Ten-eleven Translocation Methylcytosine Research Articles

DNA Hydroxymethylation by Ten-eleven Translocation Methylcytosine Dioxygenase 1 and 3 Regulates Nociceptive Sensitization in a Chronic Inflammatory Pain Model

Ten-eleven translocation methylcytosine dioxygenase converts 5-methylcytosine in DNA to 5-hydroxymethylcytosine, which plays an important role in gene transcription. Although 5-hydroxymethylcytosine is enriched in mammalian neurons, its regulatory function in nociceptive information processing is unknown. The global levels of 5-hydroxymethylcytosine and ten-eleven translocation methylcytosine dioxygenase were measured in spinal cords in mice treated with complete Freund's adjuvant. Immunoblotting, immunohistochemistry, and behavioral tests were used to explore the downstream ten-eleven translocation methylcytosine dioxygenase-dependent signaling pathway. Complete Freund's adjuvant-induced nociception increased the mean levels (± SD) of spinal 5-hydroxymethylcytosine (178 ± 34 vs. 100 ± 21; P = 0.0019), ten-eleven translocation methylcytosine dioxygenase-1 (0.52 ± 0.11 vs. 0.36 ± 0.064; P = 0.0088), and ten-eleven translocation methylcytosine dioxygenase-3 (0.61 ± 0.13 vs. 0.39 ± 0.08; P = 0.0083) compared with levels in control mice (n = 6/group). The knockdown of ten-eleven translocation methylcytosine dioxygenase-1 or ten-eleven translocation methylcytosine dioxygenase-3 alleviated thermal hyperalgesia and mechanical allodynia, whereas overexpression cytosinethem in naïve mice (n = 6/group). Down-regulation of spinal ten-eleven translocation methylcytosine dioxygenase-1 and ten-eleven translocation methylcytosine dioxygenase-3 also reversed the increases in Fos expression (123 ± 26 vs. 294 ± 6; P = 0.0031; and 140 ± 21 vs. 294 ± 60; P = 0.0043, respectively; n = 6/group), 5-hydroxymethylcytosine levels in the Stat3 promoter (75 ± 16.1 vs. 156 ± 28.9; P = 0.0043; and 91 ± 19.1 vs. 156 ± 28.9; P = 0.0066, respectively; n = 5/group), and consequent Stat3 expression (93 ± 19.6 vs. 137 ± 27.5; P = 0.035; and 72 ± 15.2 vs. 137 ± 27.5; P = 0.0028, respectively; n = 5/group) in complete Freund's adjuvant-treated mice. This study reveals a novel epigenetic mechanism for ten-eleven translocation methylcytosine dioxygenase-1 and ten-eleven translocation methylcytosine dioxygenase-3 in the modulation of spinal nociceptive information via targeting of Stat3.

Abstract 4460: TET1 over expression contributes to lung cancer malignancy

Abstract Epigenetic alterations are a leading cause of lung carcinogenesis and malignancy. While the role of enzymes catalyzing cytosine-guanine (CpG) island methylation and chromatin modifications has been extensively studied, the role of the recently discovered demethylating enzymes represented by ten-eleven translocation methylcytosine dioxygenases (TETs) remains unknown. The goal of this study was to investigate the expression and functional significance of the TET1 gene in non-small cell lung cancer (NSCLC). QRT-PCR revealed 2- to 30-fold over expression of TET1 in 52% of squamous cell carcinoma (SCC) and 40% of adenocarcinoma (AdC) tumors compared to paired normal lung tissue. This result was validated using The Cancer Genome Atlas (TCGA) database with TET1 over expressed in 72% of SCCs and 53% of AdCs. Elevated levels of TET1 transcript were associated with increased protein levels in NSCLC cell lines compared to normal human bronchial epithelial cells (HBECs). A reporter assay analysis defined the promoter region critical for elevated transcription of the TET1 gene using lung cancer cell lines, and studies to identify the transcription factors underlying the increase in expression are underway. Transient knock down of TET1 expression in NSCLC cell lines caused global changes in the transcriptome, reduced cell proliferation, colony formation, and inhibited growth of tumor xenografts in nude mice. Cellular senescence mediated by p21 signaling and associated with β-galactosidase activation was identified as one mechanism underlying growth inhibition. This study suggests that TET1 may function as an oncogene contributing to the lung cancer malignant phenotype and thus, could serve as a potential new target for treatment. (Supported by R01 CA183296) Citation Format: Piotr Teodor Filipczak, Shuguang Leng, Carmen S. Tellez, Steven A. Belinsky. TET1 over expression contributes to lung cancer malignancy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4460. doi:10.1158/1538-7445.AM2017-4460

