Analysis of TET expression/activity and 5mC oxidation during normal and malignant germ cell development.

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 oxi...

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Autores principales:	Daniel Nettersheim, Lukas C Heukamp, Florian Fronhoffs, Marc J Grewe, Natalie Haas, Anke Waha, Friedemann Honecker, Andreas Waha, Glen Kristiansen, Hubert Schorle
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2013
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/711dc364fc6d4da4ba80e750dcaf39db
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spelling	oai:doaj.org-article:711dc364fc6d4da4ba80e750dcaf39db2021-11-18T08:40:24ZAnalysis of TET expression/activity and 5mC oxidation during normal and malignant germ cell development.1932-620310.1371/journal.pone.0082881https://doaj.org/article/711dc364fc6d4da4ba80e750dcaf39db2013-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/24386123/?tool=EBIhttps://doaj.org/toc/1932-6203During 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.Daniel NettersheimLukas C HeukampFlorian FronhoffsMarc J GreweNatalie HaasAnke WahaFriedemann HoneckerAndreas WahaGlen KristiansenHubert SchorlePublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 8, Iss 12, p e82881 (2013)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Daniel Nettersheim Lukas C Heukamp Florian Fronhoffs Marc J Grewe Natalie Haas Anke Waha Friedemann Honecker Andreas Waha Glen Kristiansen Hubert Schorle Analysis of TET expression/activity and 5mC oxidation during normal and malignant germ cell development.
description	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.
format	article
author	Daniel Nettersheim Lukas C Heukamp Florian Fronhoffs Marc J Grewe Natalie Haas Anke Waha Friedemann Honecker Andreas Waha Glen Kristiansen Hubert Schorle
author_facet	Daniel Nettersheim Lukas C Heukamp Florian Fronhoffs Marc J Grewe Natalie Haas Anke Waha Friedemann Honecker Andreas Waha Glen Kristiansen Hubert Schorle
author_sort	Daniel Nettersheim
title	Analysis of TET expression/activity and 5mC oxidation during normal and malignant germ cell development.
title_short	Analysis of TET expression/activity and 5mC oxidation during normal and malignant germ cell development.
title_full	Analysis of TET expression/activity and 5mC oxidation during normal and malignant germ cell development.
title_fullStr	Analysis of TET expression/activity and 5mC oxidation during normal and malignant germ cell development.
title_full_unstemmed	Analysis of TET expression/activity and 5mC oxidation during normal and malignant germ cell development.
title_sort	analysis of tet expression/activity and 5mc oxidation during normal and malignant germ cell development.
publisher	Public Library of Science (PLoS)
publishDate	2013
url	https://doaj.org/article/711dc364fc6d4da4ba80e750dcaf39db
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Analysis of TET expression/activity and 5mC oxidation during normal and malignant germ cell development.

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