Tet oxidizes thymine to 5-hydroxymethyluracil in mouse embryonic stem cell DNA.
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.
1] Center for Integrated Protein Science at the Department of Chemistry, Ludwig-Maximilians-Universität München, München, Germany. [2].
Toni
T
Pfaffeneder
1] Center for Integrated Protein Science at the Department of Chemistry, Ludwig-Maximilians-Universität München, München, Germany. [2].
Fabio
F
Spada
1] Center for Integrated Protein Science at the Department of Chemistry, Ludwig-Maximilians-Universität München, München, Germany. [2].
Mirko
M
Wagner
Center for Integrated Protein Science at the Department of Chemistry, Ludwig-Maximilians-Universität München, München, Germany.
Caterina
C
Brandmayr
Center for Integrated Protein Science at the Department of Chemistry, Ludwig-Maximilians-Universität München, München, Germany.
Silvia K
SK
Laube
Center for Integrated Protein Science at the Department of Chemistry, Ludwig-Maximilians-Universität München, München, Germany.
David
D
Eisen
Charité Universitätsklinikum, Otto-Heubner-Centrum für Kinder und Jugendmedizin, Klinik für Allgemeine Pädiatrie, Labor für Pädiatrische Molekularbiologie, Berlin, Germany.
Matthias
M
Truss
Center for Integrated Protein Science at the Department of Chemistry, Ludwig-Maximilians-Universität München, München, Germany.
Jessica
J
Steinbacher
Center for Integrated Protein Science at the Department of Chemistry, Ludwig-Maximilians-Universität München, München, Germany.
Benjamin
B
Hackner
Center for Integrated Protein Science at the Department of Chemistry, Ludwig-Maximilians-Universität München, München, Germany.
Olga
O
Kotljarova
Department of Biomedicine, University of Basel, Basel, Switzerland.
David
D
Schuermann
Center for Integrated Protein Science at the Department of Pharmacy-Center for Drug Research, Ludwig-Maximilians-Universität München, München, Germany.
Stylianos
S
Michalakis
Center for Integrated Protein Science at the Department of Chemistry, Ludwig-Maximilians-Universität München, München, Germany.
Olesea
O
Kosmatchev
Center for Integrated Protein Science at the Department of Chemistry, Ludwig-Maximilians-Universität München, München, Germany.
Stefan
S
Schiesser
Center for Integrated Protein Science at the Department of Chemistry, Ludwig-Maximilians-Universität München, München, Germany.
Barbara
B
Steigenberger
Center for Integrated Protein Science at the Department of Chemistry, Ludwig-Maximilians-Universität München, München, Germany.
Nada
N
Raddaoui
Center for Integrated Protein Science at the Department of Chemistry, Ludwig-Maximilians-Universität München, München, Germany.
Gengo
G
Kashiwazaki
Center for Integrated Protein Science at the Department of Biology, Ludwig-Maximilians-Universität München, Planegg-Martinsried, Germany.
Udo
U
Müller
Department of Molecular Cancer Research, Cancer Genomics Netherlands, Utrecht, The Netherlands.
Cornelia G
CG
Spruijt
1] Department of Molecular Cancer Research, Cancer Genomics Netherlands, Utrecht, The Netherlands. [2].
Michiel
M
Vermeulen
Center for Integrated Protein Science at the Department of Biology, Ludwig-Maximilians-Universität München, Planegg-Martinsried, Germany.
Heinrich
H
Leonhardt
Department of Biomedicine, University of Basel, Basel, Switzerland.
Primo
P
Schär
Center for Integrated Protein Science at the Department of Chemistry, Ludwig-Maximilians-Universität München, München, Germany.
Markus
M
Müller
Center for Integrated Protein Science at the Department of Chemistry, Ludwig-Maximilians-Universität München, München, Germany.
Thomas
T
Carell
D044503:5-Methylcytosine / Q000031:analogs & derivatives; D000818:Animals; D001483:Base Sequence; D002247:Carbon Isotopes; D042002:Chromatin Assembly and Disassembly; D002853:Chromatography, Liquid; D003596:Cytosine / Q000031:analogs & derivatives / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D049308:Dioxygenases; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism*; D015870:Gene Expression; D051379:Mice; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010432:Pentoxyl / Q000031:analogs & derivatives* / Q000378:metabolism; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization; D013941:Thymine / Q000378:metabolism*; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
C560973:5-formylcytosine; D002247:Carbon Isotopes; D004268:DNA-Binding Proteins; D011518:Proto-Oncogene Proteins; C540839:TET1 protein, mouse; D014157:Transcription Factors; C011865:5-hydroxymethylcytosine; C008055:5-hydroxymethyluracil; D044503:5-Methylcytosine; D010432:Pentoxyl; D003596:Cytosine; D004247:DNA; D049308:Dioxygenases; C551914:Tet2 protein, mouse; D013941:Thymine