Molecular cloning of chicken TET family genes and role of chicken TET1 in erythropoiesis

Ten-eleven translocation (TET) methylcytosine dioxygenase has potential as an active eraser to regulate the genomic DNA methylation status. We herein cloned chicken TET (cTET) family genes, and confirmed their functions. Quantitative reverse-transcription PCR showed that cTET1 was strongly expressed in erythrocytes throughout development. This cTET1 expression pattern, together with the results of methylated or hydroxymethylated DNA immunoprecipitation, suggests that cTET1 contributes to demethylation around the promoter region of the definitive-type β-globin gene βΑ in erythroid cells. The knockdown of cTET1 in T2ECs chicken erythroid progenitor cells suppressed the induction of βΑ expression under differentiation conditions. These results suggest that cTET1 plays an important role in erythroid cell differentiation.

Ten-Eleven Translocation-2 (Tet2) Is Involved in Myogenic Differentiation of Skeletal Myoblast Cells in Vitro

Muscle cell differentiation is a complex process that is principally governed by related myogenic regulatory factors (MRFs). DNA methylation is considered to play an important role on the expression of MRF genes and on muscle cell differentiation. However, the roles of enzymes specifically in myogenesis are not fully understood. Here, we demonstrate that Tet2, a ten-eleven translocation (Tet) methylcytosine dioxygenase, exerts a role during skeletal myoblast differentiation. By using an immunostaining method, we found that the levels of 5-hydroxymethylcytosine (5-hmC) were much higher in differentiated myotubes than in undifferentiated C2C12 myoblasts. Both Tet1 and Tet2 expression were upregulated after differentiation induction of C2C12 myoblasts. Knockdown of Tet2, but not Tet1, significantly reduced the expression of myogenin as well as Myf6 and myomaker, and impaired myoblast differentiation. DNA demethylation of myogenin and myomaker promoters was negatively influenced by Tet2 knockdown as detected by bisulfite sequencing analysis. Furthermore, although vitamin C could promote genomic 5hmC generation, myogenic gene expression and myoblast differentiation, its effect was significantly attenuated by Tet2 knockdown. Taken together, these results indicate that Tet2 is involved in myoblast differentiation through promoting DNA demethylation and myogenic gene expression.

Epigenetic targeting of clear cell renal cell carcinoma (ccRCC) with ascorbic acid (AA) via upregulation of Ten-Eleven Translocation (TET) methylcytosine dioxygenase activity.

