title: Tissue distribution of 5-hydroxymethylcytosine and search for active demethylation intermediates.
abstract: 5-Hydroxymethylcytosine (hmC) was recently detected as the sixth base in mammalian tissue at so far controversial levels. The function of the modified base is currently unknown, but it is certain that the base is generated from 5-methylcytosine (mC). This fuels the hypothesis that it represents an intermediate of an active demethylation process, which could involve further oxidation of the hydroxymethyl group to a formyl or carboxyl group followed by either deformylation or decarboxylation. Here, we use an ultra-sensitive and accurate isotope based LC-MS method to precisely determine the levels of hmC in various mouse tissues and we searched for 5-formylcytosine (fC), 5-carboxylcytosine (caC), and 5-hydroxymethyluracil (hmU) as putative active demethylation intermediates. Our data suggest that an active oxidative mC demethylation pathway is unlikely to occur. Additionally, we show using HPLC-MS analysis and immunohistochemistry that hmC is present in all tissues and cell types with highest concentrations in neuronal cells of the CNS.
authors.affiliation: Department of Chemistry, Ludwig-Maximilians-University, Center for Integrated Protein Science, Munich, Germany.
authors.forename: Daniel
authors.initials: D
authors.lastname: Globisch
authors.forename: Martin
authors.initials: M
authors.lastname: Münzel
authors.forename: Markus
authors.initials: M
authors.lastname: Müller
authors.forename: Stylianos
authors.initials: S
authors.lastname: Michalakis
authors.forename: Mirko
authors.initials: M
authors.lastname: Wagner
authors.forename: Susanne
authors.initials: S
authors.lastname: Koch
authors.forename: Tobias
authors.initials: T
authors.lastname: Brückl
authors.forename: Martin
authors.initials: M
authors.lastname: Biel
authors.forename: Thomas
authors.initials: T
authors.lastname: Carell
mesh_terms: D044503:5-Methylcytosine / Q000031:analogs & derivatives; D000818:Animals; D002138:Calibration; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002853:Chromatography, Liquid / Q000379:methods; D003596:Cytosine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D019175:DNA Methylation; D007150:Immunohistochemistry / Q000379:methods; D013058:Mass Spectrometry / Q000379:methods; D051379:Mice; D008956:Models, Chemical; D010100:Oxygen / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D014018:Tissue Distribution
chemical_list: C011865:5-hydroxymethylcytosine; D044503:5-Methylcytosine; D003596:Cytosine; D010100:Oxygen