Member Of Ten-eleven Translocation Family Research Articles

Pan-cancer analyses reveal the molecular and clinical characteristics of TET family members and suggests that TET3 maybe a potential therapeutic target.

The Ten-Eleven Translocation (TET) family genes are implicated in a wide array of biological functions across various human cancers. Nonetheless, there is a scarcity of studies that comprehensively analyze the correlation between TET family members and the molecular phenotypes and clinical characteristics of different cancers. Leveraging updated public databases and employing several bioinformatics analysis methods, we assessed the expression levels, somatic variations, methylation levels, and prognostic values of TET family genes. Additionally, we explored the association between the expression of TET family genes and pathway activity, tumor microenvironment (TME), stemness score, immune subtype, clinical staging, and drug sensitivity in pan-cancer. Molecular biology and cytology experiments were conducted to validate the potential role of TET3 in tumor progression. Each TET family gene displayed distinct expression patterns across at least ten detected tumors. The frequency of Single Nucleotide Variant (SNV) in TET genes was found to be 91.24%, primarily comprising missense mutation types, with the main types of copy number variant (CNV) being heterozygous amplifications and deletions. TET1 gene exhibited high methylation levels, whereas TET2 and TET3 genes displayed hypomethylation in most cancers, which correlated closely with patient prognosis. Pathway activity analysis revealed the involvement of TET family genes in multiple signaling pathways, including cell cycle, apoptosis, DNA damage response, hormone AR, PI3K/AKT, and RTK. Furthermore, the expression levels of TET family genes were shown to impact the clinical staging of tumor patients, modulate the sensitivity of chemotherapy drugs, and thereby influence patient prognosis by participating in the regulation of the tumor microenvironment, cellular stemness potential, and immune subtype. Notably, TET3 was identified to promote cancer progression across various tumors, and its silencing was found to inhibit tumor malignancy and enhance chemotherapy sensitivity. These findings shed light on the role of TET family genes in cancer progression and offer insights for further research on TET3 as a potential therapeutic target for pan-cancer.

TET Family Members Are Integral to Porcine Oocyte Maturation and Parthenogenetic Pre-Implantation Embryogenesis.

The ten-eleven translocation (TET) enzyme family, which includes TET1/2/3, participates in active DNA demethylation in the eukaryotic genome; moreover, TET1/2/3 are functionally redundant in mice embryos. However, the combined effect of TET1/2/3 triple-gene knockdown or knockout on the porcine oocytes or embryos is still unclear. In this study, using Bobcat339, a specific small-molecule inhibitor of the TET family, we explored the effects of TET enzymes on oocyte maturation and early embryogenesis in pigs. Our results revealed that Bobcat339 treatment blocked porcine oocyte maturation and triggered early apoptosis. Furthermore, in the Bobcat339-treated oocytes, spindle architecture and chromosome alignment were disrupted, probably due to the huge loss of 5-hydroxymethylcytosine (5hmC)and concurrent increase in 5-methylcytosine (5mC). After Bobcat339 treatment, early parthenogenetic embryos exhibited abnormal 5mC and 5hmC levels, which resulted in compromised cleavage and blastocyst rate. The mRNA levels of EIF1A and DPPA2 (ZGA marker genes) were significantly decreased, which may explain why the embryos were arrested at the 4-cell stage after Bobcat339 treatment. In addition, the mRNA levels of pluripotency-related genes OCT4 and NANOG were declined after Bobcat339 treatment. RNA sequencing analysis revealed differentially expressed genes in Bobcat339-treated embryos at the 4-cell stage, which were significantly enriched in cell proliferation, cell component related to mitochondrion, and cell adhesion molecule binding. Our results indicated that TET proteins are essential for porcine oocyte maturation and early embryogenesis, and they act by mediating 5mC/5hmC levels and gene transcription.

A novel epigenetic marker, Ten-eleven translocation family member 2 (TET2), is identified in the intractable epileptic brain and regulates ATP binding cassette subfamily B member 1 (ABCB1) in the blood–brain barrier

ABSTRACT Drug-resistant epilepsy (DRE) is a chronic condition derived from spontaneous changes and regulatory effects in the epileptic brain. As demethylation factors, ten-eleven translocation (TET) family members have become a focus in recent studies of neurological disorders. Here, we quantified and localized TET1, TET2 and 5-hydroxymethylcytosine (5-hmC) in the temporal lobe cortex of DRE patients (n = 27) and traumatic brain hemorrhage controls (n = 10) by immunochemical staining. TET2 and ATP binding cassette subfamily B member 1 (ABCB1) expression patterns were determined in the isolated brain capillaries of DRE patients. TET2 expression was significantly increased in the temporal cortical tissue of DRE patients with or without hippocampal sclerosis (HS) compared to control patients, while TET1 and 5-hmC showed no differences in expression. We also found that a particularly strong expression of TET2 in the vascular tissue of DRE patients. ABCB1 and TET2 have evidently higher expression in the vascular endothelium from the neocortex of DRE patients. In blood–brain barrier (BBB) model, TET2 depletion can cause attenuated expression and function of ABCB1. Data from a cohort study and experiments in a BBB model suggest that TET2 has a specific regulatory effect on ABCB1, which may serve as a potential mechanism and target in DRE.

