TET Proteins Research Articles

The Role of TET Proteins During Development

The Role of TET Proteins During Development

Wilms' Tumor Protein 1 and Enzymatic Oxidation of 5-Methylcytosine in Brain Tumors: Potential Perspectives.

The patterns of 5-methylcytosine (5mC) and its oxidized derivatives, 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine (5caC) are reportedly altered in a range of cancers. Likewise, Wilms' Tumor protein 1 (WT1), a transcription factor essential for urogenital, epicardium, and kidney development exhibits aberrant expression in multiple tumors. Interestingly, WT1 directly interacts with TET proteins that catalyze the enzymatic oxidation of 5mC and exhibits high affinity for 5caC-containing DNA substrates in vitro. Here we review recent developments in the fields of Tet-dependent 5mC oxidation and WT1 biology and explore potential perspectives for studying the interplay between TETs and WT1 in brain tumors.

Individual retrotransposon integrants are differentially controlled by KZFP/KAP1-dependent histone methylation, DNA methylation and TET-mediated hydroxymethylation in na\xefve embryonic stem cells

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.

PRDM14 Is a Unique Epigenetic Regulator Stabilizing Transcriptional Networks for Pluripotency.

PR-domain containing protein 14 (PRDM14) is a site-specific DNA-binding protein and is required for establishment of pluripotency in embryonic stem cells (ESCs) and primordial germ cells (PGCs) in mice. DNA methylation status is regulated by the balance between de novo methylation and passive/active demethylation, and global DNA hypomethylation is closely associated with cellular pluripotency and totipotency. PRDM14 ensures hypomethylation in mouse ESCs and PGCs through two distinct layers, transcriptional repression of the DNA methyltransferases Dnmt3a/b/l and active demethylation by recruitment of TET proteins. However, the function of PRDM14 remains unclear in other species including humans. Hence, here we focus on the unique characteristics of mouse PRDM14 in the epigenetic regulation of pluripotent cells and primordial germ cells. In addition, we discuss the expression regulation and function of PRDM14 in other species compared with those in mice.

Profiling of oxBS-450K 5-hydroxymethylcytosine in human placenta and brain reveals enrichment at imprinted loci

ABSTRACTDNA methylation (5-methylcytosine, 5 mC) is involved in many cellular processes and is an epigenetic mechanism primarily associated with transcriptional repression. The recent discovery that 5 mC can be oxidized to 5-hydromethylcytosine (5hmC) by TET proteins has revealed the “sixth base” of DNA and provides additional complexity to what was originally thought to be a stable repressive mark. However, our knowledge of the genome-wide distribution of 5hmC in different tissues is currently limited. Here, we sought to define loci enriched for 5hmC in the placenta genome by combining oxidative bisulphite (oxBS) treatment with high-density Illumina Infinium HumanMethylation450 methylation arrays and to compare our results with those obtained in brain. Despite identifying over 17,000 high-confidence CpG sites with consistent 5hmC enrichment, the distribution of this modification in placenta is relatively sparse when compared to cerebellum and frontal cortex. Supported by validation using allelic T4 β-glucosyltransferase assays we identify 5hmC at numerous imprinted loci, often overlapping regions associated with parent-of-origin allelic 5 mC in both placenta and brain samples. Furthermore, we observe tissue-specific monoallelic enrichment of 5hmC overlapping large clusters of imprinted snoRNAs-miRNAs processed from long noncoding RNAs (lncRNAs) within the DLK1-DIO3 cluster on chromosome 14 and SNRPN-UBE3A domain on chromosome 15. Enrichment is observed solely on the transcribed alleles suggesting 5hmC is positively associated with transcription at these loci. Our study provides an extensive description of the 5hmC/5 mC landscape in placenta with our data available at www.humanimprints.net, which represents the most comprehensive resource for exploring the epigenetic profiles associated with human imprinted genes.

Tet2 promotes pathogen infection-induced myelopoiesis through mRNA oxidation.

