Expression Of Tet Genes Research Articles

Integrated mitoepigenetic signalling mechanisms associated with airborne particulate matter exposure: A cross-sectional pilot study

Airborne particulate matter (PM) is known to be associated with various chronic human ailments, however, precise underlying molecular mechanisms remains elusive. Based on the place of residence and amount of PM exposure, the study groups were divided into high-risk group and low-risk group. Different parameters related to oxidative DNA damage and repair, integrated stress response, mito-nuclear crosstalk, mitochondrial biogenesis, functioning and epigenetic machinery were assessed. The results of our pilot study showed that in comparison to the low-risk group, the levels of mitochondrial stress and oxidative DNA damage were significantly higher in the high-risk group. In addition, the upregulated expression levels of OMA1, DELE1, and HRI genes in the high-risk group confirmed the onset of an integrated stress response. These disturbances in the mitochondrial genome were further found to be associated with the vital processes of mitochondrial biogenesis. The high-risk group reported significant alterations in the mtDNA copy number and expression of mitochondrial fission and fusion genes. Importantly, we also observed higher expression levels of mitochondrial transcription factor TFAM, a key regulator of mtDNA replication and transcription in the high-risk group. Moreover, these changes were accompanied by the disturbances in the expression of TET genes and levels of mtDNA methylation. This impaired mtDNA methylation profile further correlated with the disturbed activity of electron chain complexes and expression of the mitochondria function genes in the high-risk group. Altogether, our observations provide vital molecular insights of the integrated mitoepigenetic signalling network in the airborne PM exposed individuals, hitherto unreported.

TET repression and increased DNMT activity synergistically induce aberrant DNA methylation.

Chronic inflammation is deeply involved in various human disorders, such as cancer, neurodegenerative disorders, and metabolic disorders. Induction of epigenetic alterations, especially aberrant DNA methylation, is one of the major mechanisms, but how it is induced is still unclear. Here, we found that expression of TET genes, methylation erasers, was downregulated in inflamed mouse and human tissues, and that this was caused by upregulation of TET-targeting miRNAs such as MIR20A, MIR26B, and MIR29C, likely due to activation of NF-κB signaling downstream of IL-1β and TNF-α. However, TET knockdown induced only mild aberrant methylation. Nitric oxide (NO), produced by NOS2, enhanced enzymatic activity of DNA methyltransferases (DNMTs), methylation writers, and NO exposure induced minimal aberrant methylation. In contrast, a combination of TET knockdown and NO exposure synergistically induced aberrant methylation, involving genomic regions not methylated by either alone. The results showed that a vicious combination of TET repression, due to NF-κB activation, and DNMT activation, due to NO production, is responsible for aberrant methylation induction in human tissues.

5-Azacytydine and resveratrol reverse senescence and ageing of adipose stem cells via modulation of mitochondrial dynamics and autophagy.

Obesity and endocrine disorders have become prevalent issues in the field of both human and veterinary medicine. Equine metabolic syndrome is a complex disorder involving alternation in metabolism and chronic systemic inflammation. It has been shown that unfavourable microenvironment of inflamed adipose tissue negatively affects adipose stem cell population (ASC) residing within, markedly limiting their therapeutic potential. ASCsEMS are characterized by increased senescence apoptosis, excessive accumulation of reactive oxygen species (ROS), mitochondria deterioration and “autophagic flux.” The aim of the present study was to evaluate whether treatment of ASCsEMS with a combination of 5‐azacytydine (AZA) and resveratrol (RES) would reverse aged phenotype of these cells. For this reason, we performed the following analyzes: molecular biology (RT‐PCR), microscopic (immunofluorescence, TEM) and flow cytometry (JC‐1, ROS, Ki67). We evaluated the mitochondrial status, dynamics and clearance as well as autophagic pathways. Furthermore, we investigated epigenetic alternations in treated cells by measuring the expression of TET genes and analysis of DNA methylation status. We have demonstrated that AZA/RES treatment of ASCsEMS is able to rejuvenate these cells by modulating mitochondrial dynamics, in particular by promoting mitochondrial fusion over fission. After AZA/RES treatment, ASCsEMS were characterized by increased proliferation rate, decreased apoptosis and senescence and lower ROS accumulation. Our findings offer a novel approach and potential targets for the beneficial effects of AZA/RES in ameliorating stem cell dysfunctions.

