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

Nat Chem Biol. 2014 Jul;10(7):574-81. doi: 10.1038/nchembio.1532. Epub 2014 May 18.

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.

MeSH terms

  • 5-Methylcytosine / analogs & derivatives
  • Animals
  • Base Sequence
  • Carbon Isotopes
  • Chromatin Assembly and Disassembly
  • Chromatography, Liquid
  • Cytosine / analogs & derivatives
  • Cytosine / metabolism
  • DNA / metabolism*
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism*
  • Embryonic Stem Cells / cytology
  • Embryonic Stem Cells / metabolism*
  • Gene Expression
  • Mice
  • Molecular Sequence Data
  • Oxidation-Reduction
  • Pentoxyl / analogs & derivatives*
  • Pentoxyl / metabolism
  • Protein Binding
  • Proto-Oncogene Proteins / genetics
  • Proto-Oncogene Proteins / metabolism*
  • Spectrometry, Mass, Electrospray Ionization
  • Thymine / metabolism*
  • Transcription Factors / genetics
  • Transcription Factors / metabolism

Substances

  • 5-formylcytosine
  • Carbon Isotopes
  • DNA-Binding Proteins
  • Proto-Oncogene Proteins
  • TET1 protein, mouse
  • Tet2 protein, mouse
  • Transcription Factors
  • 5-hydroxymethylcytosine
  • 5-hydroxymethyluracil
  • 5-Methylcytosine
  • Pentoxyl
  • Cytosine
  • DNA
  • Thymine