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.
Highlights
In the past decade, epigenetic profiles of various cell types and tissues were analyzed with great ambition
The oxidation pathway involves further oxidation steps of 5hmC to 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) by the TET proteins. 5fC and 5caC can be replaced by an unmodified cytosine by the base excision repair (BER) pathway, involving the enzyme thymine DNA glycosylase (TDG) [9] [10]
The declining intensity of 5hmC, 5fC and 5caC staining during proceeding spermatogenesis suggests that either active DNA demethylation (ADD) is active in spermatogonia, but downregulated with onset of spermatogenesis or 5hmC, 5fC and 5caC are part of normal 5mC turnover in spermatogonia which is somehow blocked/ reduced during spermatogenesis
Summary
Epigenetic profiles of various cell types and tissues were analyzed with great ambition. The protein family of DNA methyltransferases (DNMTs) catalyzes the methylation of cytosines. The oxidation pathway involves further oxidation steps of 5hmC to 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC) by the TET proteins. 5fC and 5caC can be replaced by an unmodified cytosine by the base excision repair (BER) pathway, involving the enzyme thymine DNA glycosylase (TDG) [9] [10]. The deamination pathway involves deaminases of the AID or APOBEC family to deaminate 5mC or 5hmC to thymidine or 5-hydroxymethyluracil, respectively. Both deamination products are thought to be replaced by cytosines by the BER pathway, involving TDG, MBD4 or the single strand selective monofunctional uracil DNA glycosylase (SMUG1) [8]
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