Pregnancy-Induced Physiologic Adaptation of the Abdominal Aorta Is Associated with Changes in Gene Expression and Genomic Methylation

Background/Aims: Ten-eleven translocation 2 (Tet2), a DNA demethylase enzyme, has been identified as a master epigenetic regulator of vascular smooth muscle cell plasticity. We hypothesized that pregnancy will induce significant adaptive changes in aortic biomechanics that correlate with the Tet family gene expression. Methods: Abdominal aortas from pregnant and nonpregnant mice were dissected and cannulated. Intraluminal pressure was adjusted using a pressure-servo system while using a video dimension analyzer to measure the lumen diameter. Quantitative polymerase chain reaction and immunoblot was used to analyze the expression of Tet genes. Global genomic methylation was assessed with the luminometric methylation assay. Results: Compared to the nonpregnant (NP, 706 ± 8 µm) control group, the aortic luminal diameter was significantly increased in both E18.5 (836 ± 14 µm) and PP30 (889 ± 16 µm) mice. Distensibility was reduced in E18.5 (90 ± 4%) mice and returned to NP values (108 ± 2%) in PP30 (108 ± 3%) mice. Tet2 transcription decreased at the beginning of pregnancy and subsequently increased in late gestation, inversely corresponding to changes in global methylation. Conclusion: Physiologic changes in the aorta were accompanied by changes in gene expression and genomic methylation, suggesting an epigenetic component to maternal vascular remodeling during pregnancy.

TET2 facilitates PPARγ agonist–mediated gene regulation and insulin sensitization in adipocytes

Emerging evidence indicates that epigenetic mechanisms like DNA methylation directly contribute to metabolic regulation. For example, we previously demonstrated that de novo DNA methyltransferase Dnmt3a plays a causal role in the development of adipocyte insulin resistance. Recent studies suggest that DNA demethylation plays an important role in the developmental process of adipocytes. However, little is known about whether DNA demethylase ten-eleven translocation (TET) proteins regulate the metabolic functions of adipocytes. MethodsThe expression of Tet genes was assessed in the fractionated adipocytes of chow- and high fat diet–fed C57/Bl6 mice using qPCR and western blotting. The effect of Tet2 gain- or loss-of-function in fully mature 3T3-L1 adipocytes in the presence/absence of Rosiglitazone (Rosi) and TNF-α on insulin sensitivity was using the insulin-stimulated glucose uptake and insulin signaling assays. Gene expression and DNA methylation analyses of PPARγ target genes was performed in the same setting. In addition, PPARγ reporter assays, co-immunoprecipitation assays, PPARγ ChIP-PCR analyses were performed. ResultsWe found that adipose expression of TET2, alone among its family members, was significantly reduced in diet-induced insulin resistance. TET2 gain-of-function was sufficient to promote insulin sensitivity while loss-of-function was necessary to facilitate insulin sensitization in response to the PPARγ agonist Rosiglitazone (Rosi) in cultured adipocytes. Consistent with this, TET2 was required for Rosi-dependent gene activation of certain PPARγ targets accompanied by changes in DNA demethylation at the promoter regions. Furthermore, TET2 was necessary to sustain PPARγ binding to target loci upon activation with Rosi via physical interaction with PPARγ. ConclusionsOur data demonstrate that TET2 works as an epigenetic regulator of Rosi-mediated insulin sensitization and transcriptional regulation in adipocytes.

Differential expression of ten-eleven translocation genes in endometrial cancers.

Ten-eleven translocation proteins are α-ketoglutarate-dependent dioxygenases involved in the conversion of 5-methylcytosines (5-mC) to 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine, and 5-carboxylcytosine that play a significant role in DNA demethylation. Deregulation of TET genes expression and changes in the level of 5-hmC are thought to be associated with the onset and progression of several types of cancer, but there are no such data related to endometrial cancer. The aim of the work was to investigate the messenger RNA expression levels of TET1, TET2, and TET3 in relation to clinicopathological characteristics of endometrial cancer as well as the correlation between expression of TET genes and the level of 5-hmC/5-mC. The prognostic significance of TETs expression for overall survival was established. We found that TET1 and TET2 messenger RNA expression was lower and TET3 was higher in cancers compared to normal tissues. Positive correlation between 5-hmC and the relative expression of TET1 and TET2 was found, but no correlation was observed in the case of TET3. Decreased expression of TET1 and TET2 was significantly associated with increased lymph node metastasis and International Federation of Gynecology and Obstetrics stage. Kaplan-Meier analysis indicated that low TET1 expression predicted poor overall survival (p = 0.038). Multivariate analysis identified the TET1 expression in endometrial cancer as an independent prognostic factor. Our results suggest that decreased expression of TET1 correlates with tumor progression and may serve as a potential prognostic biomarker in endometrial cancer.

