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Overexpression of TET dioxygenases in seminomas associates with low levels of DNA methylation and hydroxymethylation.

ABSTRACT: Germ cell tumors and particularly seminomas reflect the epigenomic features of their parental primordial germ cells (PGCs), including genomic DNA hypomethylation and expression of pluripotent cell markers. Because the DNA hypomethylation might be a result of TET dioxygenase activity, we examined expression of TET1-3 enzymes and the level of their product, 5-hydroxymethylcytosine (5hmC), in a panel of histologically characterized seminomas and non-seminomatous germ cell tumors. Expression of TET dioxygenase mRNAs was quantified by real-time PCR. TET1 expression and the level of 5hmC were examined immunohistochemically. Quantitative assessment of 5-methylcytosine (5mC) and 5hmC levels was done by the liquid chromatography-mass spectroscopy technique. We found highly increased expression of TET1 dioxygenase in most seminomas and strong TET1 staining in seminoma cells. Isocitrate dehydrogenase 1 and 2 mutations were not detected, suggesting the enzymatic activity of TET1. The levels of 5mC and 5hmC in seminomas were found decreased in comparison to non-seminomatous germ cell tumors and healthy testicular tissue. We propose that TET1 expression should be studied as a potential marker of seminomas and mixed germ cell tumors and we suggest that elevated expression of TET dioxygenase enzymes is associated with the maintenance of low DNA methylation levels in seminomas. This "anti-methylator" phenotype of seminomas is in contrast to the CpG island methylator phenotype (CIMP) observed in a fraction of tumors of various types.

SUBMITTER: Benesova M

PROVIDER: S-EPMC5503132 | biostudies-literature | 2017 Aug

REPOSITORIES: biostudies-literature

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Overexpression of TET dioxygenases in seminomas associates with low levels of DNA methylation and hydroxymethylation.

Benešová Martina M Trejbalová Kateřina K Kučerová Dana D Vernerová Zdenka Z Hron Tomáš T Szabó Arpád A Amouroux Rachel R Klézl Petr P Hajkova Petra P Hejnar Jiří J

Molecular carcinogenesis 20170524 8

Germ cell tumors and particularly seminomas reflect the epigenomic features of their parental primordial germ cells (PGCs), including genomic DNA hypomethylation and expression of pluripotent cell markers. Because the DNA hypomethylation might be a result of TET dioxygenase activity, we examined expression of TET1-3 enzymes and the level of their product, 5-hydroxymethylcytosine (5hmC), in a panel of histologically characterized seminomas and non-seminomatous germ cell tumors. Expression of TET ...[more]