479 Background: We and others have previously shown that the ccRCC epigenome is characterized by widespread DNA hypermethylation (CCR 2014, Nature 2014). Various important tumor suppressor genes (TSGs), such as SMAD7 (inhibitor of oncogenic TGF-β signaling), are under-expressed due to aberrantly methylated promoters or enhancers. We hypothesized that the hypermethylation in ccRCC could be due to low activity of the TET enzymes, which convert methylcytosine (5-mc) to hydroxymethylcytosine (5-hmc). Loss of function of TET enzymes can occur with an inactivating mutation (TET-2 is mutated in about 6% of ccRCC (Science Signaling 2013) or hypoactivity of normal TET enzymes, through inhibition by metabolic intermediates. AA is an essential co-factor for TET enzymes (JACS 2015). We hypothesized that high dose AA treatment of ccRCC could potentially increase the functional activity of TET enzymes leading to demethylation of the RCC genome, and enhance expression of TSGs. Methods: In vitro TET activity was performed on ccRCC cell line 769P with increasing doses of AA (L-AA). Genome wide quantitative 5-mc and 5-hmc was evaluated using mass spectrometry. SMAD7 expression was determined using qRT-PCR. Proliferation assay (MTT) was performed with AA in combination with pazopanib. Cell cycle and apoptosis assays were performed. Results: AA, at doses achieved only by the intravenous route (1-10mM), increased TET activity in ccRCC cell line 769P. Genome wide 5-mc was significantly reduced and 5-hmc was increased, correlating with increase in TET activity. SMAD7 expression was increased with AA treatment. High dose AA led to proliferation inhibition with a cell cycle arrest in the G1 phase, and had a synergistic effect with pazopanib. Since AA leads to the generation of H2O2 in-vitro, catalase was used as control. This did not reverse the effect of AA on epigenetic changes; and proliferation inhibition was seen despite catalase control. Conclusions: High dose AA causes TET mediated DNA demethylation of the hypermethylated RCC genome, resulting in the re-expression of TSGs, and proliferation inhibition. Sequencing and xenograft studies are underway.

Use of Chemicals to Inhibit DNA Replication, Transcription, and Protein Synthesis to Study Zygotic Genome Activation.

Maternal-to-zygotic transition is an event that developmental control of early embryos is switched from oocyte-derived factors to the zygotic genome. Ability to inhibit DNA replication, transcription, and translation is an important tool in studying events, such as zygotic genome activation, during embyogenesis. Here, we describe approaches to block DNA replication, transcription, and translation using chemical inhibitors. Then we also demonstrate how the transcript level of a maternally inherited gene, ten-eleven translocation methylcytosine dioxygenase 3, responses to the chemical treatments.

Tumor necrosis factor-α decreases EC-SOD expression through DNA methylation.

Extracellular-superoxide dismutase (EC-SOD) is a secreted antioxidative enzyme, and its presence in vascular walls may play an important role in protecting the vascular system against oxidative stress. EC-SOD expression in cultured cell lines is regulated by various cytokines including tumor necrosis factor-α (TNF-α). TNF-α is a major mediator of pathophysiological conditions and may induce or suppress the generation of various types of mediators. Epigenetics have been defined as mitotically heritable changes in gene expression that do not affect the DNA sequence, and include DNA methylation and histone modifications. The results of the present study demonstrated that TNF-α significantly decreased EC-SOD level in fibroblasts with an accompanying increase in methylated DNA. In DNA methylation and demethylation, cytosine is methylated to 5-methylcytosine (5mC) by DNA methyltransferase (DNMT), and 5mC is then converted to 5-hydroxymethylcytosine (5hmC) and cytosine in a stepwise manner by ten-eleven translocation methylcytosine dioxygenases (TETs). However, DNMT did not participate in TNF-α-induced DNA methylation within the EC-SOD promoter region. On the other hand, TNF-α significantly suppressed TET1 expression and EC-SOD mRNA levels were decreased by the silencing of TET1 in fibroblasts. These results demonstrate that the down-regulation of EC-SOD by TNF-α is regulated by DNA methylation through reductions in TET1.

Hypoxia-induced DNA hypermethylation: another reason to normalize tumor vessels

As solid tumors grow, regions within the tumors often experience a deficit in oxygen, known as hypoxia. Hypoxia results from an imbalance in oxygen supply and demand. There is an increase in oxygen demand as cells proliferate or get recruited to build the tumor biomass, while oxygen supply becomes limited as the blood vessels within the growing tumor are absent and/or become abnormal in their structure and function (1,2). In addition to causing cell death, hypoxia can also promote tumor progression, immunosuppression, and treatment resistance. Indeed, tumor hypoxia is associated with poor patient outcome (3). This somewhat counter-intuitive relationship is, in part, due to the ability of tumors to adapt to and evolve in oxygen- and nutrient-deficient microenvironments. A recent report by Thienpont et al . demonstrated that one such mechanism may be through epigenetic regulation of gene expression by the ten-eleven translocation (TET) methylcytosine dioxygenases (4). This exciting finding provides another rationale to normalize the tumor vasculature to alleviate hypoxia and its consequences.

