Publications by authors named "Olesea Kosmatchev"

5 Publications

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Active turnover of genomic methylcytosine in pluripotent cells.

Nat Chem Biol 2020 12 10;16(12):1411-1419. Epub 2020 Aug 10.

Department of Chemistry, Ludwig Maximilians University Munich and Center for Integrated Protein Science Munich (CIPSM), Munich, Germany.

Epigenetic plasticity underpins cell potency, but the extent to which active turnover of DNA methylation contributes to such plasticity is not known, and the underlying pathways are poorly understood. Here we use metabolic labeling with stable isotopes and mass spectrometry to quantitatively address the global turnover of genomic 5-methyl-2'-deoxycytidine (mdC), 5-hydroxymethyl-2'-deoxycytidine (hmdC) and 5-formyl-2'-deoxycytidine (fdC) across mouse pluripotent cell states. High rates of mdC/hmdC oxidation and fdC turnover characterize a formative-like pluripotent state. In primed pluripotent cells, the global mdC turnover rate is about 3-6% faster than can be explained by passive dilution through DNA synthesis. While this active component is largely dependent on ten-eleven translocation (Tet)-mediated mdC oxidation, we unveil additional oxidation-independent mdC turnover, possibly through DNA repair. This process accelerates upon acquisition of primed pluripotency and returns to low levels in lineage-committed cells. Thus, in pluripotent cells, active mdC turnover involves both mdC oxidation-dependent and oxidation-independent processes.
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http://dx.doi.org/10.1038/s41589-020-0621-yDOI Listing
December 2020

5-Formylcytosine to cytosine conversion by C-C bond cleavage in vivo.

Nat Chem Biol 2018 Jan 27;14(1):72-78. Epub 2017 Nov 27.

Center for Integrated Protein Science Munich CiPSM at the Department of Chemistry, Ludwig-Maximilians-Universität München, Munich, Germany.

Tet enzymes oxidize 5-methyl-deoxycytidine (mdC) to 5-hydroxymethyl-dC (hmdC), 5-formyl-dC (fdC) and 5-carboxy-dC (cadC) in DNA. It was proposed that fdC and cadC deformylate and decarboxylate, respectively, to dC over the course of an active demethylation process. This would re-install canonical dC bases at previously methylated sites. However, whether such direct C-C bond cleavage reactions at fdC and cadC occur in vivo remains an unanswered question. Here we report the incorporation of synthetic isotope- and (R)-2'-fluorine-labeled dC and fdC derivatives into the genome of cultured mammalian cells. Following the fate of these probe molecules using UHPLC-MS/MS provided quantitative data about the formed reaction products. The data show that the labeled fdC probe is efficiently converted into the corresponding labeled dC, most likely after its incorporation into the genome. Therefore, we conclude that fdC undergoes C-C bond cleavage in stem cells, leading to the direct re-installation of unmodified dC.
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http://dx.doi.org/10.1038/nchembio.2531DOI Listing
January 2018

5-Formyl- and 5-Carboxydeoxycytidines Do Not Cause Accumulation of Harmful Repair Intermediates in Stem Cells.

J Am Chem Soc 2017 08 21;139(30):10359-10364. Epub 2017 Jul 21.

Center for Integrated Protein Science at the Department of Chemistry, LMU Munich , Butenandtstrasse 5-13, Munich 81377, Germany.

5-Formyl-dC (fdC) and 5-carboxy-dC (cadC) are newly discovered bases in the mammalian genome that are supposed to be substrates for base excision repair (BER) in the framework of active demethylation. The bases are recognized by the monofunctional thymine DNA glycosylase (Tdg), which cleaves the glycosidic bond of the bases to give potentially harmful abasic sites (AP-sites). Because of the turnover of fdC and cadC during cell state transitions, it is an open question to what extent such harmful AP-sites may accumulate during these processes. Here, we report the development of a new reagent that in combination with mass spectrometry (MS) allows us to quantify the levels of AP-sites. This combination also allowed the quantification of β-elimination (βE) products, which are repair intermediates of bifunctional DNA glycosylases. In combination with feeding of isotopically labeled nucleosides, we were able to trace the intermediates back to their original nucleobases. We show that, while the steady-state levels of fdC and cadC are substantially increased in Tdg-deficient cells, those of both AP- and βE-sites are unaltered. The levels of the detected BER intermediates are 1 and 2 orders of magnitude lower than those of cadC and fdC, respectively. Thus, neither the presence of fdC nor that of cadC in stem cells leads to the accumulation of harmful AP- and βE-site intermediates.
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http://dx.doi.org/10.1021/jacs.7b04131DOI Listing
August 2017

Quantitative LC-MS Provides No Evidence for m dA or m dC in the Genome of Mouse Embryonic Stem Cells and Tissues.

Angew Chem Int Ed Engl 2017 09 30;56(37):11268-11271. Epub 2017 Mar 30.

Center for Integrated Protein Science (CiPSM) at the Department of Chemistry, LMU München, Butenandtstr. 5-13, 81377, München, Germany.

Until recently, it was believed that the genomes of higher organisms contain, in addition to the four canonical DNA bases, only 5-methyl-dC (m dC) as a modified base to control epigenetic processes. In recent years, this view has changed dramatically with the discovery of 5-hydroxymethyl-dC (hmdC), 5-formyl-dC (fdC), and 5-carboxy-dC (cadC) in DNA from stem cells and brain tissue. N -methyldeoxyadenosine (m dA) is the most recent base reported to be present in the genome of various eukaryotic organisms. This base, together with N -methyldeoxycytidine (m dC), was first reported to be a component of bacterial genomes. In this work, we investigated the levels and distribution of these potentially epigenetically relevant DNA bases by using a novel ultrasensitive UHPLC-MS method. We further report quantitative data for m dC, hmdC, fdC, and cadC, but we were unable to detect either m dC or m dA in DNA isolated from mouse embryonic stem cells or brain and liver tissue, which calls into question their epigenetic relevance.
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http://dx.doi.org/10.1002/anie.201700424DOI Listing
September 2017

Tet oxidizes thymine to 5-hydroxymethyluracil in mouse embryonic stem cell DNA.

Nat Chem Biol 2014 Jul 18;10(7):574-81. Epub 2014 May 18.

Center for Integrated Protein Science at the Department of Chemistry, Ludwig-Maximilians-Universität München, München, Germany.

Ten eleven translocation (Tet) enzymes oxidize the epigenetically important DNA base 5-methylcytosine (mC) stepwise to 5-hydroxymethylcytosine (hmC), 5-formylcytosine and 5-carboxycytosine. It is currently unknown whether Tet-induced oxidation is limited to cytosine-derived nucleobases or whether other nucleobases are oxidized as well. We synthesized isotopologs of all major oxidized pyrimidine and purine bases and performed quantitative MS to show that Tet-induced oxidation is not limited to mC but that thymine is also a substrate that gives 5-hydroxymethyluracil (hmU) in mouse embryonic stem cells (mESCs). Using MS-based isotope tracing, we show that deamination of hmC does not contribute to the steady-state levels of hmU in mESCs. Protein pull-down experiments in combination with peptide tracing identifies hmU as a base that influences binding of chromatin remodeling proteins and transcription factors, suggesting that hmU has a specific function in stem cells besides triggering DNA repair.
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http://dx.doi.org/10.1038/nchembio.1532DOI Listing
July 2014