TET2 Expression Research Articles

Tet2-mediated epigenetic drive for astrocyte differentiation from embryonic neural stem cells

DNA methylation and demethylation at CpG di-nucleotide sites plays important roles in cell fate specification of neural stem cells (NSCs). We have previously reported that DNA methyltransferases, Dnmt1and Dnmt3a, serve to suppress precocious astrocyte differentiation from NSCs via methylation of astroglial lineage genes. However, whether active DNA demethylase also participates in astrogliogenesis remains undetermined. In this study, we discovered that a Ten-eleven translocation (Tet) protein, Tet2, which was critically involved in active DNA demethylation through oxidation of 5-Methylcytosine (5mC), drove astrocyte differentiation from NSCs by demethylation of astroglial lineage genes including Gfap. Moreover, we found that an NSC-specific bHLH transcription factor Olig2 was an upstream inhibitor for Tet2 expression through direct association with the Tet2 promoter, and indirectly inhibited astrocyte differentiation. Our research not only revealed a brand-new function of Tet2 to promote NSC differentiation into astrocytes, but also a novel mechanism for Olig2 to inhibit astrocyte formation.

Cocaine-induced changes in behaviour and DNA methylation in rats are influenced by inter-individual differences in spontaneous exploratory activity.

Individual differences in behavioural traits influence susceptibility to addictive disorders. Drug addiction involves changes in gene expression, proposed to occur via DNA methylation (DNAm). To investigate DNAm changes in reward-related brain structures (nucleus accumbens (NAc), lateral habenula (LHb)) in response to cocaine exposure in rats differing in spontaneous exploratory activity. Rats were observed in the exploration box and categorised as high- (HE) or low explorers (LE). Rats were administered vehicle or cocaine (12 mg/kg, i.p.) for 7 days, followed by a 14-day withdrawal period and cocaine challenge (7 mg/kg); horizontal locomotor activity was recorded. Brain tissue was dissected after 24 h; we analysed messenger RNA (mRNA) and activity levels of epigenetic DNA modifiers (DNMTs and TETs) as well as mRNA and promoter methylation levels at selected genes previously linked to addictive behaviours. The cocaine challenge dose stimulated locomotor activity in both LE- and HE rats only when administered after a repeated cocaine schedule, suggesting development of behavioural sensitisation. Quantitative polymerase chain reaction analyses demonstrated higher basal expression of Dnmt3a, Tet2 and Tet3 in the LHb of HE- vs. LE rats, and we observed differential effects of cocaine exposure on the expression and activity of epigenetic DNA modifiers in the NAc and LHb of HE- and LE rats. Furthermore, cocaine exposure differentially altered promoter methylation levels of A2AR, Ppp1cc, and Taar7b in the NAc and LHb of HE- and LE rats. DNAm might play a role in the HE- and LE phenotypes as well as mediate behavioural effects of LE- and HE rats in response to drugs of abuse.

Cross-sectional and longitudinal associations between global DNA (hydroxy) methylation and exposure biomarkers of the Hebei Spirit oil spill cohort in Taean, Korea

The Hebei Spirit oil spill (HSOS) occurred on the west coast of South Korea (Taean county) on December 7, 2007, and studies revealed that exposure to the oil spill was associated with various adverse health issues in the inhabiting population. However, no studies evaluated the association between crude-oil exposure and epigenetic changes. This study aimed to investigate the HSOS exposure-associated longitudinal and cross-sectional variations in global DNA methylation (5-mc) and/or hydroxymethylation (5-hmc) and expression profiles of related genes in Taean cohort participants from 2009 (AH-baseline) and 2014 (AH-follow-up) relative to the reference group (AL). We measured global DNA 5-mc and 5-hmc levels and related gene expression levels in whole blood. We identified significant associations between HSOS exposure and AH-baseline-5-mc, AH-baseline-5-hmc, and AH-follow-up-5-hmc. HSOS exposure was associated with lower %5-mc content and higher %5-hmc content in the same individuals from both the cross-sectional and longitudinal studies. In addition, we found a strong correlation between 5-mc and DNMT3B expression, and between 5-hmc and TET1 expression. Our findings suggested that epigenetic changes are important biomarkers for HSOS exposure and that 5-hmc is likely to be more sensitive for environmental epidemiological studies.

