudied. To achieve selective catalytic tracking of each individual Dnmt enzyme, we engineered the catalytic centre of mouse Dnmt1 or Dnmt3A for preferential transfer of chemical moieties containing functional azide groups from synthetic AdoMet cofactor analogs. We derived a series of mouse and human cell lineages by installing the derived codons in one or both alleles of the Dnmt1 or Dnmt3A gene using precise genome editing. To enable selective catalysis-dependent azide-tagging of the DNMT-specific targets in vivo, we elaborated an electroporation-based procedure for pulse-internalization of an azide cofactor analog into the engineered cells or, most recently, its metabolic in-cell production from a corresponding synthetic analogue of methionine using a genome-engineered methionine-adenosyl transferase. The deposited azide groups were exploited as ‘click’ handles for precise mapping of the tagged methylation sites in the genome using TOP-seq profiling or for direct nanopore sequencing. Pilot studies of mouse ESCs and 3T3-L1 fibroblasts unveil detailed roles of the individual DNMTs during cell state transitions. Altogether, we present a new experimental platform for high-resolution genome-wide tracking of individual epigenetic writers in live cells providing unprecedented inroads into deciphering and altering mammalian epigenetic mechanisms, and paving new ways for developing next-generation diagnostic and therapeutic approaches.

Related references:
Staševskij et al. Mol Cell, 2017, 65: 554–64.
Stankevičius et al. Mol Cell, 2022, 82:1053–65.
Vilkaitis et al. Acc Chem Res, 2023, 56: 3188-97.
Gasiulė et al. J Amer Chem Soc, 2024, 146: 18722–29.

Keywords: Epigenetic mechanisms, DNA methylation, biomolecular engineering, epigenomic technologies

Biography: Prof. Saulius Klimašauskas graduated Organic Chemistry at Vilnius University and then worked on first characterization of bacterial DNA cytosine-N4 methyltransferases with Prof. Arvydas Janulaitis at the Institute of Applied Enzymology Fermentas in Vilnius, Lithuania to receive his Ph.D. in Bioorganic Chemistry in 1987. He was a postdoctoral scientist in structural and molecular biology of DNA cytosine-5 methyltransferases with Dr. Sir Richard J. Roberts (NL, FRS) at Cold Spring Harbor Laboratory, where he was a major contributor to the discovery of a novel type of DNA-protein interaction – DNA base flipping. After starting his own group in 1995 at the Institute of Biotechnology in Vilnius, Lithuania he grew through the ranks of Head of Laboratory, Head of Department to become a Distinguished Research Professor at the Institute of Biotechnology, Life Sciences Center, Vilnius University in 2017. Dr. Klimašauskas was a repeat HHMI International Research Scholar (1995–2005), a JSPS invited professor at Osaka University (2002) and a recipient of a prestigious ERC advanced grant (2016-2023). He was elected and a full member of the Lithuanian Academy of Sciences, a Fellow of the Royal Society of Chemistry and an EMBO member. His long standing research interests span mechanistic studies and molecular engineering of AdoMet-dependent methyltransferases and epigenetic mechanisms involving biological modification of DNA and RNA. He co-authored over 100 research and review articles in high impact journals and obtained over 10 international patentsand commercial licences on novel technologies for DNA/RNA labeling and epigenome analysis.