Varieties of RNA modification form the epitranscriptome for post-transcriptional regulation. 5-Methylcytosine (5-mC) is a sparse RNA modification in messenger RNA (mRNA) under physiological conditions. The function of RNA 5-hydroxymethylcytosine (5-hmC) oxidized by ten-eleven translocation (Tet) proteins in Drosophila has been revealed more recently. However, the turnover and function of 5-mC in mammalian mRNA have been largely unknown. Tet2 suppresses myeloid malignancies mostly in an enzymatic activity-dependent manner, and is important in resolving inflammatory response in an enzymatic activity-independent way. Myelopoiesis is a common host immune response in acute and chronic infections; however, its epigenetic mechanism needs to be identified. Here we demonstrate that Tet2 promotes infection-induced myelopoiesis in an mRNA oxidation-dependent manner through Adar1-mediated repression of Socs3 expression at the post-transcription level. Tet2 promotes both abdominal sepsis-induced emergency myelopoiesis and parasite-induced mast cell expansion through decreasing mRNA levels of Socs3, a key negative regulator of the JAK-STAT pathway that is critical for cytokine-induced myelopoiesis. Tet2 represses Socs3 expression through Adar1, which binds and destabilizes Socs3 mRNA in a RNA editing-independent manner. For the underlying mechanism of Tet2 regulation at the mRNA level, Tet2 mediates oxidation of 5-mC in mRNA. Tet2 deficiency leads to the transcriptome-wide appearance of methylated cytosines, including ones in the 3' untranslated region of Socs3, which influences double-stranded RNA formation for Adar1 binding, probably through cytosine methylation-specific readers, such as RNA helicases. Our study reveals a previously unknown regulatory role of Tet2 at the epitranscriptomic level, promoting myelopoiesis during infection in the mammalian system by decreasing 5-mCs in mRNAs. Moreover, the inhibitory function of cytosine methylation on double-stranded RNA formation and Adar1 binding in mRNA reveals its new physiological role in the mammalian system.

Tet protein function during Drosophila development.

The TET (Ten-eleven translocation) 1, 2 and 3 proteins have been shown to function as DNA hydroxymethylases in vertebrates and their requirements have been documented extensively. Recently, the Tet proteins have been shown to also hydroxylate 5-methylcytosine in RNA. 5-hydroxymethylcytosine (5hmrC) is enriched in messenger RNA but the function of this modification has yet to be elucidated. Because Cytosine methylation in DNA is barely detectable in Drosophila, it serves as an ideal model to study the biological function of 5hmrC. Here, we characterized the temporal and spatial expression and requirement of Tet throughout Drosophila development. We show that Tet is essential for viability as Tet complete loss-of-function animals die at the late pupal stage. Tet is highly expressed in neuronal tissues and at more moderate levels in somatic muscle precursors in embryos and larvae. Depletion of Tet in muscle precursors at early embryonic stages leads to defects in larval locomotion and late pupal lethality. Although Tet knock-down in neuronal tissue does not cause lethality, it is essential for neuronal function during development through its affects upon locomotion in larvae and the circadian rhythm of adult flies. Further, we report the function of Tet in ovarian morphogenesis. Together, our findings provide basic insights into the biological function of Tet in Drosophila, and may illuminate observed neuronal and muscle phenotypes observed in vertebrates.

Epigenetic Regulations in Neural Stem Cells and Neurological Diseases.

Among the regulatory mechanisms of the renewal and differentiation of neural stem cells, recent evidences support that epigenetic modifications such as DNA methylation, histone modification, and noncoding RNAs play critical roles in the regulation on the proliferation and differentiation of neural stem cells. In this review, we discussed recent advances of DNA modifications on the regulative mechanisms of neural stem cells. Among these epigenetic modifications, DNA 5-hydroxymethylcytosine (5hmC) modification is emerging as an important modulator on the proliferation and differentiation of neural stem cells. At the same time, Ten-eleven translocation (Tet) methylcytosine dioxygenases, the rate-limiting enzyme for the 5-hydroxymethylation reaction from 5-methylcytosine to 5-hydroxymethylcytosine, play a critical role in the tumorigenesis and the proliferation and differentiation of stem cells. The functions of 5hmC and TET proteins on neural stem cells and their roles in neurological diseases are discussed.

The effects of 2-hydroxyglutarate on the tumorigenesis of gliomas.