Genetic modes of epigenetic modification and its research progress

It is known that genomic information is not only contained in the DNA sequence, but also in varieties of genetic effects, namely epigenetics. Epigenetics is defined as stable inheritance without changes in DNA sequence, which mainly focuses on the modification regulations, including DNA methylation, histone modification, non-coding RNA, X chromatin inactivation, gene imprinting, chromatin remodeling and so on. In the past few decades, epigenetics has made a dramatic advance, and quantities of mechanisms underlie the epigenetic phenomena have been discovered. Among these, DNA methylation and histone modifications are two most important aspects in epigenetic phenomena. The major DNA methylation and demethylation happen on cytosines of the CpG dinucleotide in mammals. In this process, DNA methyltransferase (DNMT) family proteins and ten-eleven translocation methylcytosine dioxygenase (TET) family proteins cooperate and achieve the both methylation and demethylation progress. Several covalent modifications of histones can alter the nucleosome conformation by changing the electrostatic charge of it, which include histone methylation, acetylation, phosphorylation and ubiquitination. In histone modification procedure, a set of enzymes are involved, Polycomb group (PcG) and Trithorax group (TrxG) functioning in histone methylation, for instance. These modifications fit together and lead to distinct state of chromatin accessibility, resulting in either gene expression or repression. Long-range chromatin interactions organize chromatin into transcriptionally permissive or prohibitive, thereby regulating gene expression. Non-coding RNAs also play critical roles in gene expression process through interacts directly with DNA, RNA, proteins. The epigenetic mechanisms mentioned above work cooperatively in gene expression regulation, which is essential for normal development and survival. Moreover, the epigenetic information can be inherited from parents to offsprings. Previous studies have shed light on the mechanisms underlying the epigenetic inheritance, but many questions remain unanswered. For example, DNA methylation and histone modifications can be passed through a series of enzymatic reactions with the join of other biological molecules. However, in what way does the machinery conduct the epigenetic information passage between generations is not well understood. In addition, the environment can certainly influence gene expression in an epigenetic manner. Particular environmental factor may interact with the regulators in epigenetic modification and consequently change the phenotype. Recent studies showed abnormal epigenetic states commonly exist in diseases, which imply a correlation between epigenetic modifications and diseases. Furthermore, mutations in epigenetic regulators always cause dysregulation of epigenetic modifications and then diseases. Thus, those epigenetic regulators can be potential targets in treatment of diseases. In this review, we focused on the mechanisms of epigenetic inheritance reported in recent years. Meanwhile, we proposed some questions that researchers met during their work and drew a blueprint for the study in the future.

Epigenetic upregulation of ARL4C, due to DNA hypomethylation in the 3'-untranslated region, promotes tumorigenesis of lung squamous cell carcinoma.