Chrysin Induced Cell Apoptosis and Inhibited Invasion Through Regulation of TET1 Expression in Gastric Cancer Cells.

ObjectiveTen-eleven translocation (TET) enzymes that oxidize a 5-methylcytosine (5mC) to yield 5-hydroxymethylcytosine (5hmC) have been responsible for fine-tuning methylation patterns and exhibit role in epigenetic modifications. Chrysin, a natural flavone frequently present in honey, has been recognized to exhibit anti-tumor properties. In this study, we investigated the effects of Chrysin in the expression pattern of TET proteins in gastric cancer (GC) cells.Materials and MethodsUsing qRT-PCR and Western blot analysis, we analyzed the expression of TET1 in GC cells in vitro following treatment with Chrysin. Immunofluorescence staining detected the expression levels of 5mC and 5hmC. Flow cytometry, wound healing, and Matrigel invasion assays were performed to determine cell proliferation, cell cycle, apoptosis, and migration and invasion of GC cells following treatment with Chrysin, si-TET1, and TET1-KO. Furthermore, a xenograft model was developed to analyze the expression pattern of TET1 on tumor development in vivo.ResultsqRT-PCR and Western blot assays indicated that treatment with Chrysin significantly promoted the expression of TET1 in GC cells. Immunofluorescence study further confirmed that TET1 and 5hmC levels were significantly enhanced following treatment with Chrysin in MKN45 cells. Moreover, our results suggested that Chrysin could noticeably induce cell apoptosis and inhibit cell migration and invasion. Further, knockdown and overexpression of TET1 were conducted to investigate whether TET1 expression affected cell apoptosis, and cell migration and invasion in MKN45 cells. The results indicated that overexpression of TET1 markedly promoted cell apoptosis and inhibited cell migration and invasion. Furthermore, the TET1 gene knocked out was generated using the CRISPR/Cas9 system. Our data suggested that TET1 expression was associated with GC tumor growth in vivo.ConclusionThis study indicated that Chrysin exerted anti-tumor effects through the regulation of TET1 expression in GC and presented TET1 as a novel promising therapeutic target for GC therapy.

Regulation of DNA methyltransferase 3a Expression by Prostaglandin E2 in Human Fibroblasts

IntroductionPeriodontitis is common chronic inflammatory condition that not only results in loss of teeth, but contributes to a variety of systemic conditions, including cancer. Previously we showed that exposure of human gingival fibroblasts (HGF) derived from patients with periodontitis to PGE2 decreases expression of DNMT1, DNMT3a and TET1, but increases global levels of methylated and hydroxymethylated DNA, acting predominantly through the EP4 receptor. The current study compares the effects of PGE2 on expression of these genes in non‐inflamed fibroblasts and aims to further elucidate the signaling pathways involved.MethodsReal time PCR and ELISA are used to measure expression of DNMT1, DNMT3a, TET1 and EP receptors 1–4 in untreated HGF and IMR90 lung fibroblasts and those treated with PGE2. The effects of actinomycin D, as well as EP receptor agonists and antagonists and signaling pathway activators and inhibitors on mRNA expression of these genes will also be assessed by qPCR.ResultsResults so far show that PGE2 decreases expression of DNMT1 in IMR90 cells to an extent similar to that observed in HGF, but levels of DNMT3a are increased rather than decreased, and levels of TET1 are unaffected in IMR90 cells. Although all four EP receptors are expressed in both HGF and IMR90, the relative levels of EP4 expression are lower.ConclusionThe ability of this inflammatory mediator to influence DNA methylation is consistent with the association of chronic inflammation with increased risk of cancer. However, the fact that the effects are cell and context dependent indicate that more information about the specific signaling pathways involved in regulating expression of these important enzymes is needed. Further studies will be aimed at elucidating the roles of the PGE2 receptors and their signaling pathways in mediating the cell‐specific effects.

