Department of Biological DNA Modification
 | Head |
| Saulius KLIMAŠAUSKAS Dr. Habil., FRSC Distinguished Professor | |
| ORCID; Google Scholar; ResearcherID | |
| phone: +370 5 2234350 fax: +370 5 2234367 e-mail: saulius.klimasauskas (at) bti.vu.lt |
Research Staff PhD students
Giedrius VILKAITIS, Ph.D. Bernadeta MASIULIONYTĖ, M.Sc.
Edita KRIUKIENĖ, Ph.D. Kotryna KVEDERAVIČIŪTĖ, M.Sc.
Vaidotas STANKEVIČIUS, Ph.D. Kotryna SKARDŽIŪTĖ, M.Sc.
Miglė TOMKUVIENĖ, Ph.D. Joris BALČIŪNAS, M.Sc.
Rasa RAKAUSKAITĖ, Ph.D.
Eglė JAKUBAUSKIENĖ, Ph.D.
Inga PEČIULIENĖ, Ph.D.
Liepa GASIULĖ, Ph.D.
Milda MICKUTĖ, Ph.D.
Milda NARMONTĖ, Ph.D.
Janina LIČYTĖ, Ph.D.
Audronė RUKŠĖNAITĖ, M.Sc.
Zdislav STAŠEVSKIJ, M.Sc.
Giedrė URBANAVIČIŪTĖ, M.Sc.
Research Overview
AdoMet-dependent methyltransferases (MTases), which represent more than 3% of the proteins in the cell, catalyze the transfer of the methyl group from S-adenosyl-L-methionine (AdoMet) to N-, C-, O- or S-nucleophiles in DNA, RNA, proteins or small biomolecules.
In DNA, enzymatic methylation of nucleobases serves to expand the information content of the genome in organisms ranging from bacteria to mammals. Postreplicative methylation is accomplished by DNA methyltransferases yielding 5-methylcytosine, N4-methylcytosine or N6-methyladenine (Kweon et al., 2019). Genomic DNA methylation is a key epigenetic regulatory mechanism in high eukaryotes. Aberrant DNA methylation correlates with a number of pediatric syndromes and cancer, or predisposes individuals to various other human diseases. However, research into the epigenetic misregulation and its diagnostics is hampered by the limitations of available analytical techniques.
Targeted covalent labeling of biopolymers
Besides their diverse biological roles, DNA MTases are attractive models to study the structural aspects of DNA-protein interaction. Bacterial enzymes recognize an impressive variety (over 200) of short sequences in DNA. Following detailed mechanistic and structural studies of MTases, we turned to repurposing these enzymes sequence-specific covalent modification of DNA and other biopolymers. Our strategy is based on designing novel synthetic analogues of the natural cofactor AdoMet. We have synthesized a series of model AdoMet analogs with sulfonium-bound extended side chains replacing the methyl group. This novel enabling technology named mTAG (methyltransferase-directed Transfer of Activated Groups) is a convenient and robust technique that is suitable for routine laboratory use. In particular, we demonstrated that propargylic side chains can be efficiently transferred by DNA MTases with high sequence- and base-specificity (Lukinavičius et al., 2007, 2012 and 2013; Masevičius et al., 2016; Tomkuvienė et al., 2016; Tomkuvienė et al., 2019 and 2020) offering many potential applications for genomic (Neely et al., 2010) and epigenomic (see below) studies. Moreover, the newly developed cofactors are suitable for targeted transfer of functional groups or other chemical entities to RNA (Tomkuvienė et al., 2012; Plotnikova et al., 2014; Osipenko et al., 2017; Mickutė et al., 2018 and 2021) using appropriate MTases as catalysts.
