TET Proteins Research Articles

Loss of ten-eleven translocation 2 induces cardiac hypertrophy and fibrosis through modulating ERK signaling pathway

The ten-eleven translocation (Tet) family of dioxygenases convert 5-methylcytosine to 5-hydroxymethylcytosine (5hmC). Previous studies have shown that 5hmC-mediated epigenetic modifications play essential roles in diverse biological processes and diseases. Here, we show that Tet proteins and 5hmC display dynamic features during postnatal cardiac development and that Tet2 is the predominant dioxygenase present in heart. Tet2 knockout results in abnormal cardiac function, progressive cardiac hypertrophy and fibrosis. Mechanistically, Tet2 deficiency leads to reduced hydroxymethylation in the cardiac genome and alters the cardiac transcriptome. Mechanistically, Tet2 loss leads to a decrease of Hspa1b expression, a regulator of the extracellular signal-regulated protein kinase (Erk) signaling pathway, which leads to over-activation of Erk signaling. Acute Hspa1b knock down (KD) increased the phosphorylation of Erk and induced hypertrophy of cardiomyocytes, which could be blocked by Erk signaling inhibitor. Consistently, ectopic expression of Hspa1b was able to rescue the deficits of cardiomyocytes induced by Tet2 depletion. Taken together, our study's results reveal the important roles of Tet2-mediated DNA hydroxymethylation in cardiac development and function.

The Potential Equivalents of TET2 Mutations.

Simple SummaryIn acute myeloid leukemia (AML) TET2 mutations have been observed to be mutually exclusive with IDH1, IDH2, and WT1 mutations, all of them showing a similar impact on the transcription profile. Because of this, it is possible that TET2/IDH1/2/WT1 mutated AML could be considered as having similar characteristics between each other. Nonetheless, other genes also interact with TET2 and influence its activity. Because of this, it is possible that other signatures exist that would mimic the effect of TET2 mutations. Thus, in this review, we searched the literature for the genes that were observed to interact with TET2 and classified them in the following manner: transcription alteration, miRs, direct interaction, posttranslational changes, and substrate reduction.TET2 is a dioxygenase dependent on Fe2+ and α-ketoglutarate which oxidizes 5-methylcytosine (5meC) to 5-hydroxymethylcytosine (5hmeC). TET proteins successively oxidize 5mC to yield 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). Among these oxidized methylcytosines, 5fC and 5caC are directly excised by thymine DNA glycosylase (TDG) and ultimately replaced with unmethylated cytosine. Mutations in TET2 have been shown to lead to a hypermethylated state of the genome and to be responsible for the initiation of the oncogenetic process, especially in myeloid and lymphoid malignancies. Nonetheless, this was also shown to be the case in other cancers. In AML, TET2 mutations have been observed to be mutually exclusive with IDH1, IDH2, and WT1 mutations, all of them showing a similar impact on the transcription profile of the affected cell. Because of this, it is possible that TET2/IDH1/2/WT1 mutated AML could be considered as having similar characteristics between each other. Nonetheless, other genes also interact with TET2 and influence its effect, thus making it possible that other signatures exist that would mimic the effect of TET2 mutations. Thus, in this review, we searched the literature for the genes that were observed to interact with TET2 and classified them in the following manner: transcription alteration, miRs, direct interaction, posttranslational changes, and substrate reduction. What we propose in the present review is the potential extension of the TET2/IDH1/2/WT1 entity with the addition of certain expression signatures that would be able to induce a similar phenotype with that induced by TET2 mutations. Nonetheless, we recommend that this approach be taken on a disease by disease basis.

Molecular mechanism for vitamin C-derived C5-glyceryl-methylcytosine DNA modification catalyzed by algal TET homologue CMD1

C5-glyceryl-methylcytosine (5gmC) is a novel DNA modification catalyzed by algal TET homologue CMD1 using vitamin C (VC) as co-substrate. Here, we report the structures of CMD1 in apo form and in complexes with VC or/and dsDNA. CMD1 exhibits comparable binding affinities for DNAs of different lengths, structures, and 5mC levels, and displays a moderate substrate preference for 5mCpG-containing DNA. CMD1 adopts the typical DSBH fold of Fe2+/2-OG-dependent dioxygenases. The lactone form of VC binds to the active site and mono-coordinates the Fe2+ in a manner different from 2-OG. The dsDNA binds to a positively charged cleft of CMD1 and the 5mC/C is inserted into the active site and recognized by CMD1 in a similar manner as the TET proteins. The functions of key residues are validated by mutagenesis and activity assay. Our structural and biochemical data together reveal the molecular mechanism for the VC-derived 5gmC DNA modification by CMD1.