Mutation of the isocitrate-dehydrogenase (IDH) enzymes is one of the central research topics regarding gliomagenesis. Indeed, 70% of gliomas are associated with a gain-of-function IDH mutation and consequently synthesize the oncometabolite, 2-hydroxyglutarate (2-HG). This review aims to elucidate the effects of 2-HG on gliomagenesis. 2-HG promotes tumorigenesis by impacting metabolism, vascularization and altering the epigenome of glioma cells. Glioma metabolism and vascularization is altered by 2-HG’s effect on the stability of hypoxia-inducible factor (HIF) and inhibition of endostatin. However, 2-HG’s impacts on epigenetic mechanisms are more profound to gliomagenesis. Through competitive inhibition of JHDMs and TET proteins, 2-HG orchestrates histone and DNA hypermethylation, which is associated with gene silencing and dedifferentiation of cells. The hypermethylator phenotype induced by 2-HG also results in alterations of the interaction of the immune system with the tumour. Additionally, this study reviews 2-HG promotion of tumorigenesis by inhibiting repair of DNA alkylation damage through competitive inhibition of AlkB proteins.

Insight into wild-type and T1372E TET2-mediated 5hmC oxidation using ab initio QM/MM calculations.

Ten-eleven translocation 2 (TET2) is an Fe/α-ketoglutarate (α-KG) dependent enzyme that dealkylates 5-methylcytosine (5mC). The reaction mechanism involves a series of three sequential oxidations that convert 5mC to 5-hydroxy-methylcytosine (5hmC), 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). Our previous biochemical and computational studies uncovered an active site scaffold that is required for wild-type (WT) stepwise oxidation (Nat. Chem. Bio., 13, 181). We showed that the mutation of a single residue, T1372 to some amino acids, such as Glu, can impact the iterative oxidation steps and stop the oxidation of 5hmC to 5fC/caC. However, the source of the stalling at the first oxidation step by some mutant TET proteins still remains unclear. Here, we studied the catalytic mechanism of oxidation of 5hmC to 5fC by WT and T1372E TET2 using an ab initio quantum mechanical/molecular mechanical (QM/MM) approach. Our results suggest that the rate limiting step for WT TET2 involves a hydrogen atom abstraction from the hydroxyl group of 5hmC by the ferryl moiety in the WT. By contrast, our calculations for the T1372E mutant indicate that the rate limiting step for this variant corresponds to a second proton abstraction and the calculated barrier is almost twice as large as for WT TET2. Our results suggest that the large barrier for the 5hmC to 5fC oxidation in this mutant is due (at least in part) to the unfavorable orientation of the substrate in the active site. Combined electron localization function (ELF) and non-covalent interaction (NCI) analyses provide a qualitative description of the evolution of the electronic structure of the active site along the reaction path. Energy decomposition analysis (EDA) has been performed on the WT to investigate the impact of each MM residue on catalytic activity.

Nickel(ii) inhibits the oxidation of DNA 5-methylcytosine in mammalian somatic cells and embryonic stem cells.

Nickel is found widely in the environment. It is an essential microelement but also toxic. However, nickel displays only weak genotoxicity and mutagenicity. Exploration of the epigenetic toxicity of nickel is extremely interesting. Iron(ii)- and 2-oxoglutarate-dependent Tet dioxygenases are a class of epigenetic enzymes that catalyze the oxidation of DNA 5-methylcytosine (5mC). Thus, they are critical for DNA demethylation and, importantly, are involved with nuclear reprogramming, embryonic development, and regulation of gene expression. Here, we demonstrated that nickel(ii) dramatically inhibits Tet proteins-mediated oxidation of DNA 5mC in cells ranging from somatic cell lines to embryonic stem cells, as manifested by the consistent observation of a significant decrease in 5-hydroxymethylcytosine, a critical intermediate resulting from the oxidation of 5mC. The inhibitory effects of nickel(ii) were concentration- and time-dependent. Using HEK293T cells overexpressing Tet proteins and ascorbic acid-stimulated Tet-proficient ES cells, we observed that nickel(ii) significantly reduced DNA demethylation at the global level. Interestingly, we also showed that nickel(ii) might affect the naïve or ground state of pluripotent embryonic stem cells. Here we show, for the first time, that nickel(ii) represses the oxidation of DNA 5mC and potentially alters the Tet proteins-regulated DNA methylation landscape in human cells. These findings provide new insights into the epigenetic toxicology of nickel.

Research progress on 5hmC and TET dioxygenases in neurodevelopment and neurological diseases.