In the absence of the S-adenosylmethionine cofactor, bacterial cytosine-5 MTases can catalyze catalyze reversible covalent addition of exogenous aliphatic aldehydes to their target residues in DNA, thus yielding corresponding 5-hydroxyalkylcytosines (Liutkevičiūtė et al., 2009). Moreover, our further studies demonstrated the ability of the MTases to direct condensation of aliphatic thiols and selenols with 5-hydroxymethylcytosine in DNA to yield 5-alkylchalcogenomethyl derivatives (Liutkevičiūtė et al., 2011) or decarboxylation of 5-carboxylcytosines (Liutkevičiūtė et al., 2014) in DNA. These atypical reactions demonstrate a surprizing catalytic versatility of these enzymes and pave new ways for the sequence-specific derivatization and analysis of 5-hydroxymethylcytosine in mammalian DNA (Kriukienė et al., 2012; Gibas et al. 2020; Gordevičius et al., 2020).
Novel approaches to epigenome profiling
Genomic DNA methylation is a prevalent epigenetic modification in mammals, which is brought about by three known DNA cytosine-5 methyltransferases (DNMTs). Although DNA methylation has been extensively investigated, many mechanistic aspects of the DNMT action remain obscure due limitations of current analytical techniques. We therefore aim to develop new experimental approaches to genome-wide profiling of DNA methylation for epigenome studies and improved diagnostics. Our approach is based on selective mTAG labeling and enrichment of unmethylated CpG sites (Kriukienė et al. 2013; Labrie et al., 2016) in the genome followed by analysis of the enriched fractions on tiling microarrays (in collaboration with Prof. Art Petronis, CAMH, Toronto, Canada). Recently, we have advanced DNA methylome profiling by developing a high-resolution economical technique named Tethered Oligonucleotide-Primed sequencing, TOP-seq, which exploits non-homologous priming of the DNA polymerase at covalently tagged CpG or hmCpG sites to directly produce adjoining regions for their sequencing and precise genomic mapping (Staševskij et al., 2017; Gibas et al. 2020; Ličytė et al. 2020; Gordevičius et al., 2020).
Single-cell temporal tracking of epigenetic DNA marks (EpiTrack) 
Our ERC-supported studies (Single-cell temporal tracking of epigenetic DNA marks, EpiTrack) aimed to gain in-depth understanding of how the genomic methylation patterns are established and how they govern cell plasticity and variability during differentiation and development. These questions were addressed by precise determination of where and when methylation marks are deposited by the individual DNMTs, and how these methylation marks affect gene expression. To achieve this goal, we use metabolic engineering of mouse cells to permit SAM analog-based chemical pulse-tagging of their methylation sites in vivo to unveil, with unprecedented detail, the dynamics and variability of DNA methylation during differentiation of mouse embryonic cells to somatic lineages.
Methylation of small non-coding RNA
MicroRNAs and siRNAs are small non-coding double-stranded RNA molecules that control gene activity in a homology-dependent manner - a process named RNA interference. Since their discovery in 1993, numerous microRNAs have been identified and recognized as important regulators of gene expression in both plants and animals. Many microRNAs have well-defined developmental and tissue-specific expression pattern, but a great number of microRNAs and their roles are still unknown.
HEN1 methyltransferases from plants and animals catalyze the transfer methyl groups from AdoMet onto the 2'OH group of the 3'-terminal nucleotide of small RNAs, like miRNA, siRNA/siRNA or piRNA. The methylation is imperative in the biogenesis of microRNA in plants and piRNA in animals. A number of chemo-enzymatic approaches have been developed in our laboratory for examining and exploiting the unique properties of the HEN1 methyltransferases (Plotnikova et al., 2013; Baranauskė et al., 2015; Osipenko et al., 2017; Mickutė et al., 2018, 2021).
Recent publications
L. Gasiulė, V. Stankevičius, K. Kvederavičiūtė, J. M. Rimšelis, V. Klimkevičius, G. Petraitytė, A. Rukšėnaitė, V. Masevičius, S. Klimašauskas
Engineered methionine adenosyltransferase cascades for metabolic labeling of individual DNA methylomes in live cells.
J. Am. Chem. Soc.,2024, 146(27): 18722-18729.
E. Kriukienė, M. Tomkuvienė, S. Klimašauskas
5-Hydroxymethylcytosine: the many faces of the sixth base of mammalian DNA.
Chem. Soc. Rev., 2024, 53: 2264–2283.