TET-mediated regulation of effector CD4 T cells

Abstract Functionally distinct subsets of pathogen-specific CD4 helper T cells, including T follicular helper (Tfh) and T helper type 1 (Th1) subsets, are critical for orchestrating the induction and maturation of innate and adaptive immune responses. Proper genetic programming of Tfh and Th1 differentiation is required for regulating immune responses to clear primary infections and promote long-lived memory CD4 T cell-mediated protection against pathogen re-exposures. Genetic approaches to study CD4 T cell differentiation have been largely focused on transcriptional programming, but gene regulation is also critically tied to epigenetic modifications, which represents a major knowledge gap in our understanding of CD4 T cell biology. Ten-eleven-translocation (TET) family enzymes facilitate DNA demethylation to influence chromatin accessibility and promote gene expression. Prior work has shown that TET2 activity has been linked to optimal Th1 responses in vitro. TET1 and TET3 proteins mediate Cd4 locus demethylation and are required for stable CD4 expression during thymocyte development. However, the precise role of either TET1 or TET3 activity to mediate CD4 T cell fate and function in vivo has not been directly investigated. Thus, we hypothesized that TET1 and TET3 activity in peripheral, mature CD4 T cells is required for regulating T helper cell differentiation and function. We used conditional genetic and chimeric approaches, coupled with robust systems of infectious disease immunology, to directly test our hypothesis. Our data show substantial CD4 T cell developmental skewing towards Tfh fates in the absence of specific TET family members. This suggests that TET1 and TET3 enzymes play a role in governing Tfh and Th1 CD4 cell fate.

44 Misregulation of ten-eleven translocation 3 CXXC domain leads to abnormal formation of 5-hydroxymethylcytosine and expression of pluripotency genes in pig embryos

Ten-eleven translocation (TET) methylcytosine dioxygenases are considered to play an important role in regulation of DNA methylation patterns by converting 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). TET3 protein, a member of TET family, is enriched in mature oocytes and early stage embryos and contributes to DNA demethylation of the paternal genome in zygotes. N-terminal CXXC domain of TET3 is thought to be important in catalysing 5mC oxidation through its DNA binding potential. However, it is not clear whether specific DNA binding of CXXC domain is required for 5hmC conversion in mammalian embryos. Here, we investigated the role of TET3 CXXC domain in controlling 5hmC formation in fertilized pig embryos by injecting TET3 CXXC domain into mature pig oocytes as a dominant negative to inhibit the direct binding of TET3 to the genome through the CXXC domain. The CXXC domain of pig TET3 was identified through bioinformatics comparison of TET3 sequences among different species and cloned from mature pig oocyte-derived cDNA. To construct the green fluorescent protein (GFP)-CXXC fusion protein, CXXC sequence was subcloned into N-terminal GFP fusion vector, and then mRNA was synthesised by in vitro transcription. The GFP-CXXC mRNA (100 ng/µL) was injected into oocytes matured in vitro for 36 to 37h. Then, the oocytes were fertilized at 42h post-maturation. Water-injected oocytes served as a control. At 17h post-fertilization, zygotes were collected to analyse 5hmC level by immunocytochemistry. The level of 5hmC was analysed using ImageJ (https://imagej.nih.gov/ij/). Expression of pluripotency-related genes at Day 7 blastocysts was examined through quantitative RT-PCR; ΔΔCt method was used to analyse the quantitative RT-PCR data and Student’s t-test was used for statistical analysis. All experiments were conducted at least three times and P-values of less than 0.05 were considered significant. The GFP-CXXC was exclusively localised in pronuclei, indicating that the CXXC domain may lead to nuclear localization of TET3. The level of 5hmC in zygotes was not altered by the overexpression of GFP-CXXC. Interestingly, in 2-cell stage embryos, the 5hmC level was reduced in GFP-CXXC injected embryos compared with the control group, suggesting that CXXC domain is important for 5hmC formation post-DNA replication. There was an increase in transcript abundance of NANOG and ESRRB in blastocysts developed from GFP-CXXC injected oocytes compared with control blastocysts (P<0.05). There was no difference in the expression of POU5F1 and SOX2. In this study, we found that CXXC domain of TET3 is critical in maintaining the level of 5hmC formation at 2-cell stage and proper level of pluripotency-related genes (NANOG and ESRRB) in blastocysts. Future studies will focus on elucidating mechanisms behind the changes after overexpression of GFP-CXXC in pig embryos.