ADP-ribosylation factor (ARF)-like 4c (ARL4C) expression, induced by a combination of Wnt/β-catenin and EGF/Ras signaling, has been demonstrated to form epithelial morphogenesis. ARL4C overexpression, due to Wnt/β-catenin and EGF/Ras signaling alterations, was involved in tumorigenesis. It was also reported that ARL4C expression correlates with DNA hypomethylation in the 3’-untranslated region (UTR) of ARL4C gene during lymphogenesis. The current study was conducted to investigate the expression and functions of ARL4C due to DNA hypomethylation in lung and tongue cancers. Immunohistochemical analyses of tissue specimens obtained from lung and tongue squamous cell carcinoma (SCC) patients revealed that ARL4C is not observed in non-tumor regions, but is strongly expressed at high frequencies in tumor lesions. Although inhibition of Wnt/β-catenin or Ras/MAP kinase signaling did not decrease ARL4C expression in NCI-H520 lung SCC cells, ARL4C DNA was clearly hypomethylated in the 3’-UTR. Ten-eleven translocation methylcytosine dioxygenase (TET) enzyme, which mediates DNA demethylation, was highly expressed in NCI-H520 cells. Knockout of TET family proteins (TET1-3) in NCI-H520 cells reduced 5-hydroxymethylcytosine (5hmC) levels and promoted DNA methylation in the 3’-UTR, leading to the decrease in ARL4C expression and ARL4C-mediated cellular migration. In tumor lesions of ARL4C-positive lung SCC, 5hmC was frequently detected and DNA methylation in the 3’-UTR of ARL4C gene was lower than in non-tumor regions, which were consistent with the Cancer Genome Atlas dataset. These results suggest that ARL4C is expressed due to hypomethylation in the 3’-UTR for certain types of cancers and that ARL4C methylation status is involved in cancer cell function.

Abstract 4440: Diffuse intrinsic pontine glioma epigenetic marks change depending on cell culture conditions: Implications for investigating epigenetically directed therapy

Abstract Methylation of cytosine is viewed as repressive of transcription. 5-methylcytosine (5mC) can be removed from DNA by activity of ten-eleven translocation (TET) methylcytosine dioxygenase. The first step in erasing the repressive 5mC mark is conversion to 5-hydroxymethylcytosine (5hmC) by TET. Subsequent steps lead to replacement of 5hmC by cytosine, providing a mechanism by which 5mC-mediated repression can be erased. We have previously identified 5hmC and 5mC as being imbalanced in primary tumor tissue samples from the universally fatal pediatric malignancy diffuse intrinsic pontine glioma (DIPG) compared normal brain and glioblastoma (GBM). The aberration in DNA methylation along with known aberrant histone methylation in DIPGs, likely creates an active transcriptional/translational state and potentiates tumor growth. We hypothesized that the elevation of 5hmC in DIPG is secondary to increased TET activity. We found two different epigenetic signatures for DIPG cells depending on their culture conditions. When cultured in serum-free media as neurospheres, to our surprise, we found cells had low 5hmC, in contrast to what we observed in primary tumor and in orthotopic xenograft tumors. We hypothesized that 5hmC status may vary depending on differentiation state, and found that when DIPG cells were differentiated on matrigel for 14 days in the presence of serum, 5hmC levels increased and were similar to that observed in primary tissue and xenograft tumors. Differentiation was confirmed by upregulation of GFAP (glial marker) and MAP2 (neuronal marker) expression and the elaboration of long processes. We also found that DIPG cells in the serum/matrigel/differentiated state had increased TET1 and TET2 mRNA expression relative to undifferentiated neurospheres (2.6 and 2.1 fold increase respectively for TET1 and TET2 mRNA expression). As a control for adherent versus nonadherent growth, we also plated DIPG cells on laminin in EGF/FGF media, and we found that the serum-differentiated cells upregulated TET1 and TET2 mRNA 6.7 and 3.3 fold respectively, compared to laminin-plated cells, providing further support that differentiation affects epigenetic marks in DIPG. In contrast to DIPG, GBM cells did not exhibit similar changes in TET mRNA expression when differentiated, emphasizing the uniqueness of the DIPG epigenetic signature. Our data shows that neurosphere cell culture models of DIPG may not be true epigenetic representations of DIPG tumor in vivo and the predicted response to potential therapeutic options may differ based on the epigenetic state used for preclinical testing. Citation Format: Sama Ahsan, Michael Haffner, Charles Eberhart, Eric Raabe. Diffuse intrinsic pontine glioma epigenetic marks change depending on cell culture conditions: Implications for investigating epigenetically directed therapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4440.

