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

Toni Pfaffeneder, Fabio Spada, Mirko Wagner, Caterina Brandmayr, Silvia K Laube, David Eisen, Matthias Truss, Jessica Steinbacher, Benjamin Hackner, Olga Kotljarova, David Schuermann, Stylianos Michalakis, Olesea Kosmatchev, Stefan Schiesser, Barbara Steigenberger, Nada Raddaoui, Gengo Kashiwazaki, Udo Müller, Cornelia G Spruijt, Michiel Vermeulen, Heinrich Leonhardt, Primo Schär, Markus Müller, Thomas Carell

Index: Nat. Chem. Biol. 10(7) , 574-81, (2014)

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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.