The paper by the interdisciplinary team of VU scientists presents a new molecular tool, named Dnmt-TOP-seq, dedicated to high-resolution selective tracking of the catalytic activity of the Dnmt enzyme in live mammalian cells. It enables new detailed insights into epigenetic mechanisms that have not been possible with previously used methods.
For more than a decade, the LSC research team has been studying the enzymes - DNA methyltransferases - involved in 'writing' epigenetic marks in human cells. This research is important for early diagnosis of cancer and other serious diseases such as diabetes, multiple sclerosis, and autism, as well as for personalized medicine.
DNA methylation is one of the most important epigenetic modifications that regulate many developmental processes in mammals and is essential for the proper functioning of multicellular organisms. Aberrant DNA methylation is associated with disruptions of numerous biological processes leading to developmental defects and pathologies. In mammalian cells, the DNA methylation toolbox consists of three catalytically active DNA methyltransferases: Dnmt1, Dnmt3A, and Dnmt3B.
Dnmt1 is thought to be mainly responsible for the maintenance of pre-existing methylation profiles after DNA replication, however, its specific contribution to the formation of DNA methylation profiles still remains obscure.
“We have developed a new tool that allows us to see details of epigenetic mechanisms that were invisible with previously used methods. Epigenetic marks in DNA, which determine the identity and fate of a mammalian cell during development, are written by three independently controlled 'pens'. Therefore, conventional analytical methods can only see a final combined result of these three 'writers', but exactly what and when each of them writes into DNA, is largely unknown. Our newly discovered method allows us to selectively track the catalytic activity of one of the 'pens', the Dnmt1 methyltransferase, in living cells. Using biomolecular engineering technologies, we have enabled Dnmt1 to use a different 'ink' and permits tracing its activity separately from the other two 'pens',” explains Prof. Klimašauskas.
The Dnmt-TOP-seq approach opens the door to a wealth of new research that will provide a deeper insight into the mechanisms of cellular epigenetic regulation in the development of organisms and in human disease.
“It can be applied in biochemistry, biomedicine, nanotechnology, and other research fields, and in the development of tools for disease diagnosis. Tagging different regions of DNA chains using the corresponding methyltransferases allows the study of the complex process of protein translation in real-time,” notes GMC researcher Prof. G. Vilkaitis.
This work is part of a European Research Council (ERC) grant, the only one in Lithuania so far. In this work, the scientists used not only the CRISPR-Cas gene editing technology but also their previously developed and patented methods for specific biomolecule labeling and analysis, called mTAG (methyltransferase-directed Transfer of Activated Groups) and TOP-seq (Tethered Oligonucleotide-Primed Sequencing), which enabled highly accurate covalent DNA labeling and analysis in live mammalian cells.
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