The chromosomal protein SMCHD1 regulates DNA methylation and the 2c-like state of embryonic stem cells by antagonizing TET proteins.

5-Methylcytosine (5mC) oxidases, the ten-eleven translocation (TET) proteins, initiate DNA demethylation, but it is unclear how 5mC oxidation is regulated. We show that the protein SMCHD1 (structural maintenance of chromosomes flexible hinge domain containing 1) is found in complexes with TET proteins and negatively regulates TET activities. Removal of SMCHD1 from mouse embryonic stem (ES) cells induces DNA hypomethylation, preferentially at SMCHD1 target sites and accumulation of 5-hydroxymethylcytosine (5hmC), along with promoter demethylation and activation of the Dux double-homeobox gene. In the absence of SMCHD1, ES cells acquire a two-cell (2c) embryo-like state characterized by activation of an early embryonic transcriptome that is substantially imposed by Dux Using Smchd1/Tet1/Tet2/Tet3 quadruple-knockout cells, we show that DNA demethylation, activation of Dux, and other genes upon SMCHD1 loss depend on TET proteins. These data identify SMCHD1 as an antagonist of the 2c-like state of ES cells and of TET-mediated DNA demethylation.

TET-Mediated Epigenetic Regulation in Immune Cell Development and Disease.

TET proteins oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and further oxidation products in DNA. The oxidized methylcytosines (oxi-mCs) facilitate DNA demethylation and are also novel epigenetic marks. TET loss-of-function is strongly associated with cancer; TET2 loss-of-function mutations are frequently observed in hematological malignancies that are resistant to conventional therapies. Importantly, TET proteins govern cell fate decisions during development of various cell types by activating a cell-specific gene expression program. In this review, we seek to provide a conceptual framework of the mechanisms that fine tune TET activity. Then, we specifically focus on the multifaceted roles of TET proteins in regulating gene expression in immune cell development, function, and disease.

Deciphering the multifaceted roles of TET proteins in T‐cell lineage specification and malignant transformation

TET proteins are DNA demethylases that can oxidize 5-methylcytosine (5mC) to generate 5-hydroxymethylcytosine (5hmC) and other oxidized mC bases (oxi-mCs). Importantly, TET proteins govern cell fate decisions during development of various cell types by activating a cell-specific gene expression program. In this review, we focus on the role of TET proteins in T-cell lineage specification. We explore the multifaceted roles of TET proteins in regulating gene expression in the contexts of T-cell development, lineage specification, function, and disease. Finally, we discuss the future directions and experimental strategies required to decipher the precise mechanisms employed by TET proteins to fine-tune gene expression and safeguard cell identity.

Purification of TET Proteins.

The 5-methylcytosine (5mC) oxidation pathway mediated by TET proteins involves step-wise oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). 5fC and 5caC can be removed from DNA by base excision repair and the completion of this pathway results in "demethylation" of 5mC by converting the modified base back into cytosine. In vitro studies with TET proteins aimed at analyzing their DNA substrate specificities and their activity within defined chromatin templates are relatively limited. Here we describe purification methods for mammalian TET proteins based on expression in insect cells or in 293T cells. We also briefly summarize a method that can be used to monitor 5-methylcytosine oxidase activity of the purified TET proteins in vitro.

Correction to: TET Proteins and DNA Demethylation.

Correction to: TET Proteins and DNA Demethylation.

Tet Proteins Regulate Neutrophil Granulation in Zebrafish through Demethylation of socs3b mRNA

SUMMARYTet proteins (Tet1/2/3) convert 5-methylcytosine (5mC) to 5-hydroxy-methylcytosine (5hmC), initiating the process of active demethylation to regulate gene expression. Demethylation has been investigated primarily in the context of DNA, but recently Tet enzymes have also been shown to mediate demethylation of 5mC in RNA as an additional level of epitranscriptomic regulation. We analyzed compound tet2/3 mutant zebrafish and discovered a role for Tet enzymes in the maturation of primitive and definitive neutrophils during granulation. Transcript profiling showed dysregulation of cytokine signaling in tet mutant neutrophils, including upregulation of socs3b. We show that Tet normally demethylates socs3b mRNA during granulation, thereby destabilizing the transcript, leading to its downregulation. Failure of this process leads to accumulation of socs3b mRNA and repression of cytokine signaling at this crucial step of neutrophil maturation. This study provides further evidence for Tets as epitranscriptomic regulatory enzymes and implicates Tet2/3 in regulation of neutrophil maturation.