Perinatal Epigenetic Modulation of the Brain Renin Angiotensin System Programs Cardiometabolic Diseases

Cardiometabolic Disease (CMD) is one of the major health conditions associated with high morbidity and mortality. It is known for disturbed glycemic levels, elevated blood pressure and dyslipidemia. Epigenetic mechanisms especially DNA methylation have recently been shown to exert an essential role in the development of CMD. DNA methyltransferase (DNMT) and Ten eleven translocation (TET) are crucial enzymes controlling DNA methylation and demethylation. Pre‐autonomic neurons in the paraventricular nucleus of the hypothalamus (PVN) act as key regulators for blood pressure (BP) control. We recently reported that the renin‐angiotensin system (RAS) is an important modulator of those neurons via activation of A Disintegrin and Metalloprotease 17 (ADAM17) and Angiotensin II Type 1 receptors (AT1R) contributing to BP dysregulation. We propose that consumption of a hypercaloric diet during pregnancy leads to epigenetic modifications in the progeny’s brain, promoting autonomic dysfunction and hypersensitivity to neurogenic hypertension and metabolic disorders. In this study, C57BL6/J dams were fed a high‐fat high‐sucrose diet (HFHSD) or regular diet (RD) and mated with RD males. After weaning, offspring were put on RD for 3 months and underwent 24 h BP recording (telemetry), pharmacological assessment of autonomic function, blood glucose measurement and a glucose tolerance test before being sacrificed. RNA was extracted from the hypothalamus of HFHSD‐exposed (OH) and RD‐exposed (OR) offspring. mRNA expression for DNMT1, DNMT3a, DNMT3b, Tet1, Tet2, Tet3, AT1R and ADAM17 was quantified using real‐time PCR. Baseline BP recording showed a significant (p<0.05) and sex‐specific increase in systolic BP (140 ±2 vs. 132 ±3 mmHg) and heart rate (654 ±10 vs. 590 ±10 bpm) in OH males compared to OR controls during the active phase (6PM‐6AM). This group also exhibited significantly higher blood glucose levels (168 ±7 vs. 143 ±7 mg/dL) and sympathetic drive to the heart (Bradycardia to propranolol: −152 ±10 vs. −44 ±8 bpm). DNMT3b, Tet2, and Tet3 expression were significantly downregulated in OH mice (unpaired t‐test, p<0.05) while expression of ADAM17 and AT1R, components of the RAS, were upregulated (p=0.002 and 0.005, respectively). To elucidate the mechanisms involved in future experiments, an in vitro model was developed using N2A cells treated with a glucose‐lipid mixture and validated by the significant downregulation of gene expression for DNMT1, DNMT3a (p<0.01), Tet1, Tet3 (p<0.05) in comparison to untreated N2A cells. We conclude that consumption of HFHSD during pregnancy promotes changes in methylation profiles within the hypothalamus, leading to altered gene expression, notably of RAS genes in the brain, which in turn could trigger autonomic dysfunction and hypersensitivity to cardiometabolic disease.Support or Funding InformationAmerican Diabetes Association (1‐19‐IBS‐291), Veterans Affairs Merit Award (BX004294), National Institutes of Health (HL093178 and COBRE P30GM106392), and LSUHSC‐NO Research Enhancement Program.

Obesity‐associated Hypoxia Contributes to Aberrant Methylation of Genes Implicated in Inflammation and vascular Function

