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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The raw sequencing data, matrix table and targeted bisulfite amplicon sequencing data for Extended Data Fig. 2b are available at the Gene Expression Omnibus (GEO) under accession code GSE152174. The authors declare that all other data supporting the findings of this study are available within the paper and its Supplementary Information files.
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We are very thankful to the following colleagues: M. Möser (Max Planck Institute for Biochemistry) for K3 mPSCs (kindlin3+/+); H. Niwa and M. Okano (both at Kumamoto University) for Oct4-YFP-Puro E14tg2a and Dnmt TKO J1 mPSCs, respectively; Y. Zhang (Boston Children’s Hospital) for parental and Tet TKO E14tg2a cells; and S. Bultmann and C. Mulholland (both at Ludwig Maximilian University) for guidance on high-depth bisulfite amplicon sequencing. Anti-DCTD antibody and purified recombinant DCTD protein were generous gifts from F. Maley (New York State University). Funding was provided by the Deutsche Forschungsgemeinschaft via the programs SFB1309 (TP: A4), SFB1361 (TP: 2), GRK 2338 and SPP1784. This project has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (grant agreement no. EPiR 741912). Y.Z. is supported by the China Scholarship Council (CSC no. 201806200069).
The authors declare no competing interests.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended Data Fig. 1 Expression of pluripotency and early lineage specification factors in mPSCs under different culture conditions.
a, YFP fluorescence and bright field images of an Oct4 YFP reporter mPSC line after adaptation to naïve conditions in chemically defined medium (CDM/2i/LIF; left), followed by six days of priming under serum/CHIR/IWR-1 conditions (serum/CR; middle) or long term priming as mEpiSCs in chemically defined medium containing CHIR/IWR-1/FGF 2/activin A (CDM/CRFA; right). Scale bars = 100 µm. b, Transcript levels of pluripotency and early lineage specification factors assayed by reverse transcription-qPCR from cultures of the Oct4 YFP mPSC line shown in a under CDM/2i/LIF (naïve), serum/a2i/LIF as well as both serum/CR and CDM/CRFA primed conditions. Note that under serum/a2i/LIF conditions relatively high and low levels of naïve and primed markers are expressed, respectively, while the levels of Oct4 and Nanog transcripts are similar to those in naïve conditions. Also, transcript levels of naïve and primed pluripotency factors are similar under short term serum/CR priming conditions and long term primed EpiSCs in CDM/CRFA. Mean (bars) and individual values of fold change Log (diamonds) from three technical replicates are shown. ND = not detectable. Gapdh was used as housekeeping gene reference to calculate ΔCt values.
Extended Data Fig. 2 Global and local transitions of genomic dC modification levels upon priming of mPSCs.
a, Global levels of genomic mdC (left panel) and hmdC (right panel) assayed by UHPLC-MS2 in wt J1 mPSCs transitioning from naïve state (serum/2i/LIF; day 0) to primed state over five days in serum/CHIR/IWR-1 (serum/CR) and in the Oct4-YFP reporter mEpiSC line derived and permanently cultured in serum free CDM containing CHIR/IWR-1/FGF 2/activin A (CRFA). Mean (bars) and individual values (black squares) from three technical replicates are shown. b, High depth targeted amplicon bisulfite sequencing analysis of selected secondary imprints in mPSCs upon transition from a2i/LIF (a2iL) to serum/CR (FCR) conditions. Primary mouse embryonic fibroblasts (mEFs) are shown as reference for differentiated primary somatic cells. CpG methylation levels at individual positions were averaged over each amplicon. Size, numbers of CpG sites and genomic coordinates are reported in Supplementary Table 2. Boxes display the inter-quartile range (IQR), where lower and upper edges represent the first and third quartile, respectively, and the thicker horizontal line is the median. Whiskers mark 1.5xIQR and dots represent outliers beyond 1.5xIQR. Magenta and green box plots represent distinct amplicons within the H19 promoter region and Nespas. Results from individual culture samples are shown. Numbers of aligned reads used for quantifications (range depends on sample and CpG position): Dlk1: 5.5k-23k; H19: green 13k-35K, purple 22k-39k; Nespas: green 26k-67k; purple 32k-80k; Magel-Mrkn3: 14k-36.7k; Cdkn1c: 10k-26.7k.
Extended Data Fig. 3 Drastic reduction of global genomic mdC oxidation and increase of mdC-to-mdU conversion upon transition to primed pluripotency (related to Fig. 1).
Time course analysis of genomic dC derivatives upon metabolic labelling with m+4Met under naïve (a; a2i/LIF) and priming (b; serum/CR) conditions. Independent biological replicate of the experiment shown in Fig. 1c,d. The labelling/time course schedules are shown at the top. Global levels of unlabeled mC (pale yellow) and m+4dC (dark yellow), hm+3dC (blue) and f+2dC (green) are shown as mean (bars) and individual values (black squares) of three technical replicates. LOD = limt of detection.
Extended Data Fig. 4 Turnover of genomic mdC and its derivatives under priming conditions (related to Fig. 2).
a, b, Independent biological replicate of the experiment reported in Fig. 2c,e. a, Global profiles of the indicated labelled cytosine derivatives and dT+12 in the genome of mPSCs under priming conditions (serum/CR) upon chasing of m+4Met. Each panel shows fold change values relative to the highest level for each modification from an individual experiment. Dashes represent single technical replicate measurements (n = 3) from single samples. Full lines lines represent first order decay curves fitted to mean values (in the case of f+2dC t = 0 h was excluded from curve fitting). b, Absolute levels of genomic m+4Met from the same samples as in a. Black squares represent single technical replicate measurements (n = 3) from single samples and bars show their mean values.
Global levels of m+4dC (a; yellow) and m+4dU (b; red) in the genome of wt and Dnmt TKO J1 mPSCs after priming for five days in the presence m+4Met. In Dnmt TKO cells neither m+4dC nor m+4dU were above background levels (LOD = limit of detection). The two panels in b show results from two independent replicate cultures (n = 2). Mean (bars) and individual values (black squares) of three technical replicates are shown.
Extended Data Fig. 6 Double labelling of primed wt and CD DKO J1 with m+4Met and dC[D9] (related to Fig. 4).
a, Conversion of dC[D9] into m+4dC[D8] and potentially m + 4dU[D8] within gDNA. SAH = S adenosylhomocysteine. b, UHPLC chromatogram (UV trace) showing the retention delay of dT[D8] relative to natural dT. c, Quantification of m+4dU[D8] accumulated in wt and CD DKO PSCs upon priming in the presence of m+4Met and dC[D8]. Mean (bars) and individual values (black squares) of three technical replicates are shown.
Extended Data Fig. 7 Tet proteins do not trigger DNA repair events that contribute to turnover of mdC into dT (related to Fig. 3).
Global levels of mdC, m+4dC and m+4dU (red) in the genome of parental (Tet1-3 proficient) and Tet TKO E14tg2a mPSCs after priming for five days in the presence m+4Met. a) and b) show independent biological replicates. Mean (bars) and individual values (black squares) of three technical replicates are shown.
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Spada, F., Schiffers, S., Kirchner, A. et al. Active turnover of genomic methylcytosine in pluripotent cells. Nat Chem Biol 16, 1411–1419 (2020). https://doi.org/10.1038/s41589-020-0621-y
Nature Reviews Genetics (2021)