TET-mediated DNA demethylation plays an important role in arsenic-induced HBE cells oxidative stress via regulating promoter methylation of OGG1 and GSTP1

Environmental exposure to arsenic remains a worldwide public health challenge. Oxidative stress and aberrant DNA methylation are both characteristics of arsenic toxicology; however, the relationship between these is not well understood. Ten-eleven translocation (TET1, TET2 and TET3), which is the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), plays a central role in the DNA demethylation process. Further, it can prevent cytosine-phosphate-guanine (CpG) islands from developing abnormal hypermethylation under oxidative stress. Here, we observed that NaAsO2 could induce oxidative stress in human bronchial epithelial (HBE) cells. This was accompanied by an inhibition of TET-mediated DNA demethylation. Subsequent results showed that TET1 and TET2 siRNA led to further inhibition of genome 5hmC and a higher level of oxidative stress in NaAsO2-treated HBE cells. Conversely, l-ascorbic acid enhanced TET proteins and effectively upregulated 5hmC, which antagonized the NaAsO2-induced oxidative stress. Additionally, the TETs positively regulated the promoter methylation of the antioxidant genes 8-oxoguanine DNA glycosylase (OGG1) and glutathione S-transferase Pi 1 (GSTP1). Taken together, the results indicate that arsenic induced the inhibition of TET-mediated DNA demethylation, which induced promoter hypermethylation, inhibiting the expression of the OGG1 and GSTP1, and increasing oxidative stress in lung cells in vitro. l-ascorbic acid effectively alleviated arsenic-induced oxidative stress by restoring TET function.

The Intrinsic and Extrinsic Effects of Tet Proteins During Gastrulation

Mice deficient for all Ten-eleven translocation (TET) genes exhibit early gastrulation lethality. Yet, separating cause and effect in such embryonic failure is challenging. To isolate cell-autonomous effects of TET loss, we used temporal single-cell atlases from embryos with partial or complete mutant contributions. Strikingly, when developing within a wild-type embryo, TET mutant cells retain near-complete differentiation potential, while embryos solely comprised of mutant cells are defective in epiblast to ectoderm transition with degenerated mesoderm potential. We map de-repressions of early epiblast factors and failure to activate signaling from nascent mesoderm as intrinsic drivers of TET phenotypes. We further suggest near-complete loss of enhancer demethylation as a possible mechanism for mis-regulation in TET deficient cells. Collectively, our work demonstrates an unbiased approach for defining intrinsic and extrinsic embryonic gene function based on temporal differentiation atlases, and disentangles the intracellular effects of the demethylation machinery from its broader tissue-level ramifications.

Vitamin C Transporters and Their Implications in Carcinogenesis.

Vitamin C is implicated in various bodily functions due to its unique properties in redox homeostasis. Moreover, vitamin C also plays a great role in restoring the activity of 2-oxoglutarate and Fe2+ dependent dioxygenases (2-OGDD), which are involved in active DNA demethylation (TET proteins), the demethylation of histones, and hypoxia processes. Therefore, vitamin C may be engaged in the regulation of gene expression or in a hypoxic state. Hence, vitamin C has acquired great interest for its plausible effects on cancer treatment. Since its conceptualization, the role of vitamin C in cancer therapy has been a controversial and disputed issue. Vitamin C is transferred to the cells with sodium dependent transporters (SVCTs) and glucose transporters (GLUT). However, it is unknown whether the impaired function of these transporters may lead to carcinogenesis and tumor progression. Notably, previous studies have identified SVCTs’ polymorphisms or their altered expression in some types of cancer. This review discusses the potential effects of vitamin C and the impaired SVCT function in cancers. The variations in vitamin C transporter genes may regulate the active transport of vitamin C, and therefore have an impact on cancer risk, but further studies are needed to thoroughly elucidate their involvement in cancer biology.

TET is targeted for proteasomal degradation by the PHD-pVHL pathway to reduce DNA hydroxymethylation

Hypoxia-inducible factors are heterodimeric transcription factors that play a crucial role in a cell's ability to adapt to low oxygen. The von Hippel-Lindau tumor suppressor (pVHL) acts as a master regulator of HIF activity, and its targeting of prolyl hydroxylated HIF-α for proteasomal degradation under normoxia is thought to be a major mechanism for pVHL tumor suppression and cellular response to oxygen. Whether pVHL regulates other targets through a similar mechanism is largely unknown. Here, we identify TET2/3 as novel targets of pVHL. pVHL induces proteasomal degradation of TET2/3, resulting in reduced global 5-hydroxymethylcytosine levels. Conserved proline residues within the LAP/LAP-like motifs of these two proteins are hydroxylated by the prolyl hydroxylase enzymes (PHD2/EGLN1 and PHD3/EGLN3), which is prerequisite for pVHL-mediated degradation. Using zebrafish as a model, we determined that global 5-hydroxymethylcytosine levels are enhanced in vhl-null, egln1a/b-double-null, and egln3-null embryos. Therefore, we reveal a novel function for the PHD-pVHL pathway in regulating TET protein stability and activity. These data extend our understanding of how TET proteins are regulated and provide new insight into the mechanisms of pVHL in tumor suppression.