Ten-Eleven Translocation Genes are Downregulated in Endometriosis.

Our previous whole genome expression analysis of endometriomas suggested dysregulation of the ten-eleven translocation genes (TET1, TET2, and TET3), involved in converting 5- methylcytosine to 5-hydroxymethylcytosine (5-hmC). The objective of this study was to validate the expression of TET genes in ectopic and eutopic endometrium and in primary cultures of human endometrial stromal fibroblasts (HESF) during in vitro decidualization and to quantify 5-hmC levels in patients with endometriosis. Blood, eutopic endometrium, and endometriotic tissues were collected at time of gynecologic surgery. HESF cultures were created from eutopic endometrium of women without (HESF-CONTROL) and with endometriosis (HESF-ENDO) and underwent in vitro decidualization. Genomic DNA from blood and tissues underwent quantification of the absolute amount of 5-hmC using ELISA. The expression of TET1, TET2, and TET3 was decreased in endometriosis compared to non-endometriosis control eutopic endometrium. Surprisingly, the global amount of 5-hmC was higher in ectopic endometrium than control eutopic endometrium, while genomic DNA from blood of women with endometriosis contained statistically significantly less 5-hmC than women without endometriosis. Expression of TET1, TET2, and TET3 was decreased in non-decidualized HESFENDO. Upon in vitro decidualization, control HESF showed decreased expression of TET3, while decidualized HESF-ENDO showed no statistically significant change in expression of TET1, TET2, or TET3. These results indicate that the TET genes are downregulated in ectopic endometrium and in HESF-ENDO, and suggest for the first time that TET genes play a role in endometriosis. High global amounts of 5-hmC in endometriotic tissues suggest unique epigenetic regulation in these tissues.

Cell Fate by SIRT6 and TETs

Cell Fate by SIRT6 and TETs

210 DNA METHYLATION AND HYDROXYMETHYLATION ANALYSIS IN A MODEL OF OOCYTE DIFFERENTIAL DEVELOPMENTAL COMPETENCE IN SHEEP

DNA methylation is an important epigenetic mark that plays a role in gene regulation by the addition of a methyl group to CpG islands in the DNA. Despite being relatively stable in somatic cells, DNA methylation is subject to reprogramming during embryo development and gametogenesis. The aim of this work was to evaluate different aspects of DNA methylation in relation to oocyte quality in the ovine species. A model of differential developmental competence consisting in ovine oocytes and in vitro produced (IVP) blastocysts derived from adult (AD) and prepubertal (PR) donors, was used. The methylation was analysed in terms of: expression of a panel of genes involved in DNA methylation [DNA methyltransferases (DNMTs)] and demethylation [ten-eleven translocation dioxygenases (TET)] in oocytes and blastocysts; global methylation and hydroxymethylation by direct immunofluorescence; locus-specific methylation of 2 imprinted genes by pyrosequencing. Gene relative quantification was performed by RNA reverse transcription followed by real-time PCR. Pools of 10 immature (GV) and in vitro-matured (MII) oocytes and (IVP) blastocysts derived from AD and PR donors (4 replicates per class) were analysed. Lower expression of TET1, TET2, and TET3 was observed in PR GV oocytes (ANOVA; P < 0.05), while no significant differences were found for the enzymes involved in methylation (DNMT1, DNMT3A, DNMT3B; ANOVA; P > 0.05). The levels of all the genes studied showed no significant differences in embryos at blastocyst stage (ANOVA; P > 0.05). Methylation and hydroxymethylation immunostaining were performed in GV and MII oocytes using anti-5-methylcytosine mouse mAb and 5-hydroxymethylcytosine rabbit pAB. High levels of DNA methylation were observed in both AD and PR GV and MII oocytes, while hydroxymethylation immunopositivity was scattered evident throughout the gamete chromatin. Pyrosequencing of bisulfite converted DNA was used to determine the methylation status within differentially methylated regions (DMR) of maternally imprinted H19 (CTCF binding site IV; 11 CpG sites) and paternally imprinted IGF2R (17CpG sites within intron 2). No differences were observed between classes of oocytes for each gene (pools of 40 oocytes per replicate, 3 replicates per class; ANOVA; P > 0.05). Our work shows no differences in the expression of the enzymes involved in methylation, in accordance with the results of global and locus specific methylation analysis. Conversely, we observed lower expression of the TET genes in PR GV oocytes (ANOVA; P > 0.05). TET1, TET2, and TET3, whose expression has never been studied in ovine, generate 5-hydroxymethlcytosine (5hmC) by oxidation of 5-methylcytosine (5mC), and are involved in active DNA demethylation during early embryo development. Our observation of lower expression of the TET genes in lower competence PR GV oocytes suggests that epigenetic mechanisms may affect oocyte quality and paves the way to better understand methylation dynamics during sheep pre-implantation development.

