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.

Abstract: Nervous System (NS) is formed by different neural cells, such as astrocytes, neurons and oligodendroglia in the central nervous system (CNS) and swan cells in the peripheric NS. Neurons are speciality cells that communicate each other and with all body cells. Oligodendroglia and Swan cells, are productors of myeline in the CNS and in peripheric NS respectively. Astrocytes are special cells in the NS with many functions, such as fooding the neurons and myeline productor cells with glucose; elimination of glutamate in synapsis; constituent of blood brain barrio (BBB); productor of cytokines and chemokines to control inflammation; take care of the microbiota; elimination bacteria and virus no autochthon inside the NS; have a role in memory and learning producing long and short term potentiation; pass to reactive astrocytes to protect neurons, oligodendroglia and swan cells; form a plate in the CNS when is broken to evite the communication between cells to protect NS from bacteria and virus. Furthermore, astrocytes control toxic action of Aβ peptide and plates in Alzheimer’s disease; control insulin receptor and increase pAKT to decrease GSK-3 activating GS (glutamate synthase) to eliminate glucose inside the cells and, of course reduce pTAU; increase IDE (inhibitor of degrading enzyme) that decrease insulin and Aβ; because noradrenalin can unit to its receptor and, to Aβ too producing GSK-3, astrocytes diminish the increase of pTAU production. All these demonstrate than astrocytes can be the more important cells in our nervous system.
Keywords: Neurons; Astrocytes; oligodendroglia; ATP increase; IDE; GSK-3; pTAU; Aβ peptide; BBB; CNS; PNS; learning; memory.

Acknowledgements: “Fineurol group”

Biography: In 1985, Prof. Valles graduated in Biological Science at the University of Valencia and in 1996 I finished my PhD in the Cytological Research Institute (IEC) looking for astrogliogenesis during brain development. In 1997, I spent three years at Hallamshire Hospital, Sheffield, UK, working in inflammation and regulation of IL-1 and IL-1 receptor. In 2000, I returned to Spain at Department of Physiology, Faculty of Medicine in University of Valencia until now. I was working in toxicity in developmental and neurodegenerative disease (such as Alzheimer’s disease) looking for oxidative stress and inflammation mechanisms. Actually, I have my own laboratory “FINEUROL” and I am working in neurodegeneration and neurodevelopment. I have lectures in the School of Medicine as a Professor.

Biography: Prof. Nagwa A Meguid is a professor of human genetics and special needs, Former Head of Human Genetics Institute, (NRC) in Egypt. She holds a Ph.D. in Human Genetics, and she is a Senior Geneticist at the Genetics Institute, Pasadena, California; a fellow of Uppsala University, Sweden and senior geneticist at Yale University USA. She wins the outstanding L’Oreal UNESCO Award for women in Science; Africa & Middle East. Her work spans a wide area that ties together work from different disciplines; genetics, psychiatry, behavioral neuroscience and women empowerment. Awarded the National State Award of Excellence in Advanced Medical science & Technology. She was given the Distinctive Arab Female Scientist Prize in Genetics by the Arabian Gulf University. Recently Schowman Arab Award in Autism2022. She used her expertise to identify and describe several novel genetic syndromes. Had more than 100 International publications in elite Journals. Role model for postgraduate students, Supervising 80 theses. Head of the laboratory of research in DNA in behavioral disorders and Founder of autism research study group in Egypt. Jury president L’OREAL-UNESCO Egypt awards for young researchers. Head of Child Brain Research Group and Council for Nutritional and Environmental Medicine, Norway.
She built a remarkable library of ASD cases, publishing more than 70 articles in International Journals handling discovery of new genes and new Biomarkers in autistic children. She published a Chapter on Autism in Egypt, in a book by Prof. Alfred Volkmar. She is the head and Founder of autism research group study at NRC, Egypt. Had two Licensed patents for autism. Developed new strategies in Information Communication Technology application for broken neglected women having disabled children combating the stigmatization and alienation of women who bear disabled children in Egypt. Chapter in Women and ICT in Africa and the Middle East: Changing Selves, Changing Societies (2016): Published by Zed Books in collaboration with the International Development Research Centre in Canada. Jury president L’OREAL-UNESCO Egypt awards for young women researchers. Head of CONEM Egypt Child Brain Research Group and member in Council for Nutritional and Environmental Medicine, Norway. Peer Reviewer for Scientific elite International Journals. Member in Bioethics Network at NRC. She used genetics to understand the links between brain structure and cognitive abilities of neuro-developmental disorders.. Her work has a strong gene-brain-behavior research focus.