The development of the nervous system is coordinately regulated by multiple interacting factors. If a certain factor is altered or mutated, the coordinated developmental processes could be disrupted, resulting in neurological diseases. The 5-hydroxymethylcytosine (5hmC) is an intermediate product of the DNA demethylation processes. 5hmC and its metabolic enzymes, the ten-eleven translocation protein-TET family of dioxygenases, have recently been identified as new epigenetic players important in the regulation of the nervous system development, as well as in cognition, memory and other neurological functions. In various studies on neurodevelopment and neurodegeneration related diseases, the levels of 5hmC and TET proteins could be differentially regulated during development and/or disease pathogenesis, suggesting the potentially critical roles of 5hmC and TETs in these neural developmental and disease processes. In this review, we summarize the recent advances in research on 5hmC and TET dioxygenases in the regulation of neurodevelopment and neurological diseases, thereby providing significant insights on the involvements of 5hmC and TETs in neurodevelopment and on establishing new therapeutic strategies for human neurological diseases.

Author Correction: TET proteins safeguard bivalent promoters from de novo methylation in human embryonic stem cells.

In the version of this article initially published, in the Methods, the Gene Expression Omnibus accession code for H3K36me3 ChIP-seq data was incorrectly given asGSM1003585 instead of GSM733725. The error has been corrected in the HTML, PDF and print versions of the article.

Publisher Correction: TET proteins safeguard bivalent promoters from de novo methylation in human embryonic stem cells.

The version of the Supplementary Text and Figures file initially posted was missing Supplementary Tables 1-6 and the Supplementary Note and used incorrect versions of the supplementary figures.

TET proteins safeguard bivalent promoters from de novo methylation in human embryonic stem cells.

The TET enzymes oxidize 5-methylcytosine to 5-hydroxymethylcytosine, which can lead to DNA demethylation. However, direct connections between TET-mediated DNA demethylation and transcriptional output are difficult to establish due to challenges of distinguishing global versus locus-specific effects. Here we show that TET1/2/3 triple knockout (TKO) human embryonic stem cells (hESCs) exhibit prominent bivalent promoter hypermethylation without an overall corresponding gene expression decrease in the undifferentiated state. Focusing on the bivalent PAX6 locus, we find increased DNMT3B binding is associated with promoter hypermethylation, which precipitates a neural differentiation defect and failure of PAX6 induction during differentiation. dCas9-mediated locus-specific demethylation and global inactivation of DNMT3B in TKO hESCs partially reverses the hypermethylation at the PAX6 promoter and improves differentiation to neuroectoderm. Taken together with further genome-wide methylation and TET1 and DNMT3B ChIP-Seq analysis, we conclude that the TET proteins safeguard bivalent promoters from de novo methylation to ensure robust lineage-specific transcription upon differentiation.

Ten-eleven translocation 2 interacts with forkhead box O3 and regulates adult neurogenesis

Emerging evidence suggests that active DNA demethylation machinery plays important epigenetic roles in mammalian adult neurogenesis; however, the precise molecular mechanisms and critical functional players of DNA demethylation in this process remain largely unexplored. Ten–eleven translocation (Tet) proteins convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and its downstream derivatives. Here we show that 5hmC is elevated during the differentiation of adult neural stem cells (aNSCs), and Tet2 is primarily responsible for modulating 5hmC dynamics. Depletion of Tet2 leads to increased aNSC proliferation and reduced differentiation in vitro and in vivo. Genome-wide transcriptional analyses reveal important epigenetic roles of Tet2 in maintaining the transcriptome landscape related to neurogenesis. Mechanistically, transcription factor forkhead box O3 (Foxo3a) physically interacts with Tet2 and regulates the expression of genes related to aNSC proliferation. These data together establish an important role for the Tet2-Foxo3a axis in epigenetically regulating critical genes in aNSCs during adult neurogenesis.

A role for TET2 in parathyroid carcinoma.

Primary hyperparathyroidism (pHPT) is rarely caused by parathyroid carcinoma (PC, <1-5% of pHPT cases). The TET proteins oxidize the epigenetic mark 5-methylcytosine to 5-hydroxymethylcytosine (5hmC) and inactivation by mutation or epigenetic deregulation of TET1 and TET2 play important roles in various cancers. Recently, we found that 5hmC was severely reduced in all of the analyzed PCs and with deranged expression of TET1 for the majority of PCs. Here, we have examined the expression of the TET2 protein in 15 5hmC-negative PCs from patients who had local invasion or metastases. Cell growth and cell migratory roles for TET2 as well as epigenetic deregulated expression were addressed. Immunohistochemistry revealed very low/undetectable expression of TET2 in all PCs and verified for two PCs that were available for western blotting analysis. Knockdown of TET2 in the parathyroid cell line sHPT-1 resulted in increased cell growth and increased cell migration. DNA sequencing of TET2 in PCs revealed two common variants and no obvious inactivating mutations. Quantitative bisulfite pyrosequencing analysis of the TET2 promoter CpG island revealed higher CpG methylation level in the PCs compared to that in normal tissues and treatment of a PC primary cell culture with the DNA methylation inhibitor 5-aza-2'-deoxycytidine caused increased expression of the methylated TET2 gene. Hence, the data suggest that deregulated expression of TET2 by DNA hypermethylation may contribute to the aberrantly low level of 5hmC in PCs and further that TET2 plays a cell growth and cell migratory regulatory role and may constitute a parathyroid tumor suppressor gene.

