title: Analysis of TET expression/activity and 5mC oxidation during normal and malignant germ cell development.
abstract: During mammalian development the fertilized zygote and primordial germ cells lose their DNA methylation within one cell cycle leading to the concept of active DNA demethylation. Recent studies identified the TET hydroxylases as key enzymes responsible for active DNA demethylation, catalyzing the oxidation of 5-methylcytosine to 5-hydroxymethylcytosine. Further oxidation and activation of the base excision repair mechanism leads to replacement of a modified cytosine by an unmodified one. In this study, we analyzed the expression/activity of TET1-3 and screened for the presence of 5 mC oxidation products in adult human testis and in germ cell cancers. By analyzing human testis sections, we show that levels of 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine are decreasing as spermatogenesis proceeds, while 5-methylcytosine levels remain constant. These data indicate that during spermatogenesis active DNA demethylation becomes downregulated leading to a conservation of the methylation marks in mature sperm. We demonstrate that all carcinoma in situ and the majority of seminomas are hypomethylated and hypohydroxymethylated compared to non-seminomas. Interestingly, 5-formylcytosine and 5-carboxylcytosine were detectable in all germ cell cancer entities analyzed, but levels did not correlate to the 5-methylcytosine or 5-hydroxymethylcytosine status. A meta-analysis of gene expression data of germ cell cancer tissues and corresponding cell lines demonstrates high expression of TET1 and the DNA glycosylase TDG, suggesting that germ cell cancers utilize the oxidation pathway for active DNA demethylation. During xenograft experiments, where seminoma-like TCam-2 cells transit to an embryonal carcinoma-like state DNMT3B and DNMT3L where strongly upregulated, which correlated to increasing 5-methylcytosine levels. Additionally, 5-hydroxymethylcytosine levels were elevated, demonstrating that de novo methylation and active demethylation accompanies this transition process. Finally, mutations of IDH1 (IDH1 (R132)) and IDH2 (IDH2 (R172)) leading to production of the TET inhibiting oncometabolite 2-hydroxyglutarate in germ cell cancer cell lines were not detected.
authors.affiliation: Institute of Pathology, Department of Developmental Pathology, University Hospital, Bonn, Germany.
authors.forename: Daniel
authors.initials: D
authors.lastname: Nettersheim
authors.affiliation: Institute of Pathology, University Hospital, Bonn, Germany.
authors.forename: Lukas C
authors.initials: LC
authors.lastname: Heukamp
authors.affiliation: Institute of Pathology, University Hospital, Bonn, Germany.
authors.forename: Florian
authors.initials: F
authors.lastname: Fronhoffs
authors.affiliation: Institute of Pathology, University Hospital, Bonn, Germany.
authors.forename: Marc J
authors.initials: MJ
authors.lastname: Grewe
authors.affiliation: Institute of Pathology, Department of Developmental Pathology, University Hospital, Bonn, Germany.
authors.forename: Natalie
authors.initials: N
authors.lastname: Haas
authors.affiliation: Institute of Neuropathology, University Hospital, Bonn, Germany.
authors.forename: Anke
authors.initials: A
authors.lastname: Waha
authors.affiliation: Department of Oncology, Haematology and Bone Marrow Transplantation with Section Pneumology, Hubertus Wald-Tumorzentrum, University Medical Center Hamburg-Eppendorf, Hamburg, Germany.
authors.forename: Friedemann
authors.initials: F
authors.lastname: Honecker
authors.affiliation: Institute of Neuropathology, University Hospital, Bonn, Germany.
authors.forename: Andreas
authors.initials: A
authors.lastname: Waha
authors.affiliation: Institute of Pathology, University Hospital, Bonn, Germany.
authors.forename: Glen
authors.initials: G
authors.lastname: Kristiansen
authors.affiliation: Institute of Pathology, Department of Developmental Pathology, University Hospital, Bonn, Germany.
authors.forename: Hubert
authors.initials: H
authors.lastname: Schorle
mesh_terms: D044503:5-Methylcytosine / Q000378:metabolism* / Q000031:analogs & derivatives; D002277:Carcinoma / Q000235:genetics / Q000378:metabolism; D045744:Cell Line, Tumor; D003596:Cytosine / Q000031:analogs & derivatives / Q000378:metabolism; D019175:DNA Methylation; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D015536:Down-Regulation; D005854:Germ Cells / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D006899:Mixed Function Oxygenases; D009373:Neoplasms, Germ Cell and Embryonal / Q000235:genetics / Q000378:metabolism; D010084:Oxidation-Reduction; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D013091:Spermatogenesis; D013737:Testis / Q000201:enzymology / Q000378:metabolism
chemical_list: C560973:5-formylcytosine; D004268:DNA-Binding Proteins; D011518:Proto-Oncogene Proteins; C011865:5-hydroxymethylcytosine; D044503:5-Methylcytosine; D003596:Cytosine; D006899:Mixed Function Oxygenases; C462539:TET1 protein, human