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

Abstract

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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Figure 1: Metabolism of cytosine derivatives, their detection by LC-UV-ESI-MS/MS and levels in mESCs and mouse cortex.
Figure 2: hmU is present at elevated levels in mESCs compared to tissue.
Figure 3: hmU and fU are thymine oxidation products in WT mESCs with no detectable contribution from hmC or fC deamination.
Figure 4: Tet1 and Tet2 generate hmU in mESCs.
Figure 5: hmU is produced during mESC differentiation.
Figure 6: Identification of hmU:A readers.

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Acknowledgements

We thank the Excellence Cluster Center for Integrated Protein Science Munich (CiPSM) and the collaborative research centers SFB749, SFB646 and SFB1032 as well as German Research Foundation (DFG) grant CA275/8-4, the Volkswagen foundation, NGFNplus (01GS0870) and the Netherlands Organization for Scientific Research (NWO-VIDI) for financial support. T.P. and S.S. thank the Fonds der Chemischen Industrie for predoctoral fellowships. C.B. thanks the Boehringer Ingelheim Fonds for a predoctoral fellowship. G.K. thanks the Japan Society for the Promotion of Science (JSPS) for a postdoctoral fellowship for research abroad. We thank M. Moser (Max Planck Institute for Biochemistry) for providing R1- and C57Bl6/129–derived mESCs, G. Höfner and K.T. Wanner for their initial help with MS as well as M. Wirsing, L. Belzner and P. Laube for providing bioinformatic tools for data processing.

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Contributions

T.P. synthesized MS standards, performed the sample preparation and ultra high-performance LC/MS/MS method development, did LC/MS analysis, interpreted data and performed statistical analysis. F.S. and N.R. performed mESC differentiation and isotope tracing experiments. M.W. and C.B. performed the HEK-293T experiments, did LC/MS analysis and interpreted data. C.B. performed qPCR and analyzed the data. S.K.L. and D.E. performed the protein pulldown studies and interpreted data. M.T. performed mESC knockdown and knockout experiments. J.S. and O. Kosmatchev did sample preparation and LC/MS analysis. B.H., S.S. and J.S. prepared MS standards. O. Kotljarova performed in vitro assays. B.S. synthesized oligonucleotides for protein capture. G.K. synthesized tandem mass tags, and S.M. provided mouse tissue samples. U.M. and H.L. constructed Tet expression plasmids. C.G.S. and M.V. performed Uhrf1/2 overexpression in HEK-293T cells. P.S. and D.S. provided plasmids and cell lines. M.M. and T.C. conceived and supervised the project, interpreted data and wrote the manuscript.

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Correspondence to Markus Müller or Thomas Carell.

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The authors declare no competing financial interests.

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Supplementary Text and Figures

Supplementary Results, Supplementary Figures 1–12, Supplementary Tables 1–22 and Supplementary Notes 1–5. (PDF 2618 kb)

Supplementary Data Set 1

5hmU reader proteins identified from 4 independent pulldown experiments. (XLSX 126 kb)

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Pfaffeneder, T., Spada, F., Wagner, M. et al. Tet oxidizes thymine to 5-hydroxymethyluracil in mouse embryonic stem cell DNA. Nat Chem Biol 10, 574–581 (2014). https://doi.org/10.1038/nchembio.1532

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