L1 retrotransposition is activated by Ten-eleven-translocation protein 1 and repressed by methyl-CpG binding proteins

ABSTRACTOne of the major functions of DNA methylation is the repression of transposable elements, such as the long-interspersed nuclear element 1 (L1). The underlying mechanism(s), however, are unclear. Here, we addressed how retrotransposon activation and mobilization are regulated by methyl-cytosine modifying ten-eleven-translocation (Tet) proteins and how this is modulated by methyl-CpG binding domain (MBD) proteins. We show that Tet1 activates both, endogenous and engineered L1 retrotransposons. Furthermore, we found that Mecp2 and Mbd2 repress Tet1-mediated activation of L1 by preventing 5hmC formation at the L1 promoter. Finally, we demonstrate that the methyl-CpG binding domain, as well as the adjacent non-sequence specific DNA binding domain of Mecp2 are each sufficient to mediate repression of Tet1-induced L1 mobilization. Our study reveals a mechanism how L1 elements get activated in the absence of Mecp2 and suggests that Tet1 may contribute to Mecp2/Mbd2-deficiency phenotypes, such as the Rett syndrome. We propose that the balance between methylation “reader” and “eraser/writer” controls L1 retrotransposition.

Nickel(II) Inhibits Tet-Mediated 5-Methylcytosine Oxidation by High Affinity Displacement of the Cofactor Iron(II).

Ten-eleven translocation (Tet) family proteins are Fe(II)- and 2-oxoglutarate-dependent dioxygenases that regulate the dynamics of DNA methylation by catalyzing the oxidation of DNA 5-methylcytosine (5mC). To exert physiologically important functions, redox-active iron chelated in the catalytic center of Tet proteins directly involves the oxidation of the multiple substrates. To understand the function and interaction network of Tet dioxygenases, it is interesting to obtain high affinity and a specific inhibitor. Surprisingly, here we found that natural Ni(II) ion can bind to the Fe(II)-chelating motif (HXD) with an affinity of 7.5-fold as high as Fe(II). Consistently, we further found that Ni(II) ion can displace the cofactor Fe(II) of Tet dioxygenases and inhibit Tet-mediated 5mC oxidation activity with an estimated IC50 of 1.2 μM. Essentially, Ni(II) can be used as a high affinity and selective inhibitor to explore the function and dynamics of Tet proteins.

Heavy Metals Induce Decline of Derivatives of 5-Methycytosine in Both DNA and RNA of Stem Cells.

Toxic heavy metals have been considered to be harmful environmental contaminations. The molecular mechanisms of heavy-metals-induced cytotoxicity and carcinogenicity are still not well elucidated. Previous reports showed exposures to toxic heavy metals can cause a change of DNA cytosine methylation (5-methylcytosine, 5-mC). However, it is still not clear whether heavy metals have effects on the recently identified new epigenetic marks in both DNA and RNA, i.e., 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-foC), and 5-carboxylcytosine (5-caC). Here, we established a chemical labeling strategy in combination with liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS/MS) analysis for highly sensitive detection of eight modified cytidines in DNA and RNA. The developed method allowed simultaneous detection of all eight modified cytidines with improved detection sensitivities of 128-443-fold. Using this method, we demonstrated that the levels of 5-hmC, 5-foC, and 5-caC significantly decreased in both the DNA and RNA of mouse embryonic stem (ES) cells while exposed to arsenic (As), cadmium (Cd), chromium (Cr), and antimony (Sb). In addition, we found that treatments by heavy metals induced a decrease of the activities of 10-11 translocation (Tet) proteins. Furthermore, we revealed that a content change of metabolites occurring in the tricarboxylic acid cycle may be responsible for the decline of the derivatives of 5-mC. Our study shed light on the epigenetic effects of heavy metals, especially for the induced decline of the derivatives of 5-mC in both DNA and RNA.