IntroductionObesity is a major risk factor for cardiovascular disease. We previously demonstrated an impaired vascular function in obese adults (OB). We now hypothesize a role of obesity‐associated hypoxia in disturbing the methylation/expression of genes involved in inflammation/vascular function. We also propose a mediating role of the hypoxia‐inducible factor, HIF1α and the DNA hydroxymethylase, TET1.MethodsWe obtained subcutaneous and visceral adipose tissue (AT) biopsies from bariatric patients (n=60; age: 36±7 yrs; BMI: 50.7±8.7 kg/m2) and non‐obese (NOB) adults having elective surgeries (n=30; age: 36±2 yrs; BMI: 25.8±1 kg/m2). AT‐isolated arterioles were tested for vasoreactivity in response to pressure gradients of Δ10‐Δ100 cmH2O. Arteriolar nitric oxide (NO) and reactive oxygen species (ROS) were measured. Protein expression of HIF1α and TET1 and methylation/expression of leptin, IL1β, IL6, IL8, IL17, CXCL5, TNF‐α, and IFNγ were measured in the AT.ResultsFlow‐induced dilation (FID) was 40–50% higher in NOB than OB adults across all pressure gradients (p<0.05). NO production was higher, and ROS generation was lower in OB arterioles compared to NOB. HIF1α and TET1 proteins were 2–4‐fold higher in OB compared to NOB adults and correlated negatively with arteriolar FID, NO, and brachial artery FID (r=0.82, 0.64, 0.91, respectively; p<0.001) and positively with ROS (r=−0.71, p<0.01). Global hydroxymethylation and adipocytokine promoter hypomethylation and increased expression were observed in OB compared to NOB.ConclusionOur results suggest that vascular dysfunction in OB adults may be attributed to aberrant DNA methylation and increased expression of adipocytokines. This conclusion was also supported by invitro mechanistic studies.Support or Funding InformationThis research was funded by the National Heart, Lung, and Blood Institute (NHLBI), grants K99HL140049 and 4 R00 HL140049‐03 (AM) and HL095701 (S.P.)

Demethylation Status of Somatic DNA Extracted From Pituitary Neuroendocrine Tumors Indicates Proliferative Behavior.

Cytosine intermediaries 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC), epigenetic hallmarks, have never been investigated in pituitary neuroendocrine tumors (PitNET). To examine methylation-demethylation status of global deoxyribonucleic acid (DNA) in PitNET tissues and to assess its correlation with clinical and biological parameters. Altogether, 57 PitNET and 25 corresponding plasma samples were collected. 5mC and 5hmC were investigated using liquid chromatography-tandem mass spectrometry. Expression of DNA methyltransferase 1 (DNMT1); tet methylcytosine dioxygenase 1 through 3 (TET1-3); and ubiquitin-like, containing PHD and RING finger domains 1 and 2 (UHRF1-2) were measured by reverse transcription-polymerase chain reaction. Levels of 5hmC and UHRF1-2 were explored by immunohistochemistry. Effect of demethylating agent decitabine was tested on pituitary cell lines. 5hmC/5mC ratio was higher in less differentiated PitNET samples. A negative correlation between Ki-67 proliferation index and 5hmC, 5hmC to 5mC ratio were revealed. Higher 5mC was observed in SF-1 + gonadotroph adenomas with a higher Ki-67 index. Expressions of TET2 and TET3 were significantly higher in adenomas with higher proliferation rate. UHRF1 showed gradually increased expression in higher proliferative adenoma samples, and a significant positive correlation was detected between UHRF2 expression and 5hmC level. Decitabine treatment significantly decreased 5mC and increased 5hmC levels in both cell lines, accompanied with decreased cell viability and proliferation. The demethylation process negatively correlated with proliferation rate and the ratio of 5hmC to 5mC was higher in less differentiated adenomas. Therefore, epigenetic markers can be potential biomarkers for PitNET behavior. Altering the epigenome in adenoma cells by decitabine decreased proliferation, suggesting that this treatment might be a novel medical treatment for PitNET.

Expression and prognosis analysis of TET family in acute myeloid leukemia.

TET family members (TETs) encode proteins that represent crucial factors in the active DNA demethylation pathway. Evidence has proved that TET2 mutation is associated with leukemogenesis, drug response, and prognosis in acute myeloid leukemia (AML). However, few studies revealed the TETs expression and its clinical significance in AML. We conducted a detailed expression and prognosis analysis of TETs expression in human AML cell lines and patients by using public databases. We observed that TETs expression especially TET2 and TET3 was closely associated with AML among various human cancers. TET1 expression was significantly reduced in AML patients, whereas TET2 and TET3 expression was significantly increased. Kaplan-Meier analysis showed that only TET3 expression was associated with overall survival (OS) and disease-free survival (DFS) among both total AML as well as non-M3 AML, and was confirmed by another independent cohort. Moreover, Cox regression analysis revealed that TET3 expression may act as an independent prognostic factor for OS and DFS in total AML. Interestingly, patients that received hematopoietic stem cell transplantation (HSCT) did not show significantly longer OS and DFS than those who did not receive HSCT in TET3 high-expressed groups; whereas, in TET3 low-expressed groups, patients that accepted HSCT showed significantly longer OS and DFS than those who did not accept HSCT. By bioinformatics analysis, TET3 expression was found positively correlated with tumor suppressor gene including CDKN2B, ZIC2, miR-196a, and negatively correlated with oncogenes such as PAX2 and IL2RA. Our study demonstrated that TETs showed significant expression differences in AML, and TET3 expression acted as a potential prognostic biomarker in AML, which may guide treatment choice between chemotherapy and HSCT.