K. Skardžiūtė, K. Kvederavičiūtė, I. Pečiulienė, M. Narmontė, P. Gibas, J. Ličytė, S. Klimašauskas, E. Kriukienė
One-pot trimodal mapping of unmethylated, hydroxymethylated and open chromatin sites unveils distinctive 5hmC roles at dynamic chromatin loci.
Cell Chem. Biol., 2024, 31(3): 607-621.e9.
G. Vilkaitis, V. Masevičius, E. Kriukienė, S. Klimašauskas
Chemical Expansion of the Methyltransferase Reaction: Tools for DNA Labeling and Epigenome Analysis.
Acc. Chem. Res., 2023, 56: 3188-3197.
M. Mickutė, R. Krasauskas, K. Kvederavičiūtė, G. Tupikaitė, A. Osipenko, A. Kaupinis, M. Jazdauskaitė, R. Mineikaitė, M. Valius, V. Masevičius, G. Vilkaitis
Interplay between bacterial 5′-NAD-RNA decapping hydrolase NudC and DEAD-box RNA helicase CsdA in stress responses.
mSystems, 2023, 8: e00718-23.
M. Malikėnas, V. Masevičius, S. Klimašauskas
Synthesis of S-adenosyl-L-methionine analogs with extended transferable groups for methyltransferase-directed labeling of DNA and RNA.
Curr. Protoc., 2023, 3: e799.
M. Tomkuvienė, M. Meier, D. Ikasalaitė, J. Wildenauer, V. Kairys, S. Klimašauskas, L. Manelytė
Enhanced nucleosome assembly at CpG sites containing an extended 5-methylcytosine analogue.
Nucleic Acids Res., 2022, 50(11): 6549–6561.
M.J. Peña-Gómez, P. Moreno-Gordillo, M. Narmontė, C.B. García-Calderón, A. Rukšėnaitė, S. Klimašauskas, I.V. Rosado
FANCD2 maintains replication fork stability during misincorporation of the DNA demethylation products 5-hydroxymethyl-2'-deoxycytidine and 5-hydroxymethyl-2'-deoxyuridine.
Cell Death Dis., 2022, 13(5): 503.
V. Stankevičius, P. Gibas, B. Masiulionytė, L. Gasiulė, V. Masevičius, S. Klimašauskas, G. Vilkaitis
Selective chemical tracking of Dnmt1 catalytic activity in live cells.
Mol. Cell, 2022, 82(5): 1053-1065. --Meet the author interview.
J. Ličytė, K. Kvederavičiūtė, A. Rukšėnaitė, E. Godliauskaitė, P. Gibas, V. Tomkutė, G. Petraitytė, V. Masevičius, S. Klimašauskas, E. Kriukienė
Distribution and regulatory roles of oxidized 5-methylcytosines in DNA and RNA of the Basidiomycete fungi Laccaria bicolor and Coprinopsis cinerea.
Open Biol., 2022, 12(3): 210302.
M. Tomkuvienė, E. Kriukienė, S. Klimašauskas
DNA labeling using DNA methyltransferases.
Adv. Exp. Med. Biol., 2022, 1389: 535–562.
M. Narmontė, P. Gibas, K. Daniūnaitė, J. Gordevičius, E. Kriukienė
Multi-omics analysis of neuroblastoma cells reveals a diversity of malignant transformations.
Front. Cell Dev. Biol., 2021, 9: 727353.
M. Mickutė, K. Kvederavičiūtė, A. Osipenko, R. Mineikaitė, S. Klimašauskas, G. Vilkaitis
Methyltransferase-directed orthogonal tagging and sequencing of miRNAs and bacterial small RNAs.
BMC Biology, 2021, 19: 129.
A.N. Tesfahun, M. Alexeeva, M. Tomkuvienė, A. Arshad, P. Guragain, A. Klungland, S. Klimašauskas, P. Ruoff, S. Bjelland
Alleviation of C-C Mismatches in DNA by the Escherichia coli Fpg Protein.
Front. Microbiol., 2021, 12: 608839.
