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 5mC 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.

DNA methyltransferase-3 like protein expression in various histological types of testicular germ cell tumor

“…The positive cells were found in 36.3% (4/11) of the seminoma elements, 57.1% of the teratoma elements and 100% of the CC elements. Although high DNMT3L mRNA expression was reported in CC cell lines (18), to our knowledge, little is known about the DNMT3L protein expression of CCs. Pure CCs are extremely rare, and only one case was obtained in the present study.…”

Re-visiting the Protamine-2 locus: deletion, but not haploinsufficiency, renders male mice infertile

“…RNA concentrations and purity ratios were determined by NanoDrop measurement (Peqlab, Erlangen, Germany). qRT-PCR was performed as described previously64. First strand cDNA synthesis was carried out using RevertAid First Strand cDNA Synthesis Kit (Fermentas, St. Leon-Rot, Germany) after DNAseI treatment of RNA.…”

“…Immunohistochemistry (IHC) against PRM1 was performed as described previously64 using the Autostainer 480 S (Medac, Hamburg, Germany). For IHC of PRM2, sections were treated for 10 min at RT with decondensing-mix containing 25 mM dithiothreitol, 0.2% Triton X-100 and 200 IU heparin/ml in PBS to enhance antigen accessibility for the primary antibody69.…”

SOX2 is essential for in vivo reprogramming of seminoma-like TCam-2 cells to an embryonal carcinoma-like fate

“…In murine embryonic stem cells (mESC), Sox2 complexes with Oct3/4 and binds to a canonical motif, thereby driving the expression of pluripotency genes [8]. Overexpression of Sox17 is able to replace Sox2 in the complex with Oct3/4, leading to a change in target site selection to a compressed binding motif [1].…”