Novel Mutations and Decreased Expression of the Epigenetic Regulator TET2 in Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is a lethal vasculopathy. Hereditary cases are associated with germline mutations in BMPR2 and 16 other genes; however, these mutations occur in <25% of patients with idiopathic PAH and are rare in PAH associated with connective tissue diseases. Preclinical studies suggest epigenetic dysregulation, including altered DNA methylation, promotes PAH. Somatic mutations of Tet-methylcytosine-dioxygenase-2 (TET2), a key enzyme in DNA demethylation, occur in cardiovascular disease and are associated with clonal hematopoiesis, inflammation, and adverse vascular remodeling. The role of TET2 in PAH is unknown. To test for a role of TET2, we used a cohort of 2572 cases from the PAH Biobank. Within this cohort, gene-specific rare variant association tests were performed using 1832 unrelated European patients with PAH and 7509 non-Finnish European subjects from the Genome Aggregation Database (gnomAD) as control subjects. In an independent cohort of 140 patients, we quantified TET2 expression in peripheral blood mononuclear cells. To assess causality, we investigated hemodynamic and histological evidence of PAH in hematopoietic Tet2-knockout mice. We observed an increased burden of rare, predicted deleterious germline variants in TET2 in PAH patients of European ancestry (9/1832) compared with control subjects (6/7509; relative risk=6; P=0.00067). Assessing the whole cohort, 0.39% of patients (10/2572) had 12 TET2 mutations (75% predicted germline and 25% somatic). These patients had no mutations in other PAH-related genes. Patients with TET2 mutations were older (71±7 years versus 48±19 years; P<0.0001), were more unresponsive to vasodilator challenge (0/7 versus 140/1055 [13.2%]), had lower pulmonary vascular resistance (5.2±3.1 versus 10.5±7.0 Wood units; P=0.02), and had increased inflammation (including elevation of interleukin-1β). Circulating TET2 expression did not correlate with age and was decreased in >86% of PAH patients. Tet2-knockout mice spontaneously developed PAH, adverse pulmonary vascular remodeling, and inflammation, with elevated levels of cytokines, including interleukin-1β. Long-term therapy with an antibody targeting interleukin-1β blockade resulted in regression of PAH. PAH is the first human disease related to potential TET2 germline mutations. Inherited and acquired abnormalities of TET2 occur in 0.39% of PAH cases. Decreased TET2 expression is ubiquitous and has potential as a PAH biomarker.

Naloxone regulates the differentiation of neural stem cells via a receptor-independent pathway.

The abilities of opioids to activate downstream signaling pathways normally depend on the binding between opioids and their receptors. However, opioids may also function in a receptor-independent manner, especially in neural stem cells (NSCs) in which the expression of opioid receptors and endogenous opioid agonists is low. When two opioids, morphine and naloxone, were used during the early stage of NSC differentiation, increased neurogenesis was observed. However, naloxone methiodide, a membrane impenetrable analog of naloxone, did not affect the NSC differentiation. The abilities of morphine and naloxone to facilitate neurogenesis were also observed in opioid receptor-knockout NSCs. Therefore, morphine and naloxone promote neurogenesis in a receptor-independent manner at least during the early stage. In addition, the receptor-independent functions of opioids were not observed in methylcytosinedioxygenase ten-eleventranslocation 1 (Tet1) knockout NSCs. When the expression of opioid receptors increased and the expression of Tet1 decreased during the late stage of NSC differentiation, morphine, but not naloxone, inhibited neurogenesis via traditional receptor-dependent and miR181a-Prox1-Notch-related pathway. In summary, the current results demonstrated the time-dependent effects of opioids during the differentiation of NSCs and provided additional insight on the complex functions of opioids.