R. Rakauskaitė, G. Urbanavičiūtė, M. Simanavičius, A. Žvirblienė, S. Klimašauskas
Selective immunocapture and light-controlled traceless release of transiently caged proteins.
STAR Protoc., 2021, 2(2): 100455.
R. Rakauskaitė, G. Urbanavičiūtė, M. Simanavičius, R. Lasickienė, A. Vaitiekaitė, G. Petraitytė, V. Masevičius, A. Žvirblienė, S. Klimašauskas
Photocage-Selective Capture and Light-Controlled Release of Target Proteins.
iScience, 2020, 23(12): 101833.
M. Tomkuvienė, D. Ikasalaitė, A. Slyvka, A. Rukšėnaitė, M. Ravichandran, T. P. Jurkowski, M. Bochtler, S. Klimašauskas
Enzymatic hydroxylation and excision of extended 5-methylcytosine analogues.
J. Mol. Biol., 2020, 423(23): 6157-6167.
J. Gordevičius, M. Narmontė, P. Gibas, K. Kvederavičiūtė, V. Tomkutė, P. Paluoja, K. Krjutškov, A. Salumets, E. Kriukienė
Identification of fetal unmodified and 5-hydroxymethylated CG sites in maternal cell-free DNA for non-invasive prenatal testing.
Clin. Epigen., 2020, 12: 153.
J. Ličytė, P. Gibas, K. Skardžiūtė, V. Stankevičius, A. Rukšėnaitė, E. Kriukienė
A Bisulfite-free Approach for Base-Resolution Analysis of Genomic 5-Carboxylcytosine.
Cell Rep., 2020, 32(11): 108155.
P. Gibas, M. Narmontė, Z. Staševskij, J. Gordevičius, S. Klimašauskas, E. Kriukienė
Precise genomic mapping of 5-hydroxymethylcytosine via covalent tether-directed sequencing.
PLOS Biol., 2020, 18(4): e3000684.
S. Gasiulė, N. Dreize, A. Kaupinis, R. Ražanskas, L. Čiupas, V. Stankevičius, Ž. Kapustina, A. Laurinavičius, M. Valius, G. Vilkaitis
Molecular Insights into miRNA-Driven Resistance to 5-Fluorouracil and Oxaliplatin Chemotherapy: miR-23b Modulates the Epithelial–Mesenchymal Transition of Colorectal Cancer Cells.
J. Clin. Med., 2019, 8(12): 2115.
S. Gasiulė, V. Stankevičius, V. Patamsytė, R. Ražanskas, G. Žukovas, Ž. Kapustina, D. Žaliaduonytė, R. Benetis, V. Lesauskaitė, G. Vilkaitis
Tissue-Specific miRNAs Regulate the Development of Thoracic Aortic Aneurysm: The Emerging Role of KLF4 Network.
J. Clin. Med., 2019, 8(10): 1609.
S.-M. Kweon, Y. Chen, E. Moon, K. Kvederavičiūtė, S. Klimašauskas, D.E. Feldman
An adversarial DNA N6-methyladenine-sensor network preserves polycomb silencing.
Mol. Cell, 2019, 74(6): 1138-1147.e6.
M. Tomkuvienė, M. Mickutė, G. Vilkaitis, S. Klimašauskas
Repurposing enzymatic transferase reactions for targeted labeling and analysis of DNA and RNA.
Curr. Opin. Biotechnol., 2019, 55: 114-123.
K. Daniūnaitė, S. Jarmalaitė, E. Kriukienė
Epigenomic technologies for diciphering circulating tumor DNA.
Curr. Opin. Biotechnol., 2019, 55: 23-29.
M. Mickutė, M. Nainytė, L. Vasiliauskaitė. A. Plotnikova, V. Masevičius, S. Klimašauskas, G. Vilkaitis
Animal Hen1 2′-O-methyltransferases as tools for 3′-terminal functionalization and labelling of single-stranded RNAs.
Nucleic Acids Res., 2018, 46: e104.