Tet2 regulates Barx2 expression in undifferentiated and early differentiated mouse embryonic stem cells

The methylcytosine oxidase TET proteins play important roles in DNA demethylation and development. In developing embryos, TET2 are upregulated during pre-implantation development, and significantly expressed in the trophectoderm and inner cell mass. In this study, we identified Barx2 as a new target of Tet2. Tet2 bound and demethylated the promoter of Barx2 in mouse embryonic stem cells (mESCs) to maintain the expression of Barx2. During mESC differentiation, Tet2 bound the promoter of Barx2 in day 4 embryonic bodies but not in day 8 EBs. However, Barx2 expression remained unchanged. Thus, Tet2 functioned as a demethylase and maintained the expression of Barx2 in undifferentiated and early differentiated mESCs.

Nuclear and Cytoplasmic Functions of Vitamin C.

Vitamin C (ascorbic acid) is a water-soluble antioxidant and a cofactor for a large number of enzymes. It is present in all tissues and especially abundant in corneal epithelium, stem cells, and neurons. Although similar to thiols in its ability to react with many reactive oxygen species (ROS), ascorbate is much better (>100× faster) than glutathione at scavenging of primary ROS (superoxide radical and singlet oxygen). Ascorbate appears to be especially important for elimination of O2•– in the nucleus which contains little or no SOD activity. Cofactor functions of ascorbate involve the maintenance of activity of Fe(II)/2-oxoglutarate-dependent dioxygenases via reduction of Fe(III). The most prominent activity of ascorbate-dependent dioxygenases in the cytoplasm is hydroxylation of prolines in proteins involved in the formation of extracellular matrix and regulation of metabolism and hypoxia responses. In the nucleus, ascorbate is important for oxidative demethylation of 5-methylcytosine in DNA (by TET proteins) and removal of methyl groups from histone lysines (by JmjC demethylases). Differentiation and other cellular reprograming processes involving DNA demethylation are especially sensitive to ascorbate insufficiency. High doses of vitamin C alone or in combinations with drugs produced cancer-suppressive effects which involved redox, immune, and epigenetic mechanisms. Solutions to vitamin C deficiency in cultured cells are discussed to improve the physiological relevance of in vitro models. An abundance of vitamin C in rodents limits their ability to fully recapitulate human sensitivity to adverse health effects of malnutrition and xenobiotics, including neurotoxicity, lung injury, and intergenerational and other epigenetic effects.

Analysis of 5-Carboxylcytosine Distribution Using DNA Immunoprecipitation.

DNA methylation (5-methylcytosine, 5mC) is involved in regulation of a wide range of biological processes. TET proteins can oxidize 5mC to 5-hydroxymethylcytosine, 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC). Although both 5fC and 5caC serve as intermediates in active demethylation pathway, growing body of experimental evidence indicate that these DNA modifications may also interact with specific sets of reader proteins and therefore may represent bona fide epigenetic marks. Despite a number of single-base resolution techniques have recently been proposed for 5fC/5caC mapping, antibody-based approaches still represent a relatively simple and plausible alternative for the analysis of genomic distribution of these DNA modifications. Here, we describe a protocol for 5caC DNA immunoprecipitation (5caC DIP) that can be used for both locus-specific and genome-wide assessment of 5caC distribution. In combination with mass spectrometry-based techniques and single base resolution mapping methods, this approach may contribute to elucidating the role of 5caC in development, differentiation, and tumorigenesis.

Analysis of 5-Hydroxymethyluracil Levels Using Flow Cytometry.

5-hydroxymethyluracil was originally identified as an oxidatively modified DNA base derivative. Recent evidence suggests that its formation may result from the oxidation of thymine in a reaction that is catalyzed by TET proteins. Alternatively, it could be generated through the deamination of 5-hydroxymethylcytosine by activation-induced cytidine deaminase. The standard method for evaluating 5-hydroxymethyluracil content is the highly sensitive and highly specific isotope-dilution automated online two-dimensional ultraperformance liquid chromatography with tandem mass spectrometry (2D-UPLC-MS/MS). Despite many advantages, this method has one great limitation. It is not able to measure compounds at a single-cell level. Our goal was to develop and optimize a method based on flow cytometry that allows the evaluation of 5-hydroxymethyluracil levels at a single cell level in peripheral leukocytes.