Molecular pathogenesis of progression to myeloid leukemia from TET-insufficient status

AbstractLoss-of-function mutations in ten-eleven translocation-2 (TET2) are recurrent events in acute myeloid leukemia (AML) as well as in preleukemic hematopoietic stem cells (HSCs) of age-related clonal hematopoiesis. TET3 mutations are infrequent in AML, but the level of TET3 expression in HSCs has been found to decline with age. We examined the impact of gradual decrease of TET function in AML development by generating mice with Tet deficiency at various degrees. Tet2f/f and Tet3f/f mice were crossed with mice expressing Mx1-Cre to generate Tet2f/wtTet3f/fMx-Cre+ (T2ΔT3), Tet2f/fTet3f/wtMx-Cre+ (ΔT2T3), and Tet2f/fTet3f/fMx-Cre+ (ΔT2ΔT3) mice. All ΔT2ΔT3 mice died of aggressive AML at a median survival of 10.7 weeks. By comparison, T2ΔT3 and ΔT2T3 mice developed AML at longer latencies, with a median survival of ∼27 weeks. Remarkably, all 9 T2ΔT3 and 8 ΔT2T3 mice with AML showed inactivation of the remaining nontargeted Tet2 or Tet3 allele, respectively, owing to exonic loss in either gene or stop-gain mutations in Tet3. Recurrent mutations other than Tet3 were not noted in any mice by whole-exome sequencing. Spontaneous inactivation of residual Tet2 or Tet3 alleles is a recurrent genetic event during the development of AML with Tet insufficiency.

Hypercholesterolemia Accelerates the Aging Phenotypes of Hematopoietic Stem Cells by a Tet1-Dependent Pathway

Hypercholesterolemia accelerates the phenotypes of aging in hematopoietic stem cells (HSCs). As yet, little is known about the underlying mechanism. We found that hypercholesterolemia downregulates Ten eleven translocation 1 (Tet1) in HSCs. The total HSC population was increased, while the long-term (LT) population, side population and reconstitution capacity of HSCs were significantly decreased in Tet1−/− mice. Expression of the Tet1 catalytic domain in HSCs effectively restored the LT population and reconstitution capacity of HSCs isolated from Tet1−/− mice. While Tet1 deficiency upregulated the expression of p19 and p21 in HSCs by decreasing the H3K27me3 modification, the restoration of Tet1 activity reduced the expression of p19, p21 and p27 by restoring the H3K27me3 and H3K36me3 modifications on these genes. These results indicate that Tet1 plays a critical role in maintaining the quiescence and reconstitution capacity of HSCs and that hypercholesterolemia accelerates HSC aging phenotypes by decreasing Tet1 expression in HSCs.

Aberrant DNA and histone methylation during zygotic genome activation in goat cloned embryos

In somatic cell nuclear transfer (SCNT) embryos, developmental defects first appear at the time of zygotic genome activation (ZGA), a process that is under the control of DNA and histone methylation. However, dynamics of 5-mC and 5-hmC during ZGA differ between porcine and bovine SCNT embryos, and histone methylation during ZGA in goat SCNT embryos remains poorly understood. Therefore, in the present study, we investigated the dynamic changes of 5-mC, 5-hmC, H3K4me2/3, and H3K9me3, as well as the expression of key genes related to these epigenetic modifications, during ZGA in goat cloned embryos. Compared with the IVF embryos, the 5-mC signal intensity was significantly increased at the 2- and 4-cell stage SCNT embryos, and the H3K4me3 and H3K9me3 signal intensity was significantly increased at 2- to 8-cell stage SCNT embryos, while the 5-hmC and H3K4me2 signal intensity was significantly lower at the 4- and 8-cell stage SCNT embryos. Of note, the H3K9me3 level was also significantly higher, whereas H3K4me3 signal intensity showed no statistical difference in the pronuclear stage SCNT embryos. Moreover, the expression of TET2, DNMT3B, KDM4A, SUV39H1, G9A, and SETDB1 was significantly increased, while the expression of UHRF1, PCNA, KDM4B, KDM4D, KDM5A, KDM5B, and KDM5C was significantly decreased at the 8-cell stage SCNT embryos. Our data revealed aberrant DNA and histone methylation during ZGA in goat cloned embryos. We further inferred that the abnormally higher level of 5-mC, H3K4me3, and H3K9me3 might serve as epigenetic barriers of the reprogramming and modifying these aberrant modifications might be a promising strategy to improve cloning efficiency in goat.

