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

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

Nature chemical biology (2014-05-20)
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
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.

MATERIALS
Product Number
Brand
Product Description

Gemcitabine impurity A, European Pharmacopoeia (EP) Reference Standard
Supelco
Thymine(Zidovudine Related Compound C), Pharmaceutical Secondary Standard; Certified Reference Material
Sigma-Aldrich
5-(Hydroxymethyl)uracil, 97%
Supelco
Cytosine, Pharmaceutical Secondary Standard; Certified Reference Material
Sigma-Aldrich
Cytosine, ≥99%
Sigma-Aldrich
Thymine, suitable for cell culture, BioReagent
Sigma-Aldrich
Thymine, ≥99%