Ascorbate Induces Ten-Eleven Translocation (Tet) Methylcytosine Dioxygenase-mediated Generation of 5-Hydroxymethylcytosine

Tet methylcytosine dioxygenase converts 5-mC to 5-hmC in DNA. Ascorbate significantly and specifically enhances Tet-mediated generation of 5-hmC. Our findings suggest that ascorbate enhances 5-hmC generation, most likely by acting as a co-factor for Tet methylcytosine dioxygenase to generate 5-hmC. The availability of ascorbate could have significant consequences for health and diseases by modulating the epigenetic control of genome activity. Ascorbate (vitamin C) is best known for its role in scurvy, in which the hydroxylation of collagen catalyzed by dioxygenases is incomplete due to ascorbate deficiency. Here, we report a novel function of ascorbate in the hydroxylation of 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC) in DNA catalyzed by Tet (ten-eleven translocation) methylcytosine dioxygenase. The content of 5-hmC is extremely low in mouse embryonic fibroblasts cultured in ascorbate-free medium. Additions of ascorbate dose- and time-dependently enhance the generation of 5-hmC, without any effects on the expression of Tet genes. Treatment with another reducer glutathione (GSH) does not change the level of 5-hmC. Further, blocking ascorbate entry into cells by phloretin and knocking down Tet (Tet1, Tet2, and Tet3) expression by short interference RNAs (siRNA) significantly inhibit the effect of ascorbate on 5-hmC. These results suggest that ascorbate enhances 5-hmC generation, most likely by acting as a co-factor for Tet methylcytosine dioxygenase to hydroxylate 5-mC. Thus, we have uncovered a novel role for ascorbate in modulating the epigenetic control of genome activity.

Sensitive enzymatic quantification of 5-hydroxymethylcytosine in genomic DNA

The recent discovery of genomic 5-hydroxymethylcytosine (hmC) and mutations affecting the respective Tet hydroxylases in leukemia raises fundamental questions about this epigenetic modification. We present a sensitive method for fast quantification of genomic hmC based on specific transfer of radiolabeled glucose to hmC by a purified glucosyltransferase. We determined hmC levels in various adult tissues and differentiating embryonic stem cells and show a correlation with differential expression of tet genes.

Aminoglycoside uptake increased by tet gene expression.

The expression of extrachromosomal tet genes not only confers tetracycline resistance but also increases the susceptibilities of gram-negative bacteria to commonly used aminoglycoside antibiotics. We investigated the possibility that tet expression increases aminoglycoside susceptibility by increasing bacterial uptake of aminoglycoside. Studies of [3H]gentamicin uptake in paired sets of Escherichia coli HB101 and Salmonella typhimurium LT2 expressing and not expressing tet showed that tet expression accelerates energy-dependent [3H]gentamicin uptake. Increased [3H]gentamicin uptake was accompanied by decreased bacterial protein synthesis and bacterial growth. Increased aminoglycoside uptake occurred whether tet expression was constitutive or induced, whether the tet gene was class B or C, and whether the tet gene was plasmid borne or integrated into the bacterial chromosome. tet expression produced no measurable change in membrane potential, suggesting that tet expression increases aminoglycoside uptake either by increasing the availability of specific carriers or by lowering the minimum membrane potential that is necessary for uptake.

An N-terminal domain of the tetracycline resistance protein increases susceptibility to aminoglycosides and complements potassium uptake defects in Escherichia coli.

Expression of extrachromosomal tet genes increased the susceptibility of gram-negative bacteria to specific aminoglycoside antibiotics. The magnitude of the increase in susceptibility was dependent on the amount and the class of the tet gene product (designated Tet) and the bacterial species in which the tet gene was expressed. Truncated Tet proteins that contained more than the first 33, but not more than the first 97, N-terminal amino acids of Tet also increased the susceptibility to aminoglycosides and complemented the potassium uptake defects in Escherichia coli. The primary structure of this N-terminal Tet fragment has the hydropathic characteristics of a multimeric, transmembrane structure and is highly conserved in three different classes of Tet proteins.