Analysis of TET2 and EZH2 gene functions in chromosome instability in acute myeloid leukemia

TET2 and EZH2 play important roles in the epigenetic regulation in many cancers. However, their specific roles in acute myeloid leukemia (AML) pathogenesis remain unknown. Here, the expression, methylation or mutation of EZH2 and TET2 was determined and further correlated with the levels of the chromosome instability (CIN) genes MAD2 and CDC20. We down-regulated EZH2 and TET2 in AML cell lines and assessed the effect on CIN using fluorescence in situ hybridization (FISH). Our results showed that TET2, EZH2, MAD2 and CDC20 were aberrantly expressed in AML patients. The expression level of MAD2 or CDC20 was positively correlated with that of TET2 or EZH2. Hypermethylation of the TET2 gene down-regulated its transcription. Down-regulation of EZH2 or TET2 expression inhibited apoptosis, affected MAD2 and CDC20 expression, and promoted CIN in AML cells. Decitabine treatment restored TET2 methylation and EZH2 transcription and ameliorated CIN in AML. Therefore, TET2 and EZH2 play a tumor-inhibiting role in AML that affects CIN via MAD2 and CDC20.

TET2-mediated Cdkn2A DNA hydroxymethylation in midbrain dopaminergic neuron injury of Parkinson's disease.

It has been reported that abnormal epigenetic modification is associated with the occurrence of Parkinson's disease (PD). Here, we found that a ten-eleven translocation 2 (TET2), a staff of the DNA hydroxylases family, was increased in dopaminergic neurons in vitro and in vivo. Genome-wide mapping of DNA 5-hydroxymethylcytosine (5-hmC)-sequencing has revealed an aberrant epigenome 5-hmC landscape in 1-methyl-4-phenylpyridinium iodide (MPP+)-induced SH-SY5Y cells. The TET family of DNA hydroxylases could reverse DNA methylation by oxidization of 5-methylcytosine (5-mC) to 5-hmC. However, the relationship between modification of DNA hydroxymethylation and the pathogenesis of PD is not clear. According to the results of 5-hmC-sequencing studies, 5-hmC was associated with gene-rich regions in the genomes related to cell cycle, especially gene-cyclin-dependent kinase inhibitor 2A (Cdkn2A). Downregulation of TET2 expression could significantly rescue MPP+-stimulated SH-SY5Y cell damage and cell cycle arrest. Meanwhile, knockdown of Tet2 expression in the substantia nigra pars compacta of MPTP-induced PD mice resulted in attenuated MPTP-induced motor deficits and dopaminergic neuronal injury via p16 suppression. In this study, we demonstrated a critical function of TET2 in PD development via the CDKN2A activity-dependent epigenetic pathway, suggesting a potential new strategy for epigenetic therapy.

Double dissociation of inhibitory effects between the hippocampal TET1 and TET3 in the acquisition of morphine self-administration in rats.

The development of opioid addiction involves DNA methylation. Accordingly, the DNA demethylation, induced by ten-eleven translocation (Tet) enzymes, may represent a novel approach to prevent opioid addiction. The present study examined the role of TET1 and TET3 in the development of morphine-seeking behavior in rats. We showed that 1 day of morphine self-administration (SA) training upregulated TET3 but not TET1 expression in the hippocampal CA1. With 7 days of morphine SA training, the expression of TET3 in the CA1 returned to the baseline level, while the TET1 expression was downregulated. No change of TET1 and TET3 in the nucleus accumbens shell was observed in morphine SA trained rats, or in the yoked morphine rats, or in rats trained for saccharin SA. Furthermore, we found that knocking down TET3 expression in the CA1 accelerated the acquisition of morphine SA, while overexpression of the catalytic domain of TET1 in the CA1 attenuated the acquisition. Together, these findings suggest that TET1 and TET3 in the CA1 are important epigenetic modulators involved in the morphine-seeking behavior and provide a new strategy in the treatment of opioid addiction.

