Department of Biological DNA Modification
|Saulius KLIMAŠAUSKAS |
Dr. Habil., FRSC
|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.
Edita Kriukienė, Ph.D. Janina Ličytė, M.Sc.
Rasa Rakauskaitė, Ph.D. Milda Mickutė, M.Sc.
Viktoras Masevičius, Ph.D. Povilas Gibas, M.Sc.
Juozas Gordevičius, Ph.D. Milda Narmontė, M.Sc.
Miglė Tomkuvienė, Ph.D. Kotryna Skardžiūtė, M.Sc.
Vaidotas Stankevičius, Ph.D. Bernadeta Masiulionytė, M.Sc.
Aleksandr Osipenko, Ph.D.
Liepa (Stasė) Gasiulė, Ph.D.
Zdislav Staševskij, M.Sc.
Giedrė Urbanavičiūtė, M.Sc.
Kotryna Kvederavičiūtė, M.Sc.
Audronė Rukšėnaitė, M.Sc.
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 (). 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 () or decarboxylation of 5-carboxylcytosines () 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 ().
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).
Our current ERC-supported studies (Single-cell temporal tracking of epigenetic DNA marks, EpiTrack) seek 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 are 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. We are also working to achieve high-resolution single-cell profiling of the tagged DNA modification sites, which will 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).
M. Mickutė, K. Kvederavičiūtė, A. Osipenko, R. Mineikaitė, S. Klimašauskas and G. Vilkaitis
Methyltransferase-directed orthogonal tagging and sequencing of miRNAs and bacterial small RNAs.
BMC Biology, 2021, 19: in press
A.N. Tesfahun, M. Alexeeva, M. Tomkuvienė, A. Arshad, P. Guragain, A. Klungland, S. Klimašauskas, P. Ruoff and 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 and 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 and 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 and 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 and 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 and 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 and 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, and 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 and 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 and 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 and 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, and 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 and 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 and 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.
J. Gordevičius, A. Kriščiūnas, D.E. Groot, S.M. Yip, M. Susic, A. Kwan, R. Kustra, A.M. Joshua, K.N. Chi, A. Petronis and G. Oh.
Cell-free DNA modification dynamics in abiraterone acetate-treated prostate cancer patients.
Clin. Cancer Res., 2018, 24(14): 3317-3324.
M. Tomkuvienė, J. Ličytė, I. Olendraitė, Z. Liutkevičiūtė, B. Clouet-d'Orval and 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 and 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 and 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 and 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 and 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 and 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 and 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.
D. Esyunina, M. Turtola, D. Pupov, I. Bass, S. Klimašauskas, G. Belogurov and A. Kulbachinskiy
Lineage-specific variations in the trigger loop modulate RNA proofreading by bacterial RNA polymerases.
Nucleic Acids Res., 2016, 44(3): 1298–1308.
R. Rakauskaitė, G. Urbanavičiūtė, A. Rukšėnaitė, Z. Liutkevičiūtė, R. Juškėnas, V. Masevičius, and 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 and 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 and 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 and S. Klimašauskas
Direct decarboxylation of 5-carboxylcytosine by DNA C5-methyltransferases.
J. Am. Chem. Soc., 2014, 136(16): 5884−5887.
N. Miropolskaya, D. Esyunina, S. Klimašauskas, V. Nikiforov, I. Artsimovitch and A. Kulbachinskiy
Interplay between the trigger loop and the F loop during RNA polymerase catalysis.
Nucleic Acids Res., 2014, 42(1): 544–552.
E. Kriukienė, V. Labrie, T. Khare, G. Urbanavičiūtė, A. Lapinaitė, K. Koncevičius, D. Li, T. Wang, S. Pai,C. Ptak, J. Gordevičius, S.C. Wang, A. Petronis, and S. Klimašauskas
DNA unmethylome profiling by covalent capture of CpG sites.
Nature Commun., 2013, 4: 2190.
A. Plotnikova, S. Baranauskė, A. Osipenko, S. Klimašauskas and G. Vilkaitis
Mechanistic insights into small RNA recognition and modification by the HEN1 methyltransferase.
Biochem J., 2013, 453: 281-290.
G. Lukinavičius, M. Tomkuvienė, V. Masevičius and S. Klimašauskas
Enhanced chemical stability of AdoMet analogues for improved methyltransferase-directed labeling of DNA.
ACS Chem. Biol., 2013, 8: 1134-1139.
T. Khare, S. Pai, K. Koncevičius, M. Pal, E. Kriukienė, Z. Liutkevičiūtė, M. Irimia, P. Jia, C. Ptak, M. Xia, R. Tice, M. Tochigi, S. Moréra, A. Nazarians, D. Belsham, A.H.C. Wong, B.J. Blencowe, S.C. Wang, P. Kapranov, R. Kustra, V. Labrie, S. Klimašauskas and A. Petronis
5-hmC in the brain is abundant in synaptic genes and shows differences at the exon-intron boundary.
Nature Struct. Mol. Biol., 2012, 19: (10) 1037–1043.
G. Lukinavičius, A. Lapinaitė, G. Urbanavičiūtė, R. Gerasimaitė and S. Klimašauskas
Engineering the DNA cytosine-5 methyltransferase reaction for sequence-specific labeling of DNA.
Nucleic Acids Res., 2012, 40, (22) 11594–11602.
M. Tomkuvienė, B. Clouet-d'Orval, I. Černiauskas, E. Weinhold and S. Klimašauskas
Programmable sequence-specific click-labeling of RNA using archaeal box C/D RNP methyltransferases.
Nucleic Acids Res., 2012, 40, (14) 6765-6773.
R. Sakaguchi, A. Giessing, Q. Dai, G. Lahoud, Z. Liutkevičiūtė, S. Klimašauskas, J. Piccirilli, F. Kirpekar and Y.-M. Hou
Recognition of guanosine by dissimilar tRNA methyltransferases.
RNA, 2012, 18: 1687–1701.
Z. Liutkevičiūtė, E. Kriukienė, I. Grigaitytė, V. Masevičius and S. Klimašauskas
Methyltransferase-directed derivatization of 5-hydroxymethylcytosine in DNA.
Angew. Chem. Int. Ed., 2011, 50: 2090-2093.
R. Gerasimaitė, E. Merkienė and S. Klimašauskas
Direct observation of cytosine flipping and covalent catalysis in a DNA methyltransferase.
Nucleic Acids Res., 2011, 39: 3771-3780.