Publications by authors named "Sarah Schiffers"

6 Publications

  • Page 1 of 1

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

Analysis of an Active Deformylation Mechanism of 5-Formyl-deoxycytidine (fdC) in Stem Cells.

Angew Chem Int Ed Engl 2020 03 25;59(14):5591-5594. Epub 2020 Feb 25.

Department of Chemistry, Ludwig-Maximilians Universität München, Butenandtstr. 5-13, 81377, München, Germany.

The removal of 5-methyl-deoxycytidine (mdC) from promoter elements is associated with reactivation of the silenced corresponding genes. It takes place through an active demethylation process involving the oxidation of mdC to 5-hydroxymethyl-deoxycytidine (hmdC) and further on to 5-formyl-deoxycytidine (fdC) and 5-carboxy-deoxycytidine (cadC) with the help of α-ketoglutarate-dependent Tet oxygenases. The next step can occur through the action of a glycosylase (TDG), which cleaves fdC out of the genome for replacement by dC. A second pathway is proposed to involve C-C bond cleavage that converts fdC directly into dC. A 6-aza-5-formyl-deoxycytidine (a-fdC) probe molecule was synthesized and fed to various somatic cell lines and induced mouse embryonic stem cells, together with a 2'-fluorinated fdC analogue (F-fdC). While deformylation of F-fdC was clearly observed in vivo, it did not occur with a-fdC, thus suggesting that the C-C bond-cleaving deformylation is initiated by nucleophilic activation.
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http://dx.doi.org/10.1002/anie.202000414DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7155088PMC
March 2020

Influencing Epigenetic Information with a Hydrolytically Stable Carbocyclic 5-Aza-2'-deoxycytidine.

Angew Chem Int Ed Engl 2019 09 25;58(37):12984-12987. Epub 2019 Jul 25.

Department of Chemistry, Ludwig-Maximilians-Universität, Butenandtstrasse 5-13, Munich, Germany.

5-Aza-2'-deoxycytidine (AzadC) is an antimetabolite in clinical use, which reduces the level of the epigenetic modification 5-methyl-2'-deoxycytidine (mdC). AzadC is incorporated into the genome of proliferating cells, where it inhibits DNA methyltransferases (DNMTs), leading to a reduction of mdC. The loss of mdC, which is a transcriptional silencer in the promoter region found upstream of genes, leads to the reactivation of the corresponding gene, including tumor-suppressor genes, which elicits a beneficial effect. The problem associated with AzadC is that the compound is hydrolytically unstable. It decomposes during treatment to a variety of poorly characterized hydrolysis products. After its incorporation into the genome, this hydrolytic instability generates abasic sites. It is consequently difficult to dissect whether the activity of the compound is caused by DNMT inhibition or more generally by DNA lesion formation. We now discovered that a disarmed version of AzadC, in which the ribose oxygen was replaced by a CH group, is surprisingly stable under a variety of pH values while keeping activity against the DNMTs.
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http://dx.doi.org/10.1002/anie.201904794DOI Listing
September 2019

Isotope-dilution mass spectrometry for exact quantification of noncanonical DNA nucleosides.

Nat Protoc 2019 01;14(1):283-312

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

DNA contains not only canonical nucleotides but also a variety of modifications of the bases. In particular, cytosine and adenine are frequently modified. Determination of the exact quantity of these noncanonical bases can contribute to the characterization of the state of a biological system, e.g., determination of disease or developmental processes, and is therefore extremely important. Here, we present a workflow that includes detailed description of critical sample preparation steps and important aspects of mass spectrometry analysis and validation. In this protocol, extraction and digestion of DNA by an optimized spin-column and enzyme-based method are described. Isotopically labeled standards are added in the course of DNA digestion, which allows exact quantification by isotope dilution mass spectrometry. To overcome the major bottleneck of such analyses, we developed a short (~14-min-per-sample) ultra-HPLC (UHPLC) and triple quadrupole mass spectrometric (QQQ-MS) method. Easy calculation of the modification abundance in the genome is possible with the provided evaluation sheets. Compared to alternative methods, the quantification procedure presented here allows rapid, ultrasensitive (low femtomole range) and highly reproducible quantification of different nucleosides in parallel. Including sample preparation and evaluation, quantification of DNA modifications can be achieved in less than a week.
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http://dx.doi.org/10.1038/s41596-018-0094-6DOI Listing
January 2019

Chromatin-dependent allosteric regulation of DNMT3A activity by MeCP2.

Nucleic Acids Res 2018 09;46(17):9044-9056

Department of Biochemistry, Institute of Biochemistry and Technical Biochemistry, Faculty of Chemistry, University Stuttgart, Allmandring 31, 70569 Stuttgart, Germany.

Despite their central importance in mammalian development, the mechanisms that regulate the DNA methylation machinery and thereby the generation of genomic methylation patterns are still poorly understood. Here, we identify the 5mC-binding protein MeCP2 as a direct and strong interactor of DNA methyltransferase 3 (DNMT3) proteins. We mapped the interaction interface to the transcriptional repression domain of MeCP2 and the ADD domain of DNMT3A and find that binding of MeCP2 strongly inhibits the activity of DNMT3A in vitro. This effect was reinforced by cellular studies where a global reduction of DNA methylation levels was observed after overexpression of MeCP2 in human cells. By engineering conformationally locked DNMT3A variants as novel tools to study the allosteric regulation of this enzyme, we show that MeCP2 stabilizes the closed, autoinhibitory conformation of DNMT3A. Interestingly, the interaction with MeCP2 and its resulting inhibition were relieved by the binding of K4 unmodified histone H3 N-terminal tail to the DNMT3A-ADD domain. Taken together, our data indicate that the localization and activity of DNMT3A are under the combined control of MeCP2 and H3 tail modifications where, depending on the modification status of the H3 tail at the binding sites, MeCP2 can act as either a repressor or activator of DNA methylation.
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http://dx.doi.org/10.1093/nar/gky715DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6158614PMC
September 2018

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