Helicobacter pylori infection leads to KLF4 inactivation in gastric cancer through a TET1-mediated DNA methylation mechanism.

Krüppel‐like factor 4 (KLF4) has a tumor suppressor role in the progression of gastric cancer (GC), and inhibition or loss of KLF4 expression was identified in GC. The aim of this study was to explore the new molecular mechanism of KLF4 inactivation in gastric cancer. Herein, we report that Helicobacter pylori infection or Cag pathogenicity island protein A (CagA) gene transduction resulted in KLF4 expression downregulation and promoted gastric epithelial cell and gastric cancel cell proliferation, migration, and colony formation. Mechanistically, we found that CagA gene transduction led to DNA methylation of the KLF4 promoter, an effect that was relevant to the significant downregulation of TET1 expression. Causally, knockdown of TET1 expression decreased KLF4 expression, whereas overexpression of TET1 had the opposite effect. Clinically, we found that KLF4 expression and the 5‐hmC levels were lower in GC cells with H pylori infection than in GC cells without H pylori infection. Thus, our study not only sheds new light on how H pylori infection promotes the progression of GC but also elucidates a novel mechanism of KLF4 inactivation in GC pathogenesis. During pathogenesis, an alteration in the H pylori/CagA‐TET1‐KLF4 signaling pathway plays a critical role, suggesting that this pathway may be a prospective target for gastric carcinoma intervention and therapy.

Epigenetic induction of tumor stemness via the lipopolysaccharide-TET3-HOXB2 signaling axis in esophageal squamous cell carcinoma

BackgroundEsophageal squamous cell cancer (ESCC) is one kind of frequent digestive tumor. The inflammatory environment plays an important role in the tumorigenesis and development of ESCC. Cancer stem cells are a small group of tumor cells with stem cell characteristics, which can potentially hinder the tumor management and treatment.MethodsELISA was performed to detect the lipopolysaccharide concentration in cancer tissues. qPCR, Western blot, FACS, Immunohistochemistry, Immunofluorescence and Dot blot were applied to detect target genes expression. CCK-8, Colony-formation, Transwell, Sphere and Xenograft were conducted to investigate the function of cells, influenced by risk factors. The survival curve was drawn with the Kaplan-Meier product limit estimator. Nano-hmC-Seal-seq was utilized to detect the downstream target of TET3. ChIP-qPCR was adopted to demonstrate the transcriptional regulation of stem cell-associated genes by HOXB2.ResultsLipopolysaccharide concentration was significantly up-regulated in ESCC. High concentration of lipopolysaccharide stimulation induced the stemness of ESCC cells. TET3 expression was elevated with lipopolysaccharide stimulation via p38/ERK-MAPK pathway in ESCC and negatively correlated with patients’ survival. TET3 induced the stemness of ESCC cells. Nano-hmC-Seal-seq showed that TET3 overexpression led to a significant increase in 5hmC levels of HOXB2 gene region, which was thus identified as the downstream target of TET3. The binding of HOXB2 to NANOG and cMYC was verified by ChIP-qPCR.ConclusionsLipopolysaccharide served as a tumor promotor in ESCC by inducing cancer cell stemness through the activation of a LPS-TET3-HOXB2 signaling axis, which might provide a novel therapeutic strategy for ESCC.5MHraejBx-4JJdn73ZdPoLVideo

Role of TET Dioxygenases and DNA Hydroxymethylation in Bisphenols-Stimulated Proliferation of Breast Cancer Cells.

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 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 breast cancer cells treated with BPA or its replacement, BPS, accompanied by an 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 (dimerized and phosphorylated), which was also induced by BPA/BPS binding. Instead of a direct interaction between TET2 and , data of our Co-IP, BSP, and ChIP-qPCR experiments indicated that the activated 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 activation–DNMT-TET2-DNA hydroxymethylation in 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