TET2 in Normal and Malignant Hematopoiesis.

The ten-eleven translocation (TET) family of enzymes were originally cloned from the translocation breakpoint of t(10;11) in infant acute myeloid leukemia (AML) with subsequent genomic analyses revealing somatic mutations and suppressed expression of TET family members across a range of malignancies, particularly enriched in hematological neoplasms. The TET family of enzymes is responsible for the hydroxylation of 5-methylcytosines (5-mC) to 5-hydroxymethylcytosine (5-hmC), followed by active and passive mechanisms leading to DNA demethylation. Given the complexity and importance of DNA methylation events in cellular proliferation and differentiation, it comes as no surprise that the TET family of enzymes is intricately regulated by both small molecules and regulatory cooperating proteins. Here, we review the structure and function of TET2, its interactions with cooperating mutations and small molecules, and its role in aberrant hematopoiesis.

TET1 and TET3 are essential in induction of Th2-type immunity partly through regulation of IL-4/13A expression in zebrafish model

It has been considered that epigenetic modulation can affect a diverse array of cellular activities, in which ten eleven translocation (TET) methylcytosine dioxygenase family members refer to a group of fundamental components involved in catalyzation of 5-hydroxymethylcytosine and modification of gene expression. Even though the function of TET proteins has been gradually revealed, their roles in immune regulation are still largely unknown. Recent studies provided clues that TET2 could regulate several innate immune-related inflammatory mediators in mammals. This study sought to explore the function of TET family members in potential T-helper (Th) cell differentiation involved in adaptive immunity by utilizing a zebrafish model. As shown by results, soluble antigens could induce expression of zebrafish IL-4/13A (i.e. a pivotal Th2-type cytokine essential in Th2 cell differentiation and functions), and further trigger the expression of Th1- and Th2-related genes. It is noteworthy that this response was accompanied by the up-regulation of two TET family members (TET1 and TET3) both in immune organs (spleen and kidney) and cells (peripheral lymphocytes). Knocking-down of TET1 and TET3 will give rise to the decreased responses of IL-4/13A induction against exogenous soluble antigen stimulation, and further restrain the expression of Th2-related genes, which indicates a restrained Th2 cell differentiation. Nonetheless, TET2 did not exhibit effect on the modification of Th1/Th2 related gene expression. Hence, these data showed that TET1 and TET3 might be two significant epigenetic regulators involved in Th2 differentiation through regulation of IL-4/13A expression. This is the first report to show that TET family members play indispensable roles in Th2-type immunity, indicating an epigenetic modulation manner involved in adaptive immune regulations and responses.

TET3 inhibits TGF-β1-induced epithelial-mesenchymal transition by demethylating miR-30d precursor gene in ovarian cancer cells.

BackgroundAbnormal DNA methylation/demethylation is recognized as a hallmark of cancer. TET (ten-eleven translocation) family members are novel DNA demethylation related proteins that dysregulate in multiple malignances. However, their effects on ovarian cancer remain to be elucidated.MethodsThe changes of TET family members during TGF-β1-induced epithelial-mesenchymal transition (EMT) in SKOV3 and 3AO ovarian cancer cells were detected. TET3 was ectopically expressed in TGF-β1-treated ovarian cancer cells to examine its effect on TGF-β1-induced EMT phenotype. The downstream target of TET3 was further identified. Finally, the relationships of TET3 expression to clinic-pathological parameters of ovarian cancer were investigated with a tissue microarray using immunohistochemistry.ResultsTET3 was downregulated during TGF-β1-initiatd epithelial-mesenchymal transition (EMT) in SKOV3 and 3AO ovarian cancer cells. Overexpression of TET3 reversed TGF-β1-induced EMT phenotypes including the expression pattern of molecular markers (E-cadherin, Vimentin, N-cadherin, Snail) and migratory and invasive capabilities of ovarian cancer cells. miR-30d was identified as a downstream target of TET3, and TET3 overexpression resumed the demethylation status in the promoter region of miR-30d precursor gene, resulting in restoration of miR-30d (an EMT suppressor of ovarian cancer cells proven in our previous study) level in TGF-β1-induced EMT. We further found that TET3 expression was decreased in ovarian cancer tissues, especially in serous ovarian cancers. The overall positivity of TET3 was inversely correlated with the grade of differentiation status of ovarian cancer.ConclusionOur results revealed that TET3 acted as a suppressor of ovarian cancer by demethylating miR-30d precursor gene promoter to block TGF-β1-induced EMT.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-016-0350-y) contains supplementary material, which is available to authorized users.