M. Alexeeva, P. Guragain, A.N. Tesfahun, M. Tomkuvienė, A. Arshad, R. Gerasimaitė, A. Rukšėnaitė, G. Urbanavičiūtė, M. Bjørås, J.K. Laerdahl, A. Klungland, S. Klimašauskas, S. Bjelland
Excision of the double methylated base N4,5-dimethylcytosine from DNA by Escherichia coli Nei and Fpg proteins.
Phil. Trans. R. Soc. B, 2018, 373(1748): 20170337.
M. Tomkuvienė, J. Ličytė, I. Olendraitė, Z. Liutkevičiūtė, B. Clouet-d'Orval, S. Klimašauskas
Archaeal fibrillarin-Nop5 heterodimer 2'-O-methylates RNA independently of the C/D guide RNP particle.
RNA, 2017, 23(9): 1329-1337.
A. Osipenko, A. Plotnikova, M. Nainytė, V. Masevičius, S. Klimašauskas, G. Vilkaitis
Oligonucleotide-addressed covalent 3’-terminal derivatization of small RNA strands for enrichment and visualization.
Angew. Chem. Int. Ed., 2017, 56(23): 6507–6510.
Z. Staševskij, P. Gibas, J. Gordevičius, E. Kriukienė, S. Klimašauskas
Tethered Oligonucleotide-Primed sequencing, TOP-seq: a high resolution economical approach for DNA epigenome profiling.
Mol. Cell, 2017, 65(3): 554–564.
M. Tomkuvienė, E. Kriukienė, S. Klimašauskas
DNA labeling using DNA methyltransferases.
Adv. Exp. Med. Biol., 2016, 945: 511-535.
V. Labrie, O. J. Buske, E. Oh, R. Jeremian, C. Ptak, G. Gasiūnas, A. Maleckas, R. Petereit, A. Žvirbliene, K. Adamonis, E. Kriukienė, K. Koncevičius, J. Gordevičius, A. Nair, A. Zhang, S. Ebrahimi, G. Oh, V. Šikšnys, L. Kupčinskas, M. Brudno, A. Petronis
Lactase nonpersistence is directed by DNA-variation-dependent epigenetic aging.
Nature Struct. Mol. Biol. 2016, 23(6): 566-573.
V. Myrianthopoulos, P. F. Cartron, Z. Liutkevičiūtė, S. Klimašauskas, D. Matulis, C. Bronner, N. Martinet, E. Mikros
Tandem virtual screening targeting the SRA domain of UHRF1 identifies a novel chemical tool modulating DNA methylation.
Eur. J. Med. Chem., 2016, 114: 390–396.
V. Masevičius, M. Nainytė, S. Klimašauskas
Synthesis of S-adenosyl-L-methionine analogs with extended transferable groups for methyltransferase-directed labeling of DNA and RNA.
Curr. Protoc. Nucleic Acid Chem., 2016, 64: 1.36.1-1.36.13.
R. Rakauskaitė, G. Urbanavičiūtė, A. Rukšėnaitė, Z. Liutkevičiūtė, R. Juškėnas, V. Masevičius, S. Klimašauskas
Biosynthetic selenoproteins with geneticallyencoded photocaged selenocysteines.
Chem. Commun., 2015, 51(39): 8245-8248.
S. Baranauskė, M. Mickutė, A. Plotnikova, A. Finke, Č. Venclovas, S. Klimašauskas, G. Vilkaitis
Functional mapping of the plant small RNAmethyltransferase: HEN1 physically interacts with HYL1 and DICER-LIKE 1 proteins.
Nucleic Acids Res., 2015, 43(5): 2802-2812.
A. Plotnikova, A. Osipenko, V. Masevičius, G. Vilkaitis, S. Klimašauskas
Selective covalent labeling of miRNA and siRNA duplexes using HEN1 methyltransferase.
J. Am. Chem. Soc., 2014, 136(39): 13550–13553.
Z. Liutkevičiūtė, E. Kriukienė, J. Ličytė, M. Rudytė, G. Urbanavičiūtė, S. Klimašauskas
Direct decarboxylation of 5-carboxylcytosine by DNA C5-methyltransferases.
J. Am. Chem. Soc., 2014, 136(16): 5884−5887.