Distinct and stage-specific contributions of TET1 and TET2 to stepwise cytosine oxidation in the transition from naive to primed pluripotency

Cytosine DNA bases can be methylated by DNA methyltransferases and subsequently oxidized by TET proteins. The resulting 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) are considered demethylation intermediates as well as stable epigenetic marks. To dissect the contributions of these cytosine modifying enzymes, we generated combinations of Tet knockout (KO) embryonic stem cells (ESCs) and systematically measured protein and DNA modification levels at the transition from naive to primed pluripotency. Whereas the increase of genomic 5-methylcytosine (5mC) levels during exit from pluripotency correlated with an upregulation of the de novo DNA methyltransferases DNMT3A and DNMT3B, the subsequent oxidation steps turned out to be far more complex. The strong increase of oxidized cytosine bases (5hmC, 5fC, and 5caC) was accompanied by a drop in TET2 levels, yet the analysis of KO cells suggested that TET2 is responsible for most 5fC formation. The comparison of modified cytosine and enzyme levels in Tet KO cells revealed distinct and differentiation-dependent contributions of TET1 and TET2 to 5hmC and 5fC formation arguing against a processive mechanism of 5mC oxidation. The apparent independent steps of 5hmC and 5fC formation suggest yet to be identified mechanisms regulating TET activity that may constitute another layer of epigenetic regulation.

Forced running exercise mitigates radiation-induced cognitive deficits via regulated DNA hydroxymethylation.

Aim: Roles of forced running exercise (FE) in remediation of neurogenesis inhibition and radiation-induced cognitive dysfunction were investigated in a whole-brain irradiation mice model via the regulation of DNA 5-hydroxymethylation modification (5 hmC) and its catalytic enzymes ten-eleven translocation (Tet) proteins. Materials& methods: Hippocampal neurogenesis and cognitive function, DNA 5 hmC level and Tet expression were determined in mice. Results: The expression of DNA 5 hmC and Tet2, brain-derived neurotrophic factor significantly decreased in hippocampus postradiation. FE mitigated radiation-induced neurogenesis deficits and cognitive dysfunction. Furthermore, FE increased 5 hmC and brain-derived neurotrophic factor expression. SC1, a Tet inhibitor, reversed partly such changes. Conclusion: Tet-mediated 5 hmC modification represents a kind of diagnostic biomarkers of radiation-induced cognitive dysfunction. Targeting Tet-related epigenetic modification may be a novel therapeutic strategy for radiation-induced brain injury.

Reduction of Tet2 exacerbates early stage Alzheimer's pathology and cognitive impairments in 2×Tg-AD mice.

Abnormal modification of 5-hydroxymethylcytosine (5hmC) is closely related to the occurrence of Alzheimer's disease (AD). However, the role of 5hmC and its writers, ten-eleven translocation (Tet) proteins, in regulating the pathogenesis of AD remains largely unknown. We detected a significant decrease in 5hmC and Tet2 levels in the hippocampus of aged APPswe/PSEN1 double-transgenic (2×Tg-AD) mice that coincides with abundant amyloid-β (Aβ) plaque accumulation. On this basis, we examined the reduction of Tet2 expression in the hippocampus at early disease stages, which caused a decline of 5hmC levels and led young 2×Tg-AD mice to present with advanced stages of AD-related pathological hallmarks, including Aβ accumulation, GFAP-positive astrogliosis and Iba1-positive microglia overgrowth as well as the overproduction of pro-inflammatory factors. Additionally, the loss of Tet2 in the 2×Tg-AD mice at 5months of age accelerated hippocampal-dependent learning and memory impairments compared to age-matched control 2×Tg-AD mice. In contrast, restoring Tet2 expression in adult neural stem cells isolated from aged 2×Tg-AD mice hippocampi increased 5hmC levels and increased their regenerative capacity, suggesting that Tet2 might be an exciting target for rejuvenating the brain during aging and AD. Further, hippocampal RNA sequencing data revealed that the expression of altered genes identified in both Tet2 knockdown and control 2×Tg-AD mice was significantly associated with inflammation response. Finally, we demonstrated that Tet2-mediated 5hmC epigenetic modifications regulate AD pathology by interacting with HDAC1. These results suggest a combined approach for the regulation and treatment of AD-related memory impairment and cognitive symptoms by increasing Tet2 via HDAC1 suppression.