MiR-506 inhibits cell proliferation and invasion by targeting TET family in colorectal cancer.

Ten-eleven translocation (TET) family members have been shown to be involved in the development of many tumors. However, the biological role of the TET family and its mechanism of action in colorectal carcinogenesis and progression remain poorly understood. We measured the expression levels of TET family members in colorectal cancer (CRC) specimens, in the corresponding normal tissues and in cell lines using quantitative real-time PCR (qRT-PCR) and in situ hybridization (ISH). Both the protein function and the protein-independent role of TETs were investigated by cell viability assays and cell invasion assays using in vitro and in vivo models. We found that all three TET genes were strongly up-regulated at the transcript level in CRC samples compared to matched normal tissues. The same results were observed in colorectal cancer cell lines. Knockdown of TETs by shTET1/2/3 showed that TET family members inhibited CRC growth and metastasis. We showed that TET family member degradation by miR-506 inhibits cell proliferation and invasion in colorectal cancer. Through this study, we advance our understanding of the expression levels TETs and miR-506 in CRC and further clarify the internal regulatory mechanism of miR-506 by targeting TET during CRC processes. These findings may contribute to a novel avenue for researching and developing targeted therapies for CRC.

Global DNA hypomethylation and hypoxia-induced expression of the ten eleven translocation (TET) family, TET1, in scleroderma fibroblasts.

The precise mechanisms of tissue fibrosis have not yet been elucidated in systemic sclerosis (SSc). However, studies of the regulation of DNA methylation, the most widely studied epigenetic mechanism, have confirmed the involvement of the TET family proteins, recently identified DNA demethylases, in the pathogenesis of SSc. The mRNA levels of TET family members were compared in normal and SSc fibroblasts. The effects of hypoxia and siRNA specific to HIF-1α on TET expression were also examined. Global methylation status was analysed by LUMA. The presence of 5-hydroxymethylcytosine (5hmC) in SSc was examined by immunohistochemistry. The level of TET1 mRNA in SSc fibroblasts was elevated by 1.68 fold compared with that of normal fibroblasts, but the expression levels of TET2 and TET3 were comparable between both cell types. The expression levels of DNMT1 and DNMT3B mRNA have a tendency to elevate in SSc fibroblasts. Among TET family members, the expression of TET1 was exclusively induced by hypoxia via HIF-1α-independent pathways in SSc fibroblasts, but not in normal fibroblasts. The methylation level was decreased in SSc fibroblasts relative to normal fibroblasts, and 5hmC was present in dermal fibroblasts of skin sections from patients with SSc. TET1 expression in SSc fibroblasts was abnormally regulated in the hypoxic environment and accompanied by global DNA hypomethylation, suggesting the involvement of aberrant DNA methylation in the pathogenesis of SSc.

Clinical significance of DNA methylation mRNA levels of TET family members in colorectal cancer.

Purpose Ten eleven translocation (TET) enzyme activity is essential for active DNA demethylation in biological processes, and their altered expression has been observed in various malignancies. Therefore, we investigated DNA methylation and mRNA levels of all TETs in colorectal cancer (CRC) patients.Methods TET mRNA levels were evaluated using quantitative RT-PCR in primary cancerous and histopathologically unchanged colorectal tissues from patients who underwent radical surgical colon resection (n = 113). DNA methylation levels of the TET CpG island were assessed using bisulfite DNA sequencing and high-resolution melting analysis.ResultsWe found reduced transcript levels of TET1, TET2 and TET3 in cancerous tissue compared with their histopathologically unchanged counterparts (p = 0.000011; p = 0.000001; p = 0.00031, respectively). Importantly, multivariate Cox regression analysis revealed favorable overall survival (OS) and disease-free survival (DFS) outcomes for patients with high TET2 mRNA levels in histopathologically unchanged tissue (HROS = 0.091, 95 % CI 0.011–0.77, p = 0.028; HRDFS = 0.21, 95 % CI 0.04–1.06, p = 0.059). Moreover, we found no DNA methylation in the TET2 and TET3 promoter regions in cancerous and histopathologically unchanged tissue. In contrast, we reported TET1 DNA hypermethylation in a small fraction of patients (n = 12/113).ConclusionTo best of our knowledge, our study is the first to investigate TET mRNA levels in a cohort of CRC patients and correlate them with patients’ prognosis. Present study provides the evidence that TET2 mRNA expression may be an independent prognostic factor for disease recurrence and outcome. Additionally, our findings initially indicate the importance of DNA methylation in regulating TET1 expression.Electronic supplementary materialThe online version of this article (doi:10.1007/s00432-014-1901-2) contains supplementary material, which is available to authorized users.