PMID: 28218476

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Project description:BACKGROUND:Bisphenol A (BPA), a ubiquitous environmental endocrine disruptor targeting estrogen receptors (ERs), has been implicated in the promotion of breast cancer. Perinatal exposure of BPA could induce longitudinal alteration of DNA hydroxymethylation in imprinted loci of mouse blood cells. To date, no data has been reported on the effects of BPA on DNA hydroxymethylation in breast cells. Therefore, we asked whether BPA can induce DNA hydroxymethylation change in human breast cells. Given that dysregulated epigenetic DNA hydroxymethylation is observed in various cancers, we wondered how DNA hydroxymethylation modulates cancer development, and specifically, whether and how BPA and its analogs promote breast cancer development via DNA hydroxymethylation. OBJECTIVES:We aimed to explore the interplay of the estrogenic activity of bisphenols at environmental exposure dose levels with TET dioxygenase-catalyzed DNA hydroxymethylation and to elucidate their roles in the proliferation of ER+ breast cancer cells as stimulated by environmentally relevant bisphenols. METHODS:Human MCF-7 and T47D cell lines were used as ER-dependent breast cell proliferation models, and the human MDA-MB-231 cell line was used as an ER-independent breast cell model. These cells were treated with BPA or bisphenol S (BPS) to examine BPA/BPS-related proliferation. Ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) and enzyme-linked immunosorbent assays (ELISAs) were used to detect DNA hydroxymethylation. Crispr/Cas9 and RNA interference technologies, quantitative polymerase chain reaction (qPCR), and Western blot analyses were used to evaluate the expression and function of genes. Co-immunoprecipitation (Co-IP), bisulfite sequencing-PCR (BSP), and chromatin immunoprecipitation-qPCR (ChIP-qPCR) were used to identify the interactions of target proteins. RESULTS:We measured higher proliferation in ER+ breast cancer cells treated with BPA or its replacement, BPS, accompanied by an ER?-dependent decrease in genomic DNA hydroxymethylation. The results of our overexpression, knockout, knockdown, and inhibition experiments suggested that TET2-catalyzed DNA hydroxymethylation played a suppressive role in BPA/BPS-stimulated cell proliferation. On the other hand, we observed that TET2 was negatively regulated by the activation of ER? (dimerized and phosphorylated), which was also induced by BPA/BPS binding. Instead of a direct interaction between TET2 and ER?, data of our Co-IP, BSP, and ChIP-qPCR experiments indicated that the activated ER? increased the DNA methyltransferase (DNMT)-mediated promoter methylation of TET2, leading to an inhibition of the TET2 expression and DNA hydroxymethylation. CONCLUSIONS:We identified a new feedback circuit of ER? activation-DNMT-TET2-DNA hydroxymethylation in ER+ breast cancer cells and uncovered a pivotal role of TET2-mediated DNA hydroxymethylation in modulating BPA/BPS-stimulated proliferation. Moreover, to our knowledge, we for the first time established a linkage among chemical exposure, DNA hydroxymethylation, and tumor-associated proliferation. These findings further clarify the estrogenic activity of BPA/BPS and its profound implications for the regulation of epigenetic DNA hydroxymethylation and cell proliferation. https://doi.org/10.1289/EHP5862.

Project description:BackgroundThe KZFP/KAP1 (KRAB zinc finger proteins/KRAB-associated protein 1) system plays a central role in repressing transposable elements (TEs) and maintaining parent-of-origin DNA methylation at imprinting control regions (ICRs) during the wave of genome-wide reprogramming that precedes implantation. In naïve murine embryonic stem cells (mESCs), the genome is maintained highly hypomethylated by a combination of TET-mediated active demethylation and lack of de novo methylation, yet KAP1 is tethered by sequence-specific KZFPs to ICRs and TEs where it recruits histone and DNA methyltransferases to impose heterochromatin formation and DNA methylation.ResultsHere, upon removing either KAP1 or the cognate KZFP, we observed rapid TET2-dependent accumulation of 5hmC at both ICRs and TEs. In the absence of the KZFP/KAP1 complex, ICRs lost heterochromatic histone marks and underwent both active and passive DNA demethylation. For KAP1-bound TEs, 5mC hydroxylation correlated with transcriptional reactivation. Using RNA-seq, we further compared the expression profiles of TEs upon Kap1 removal in wild-type, Dnmt and Tet triple knockout mESCs. While we found that KAP1 represents the main effector of TEs repression in all three settings, we could additionally identify specific groups of TEs further controlled by DNA methylation. Furthermore, we observed that in the absence of TET proteins, activation upon Kap1 depletion was blunted for some TE integrants and increased for others.ConclusionsOur results indicate that the KZFP/KAP1 complex maintains heterochromatin and DNA methylation at ICRs and TEs in naïve embryonic stem cells partly by protecting these loci from TET-mediated demethylation. Our study further unveils an unsuspected level of complexity in the transcriptional control of the endovirome by demonstrating often integrant-specific differential influences of histone-based heterochromatin modifications, DNA methylation and 5mC oxidation in regulating TEs expression.

Dataset Information

Overexpression of TET dioxygenases in seminomas associates with low levels of DNA methylation and hydroxymethylation.

Publications

Overexpression of TET dioxygenases in seminomas associates with low levels of DNA methylation and hydroxymethylation.

Similar Datasets

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