Hydroquinone-induced FOXP3-ADAM17-Lyn-Akt-p21 signaling axis promotes malignant progression of human leukemia U937 cells

Hydroquinone (1,4-benzenediol; HQ), a major marrow metabolite of the leukemogen benzene, has been proven to evoke benzene-related hematological disorders and myelotoxicity in vitro and in vivo. The goal of the present study was to explore the role of FOXP3 in HQ-induced malignant progression of U937 human leukemia cells. U937 cells were treated with 5μM HQ for 24h, and the cells were re-suspended in serum-containing medium without HQ for 2days. The same procedure was repeated three times, and the resulting U937/HQ cells were maintained in cultured medium containing 5μM HQ. Proliferation and colony formation of U937/HQ cells were notably higher than those of U937 cells. Ten-eleven translocation methylcytosine dioxygenase-mediated demethylation of the Treg-specific demethylated region in FOXP3 gene resulted in higher FOXP3 expression in U937/HQ cells than in U937 cells. FOXP3-induced miR-183 expression reduced β-TrCP mRNA stability and suppressed β-TrCP-mediated Sp1 degradation, leading to up-regulation of Sp1 expression in U937/HQ cells. Sp1 up-regulation further increased ADAM17 and Lyn expression, and ADAM17 up-regulation stimulated Lyn activation in U937/HQ cells. Moreover, U937/HQ cells showed higher Lyn-mediated Akt activation and cytoplasmic p21 expression than U937 cells did. Abolishment of Akt activation decreased cytoplasmic p21 expression in U937/HQ cells. Suppression of FOXP3, ADAM17, and Lyn expression, as well as Akt inactivation, repressed proliferation and clonogenicity of U937/HQ cells. Together with the finding that cytoplasmic p21 shows anti-apoptotic and oncogenic activities in cancer cells, the present data suggest a role of FOXP3/ADAM17/Lyn/Akt/p21 signaling axis in HQ-induced hematological disorders.

TET2 Negatively Regulates Nestin Expression in Human Melanoma

Although melanoma is an aggressive cancer, the understanding of the virulence-conferring pathways involved remains incomplete. We have demonstrated that loss of ten-eleven translocation methylcytosine dioxygenase (TET2)-mediated 5-hydroxymethylcytosine (5-hmC) is an epigenetic driver of melanoma growth and a biomarker of clinical virulence. We also have determined that the intermediate filament protein nestin correlates with tumorigenic and invasive melanoma growth. Here we examine the relationships between these two biomarkers. Immunohistochemistry staining of nestin and 5-hmC in 53 clinically annotated primary and metastatic patient melanomas revealed a significant negative correlation. Restoration of 5-hmC, as assessed in a human melanoma cell line by introducing full-length TET2 and TET2-mutated constructs, decreased nestin gene and protein expression invitro. Genome-wide mapping using hydroxymethylated DNA immunoprecipitation sequencing disclosed significantly less 5-hmC binding in the 3' untranslated region of the nestin gene in melanoma compared to nevi, and 5-hmC binding in this region was significantly increased after TET2 overexpression in human melanoma cells invitro. Our findings provide evidence suggesting that nestin regulation is negatively controlled epigenetically by TET2 via 5-hmC binding at the 3' untranslated region of the nestin gene, providing one potential pathway for understanding melanoma growth characteristics. Studies are now indicated to further define the interplay between 5-hmC, nestin expression, and melanoma virulence.

Hydrogen Sulfide Maintains Mitochondrial DNA Replication via Demethylation of TFAM.

Hydrogen sulfide (H2S) exerts a wide range of actions in the body, especially in the modulation of mitochondrial functions. The normal replication of mitochondrial DNA (mtDNA) is critical for cellular energy metabolism and mitochondrial biogenesis. The aim of this study was to investigate whether H2S affects mtDNA replication and the underlying mechanisms. We hypothesize that H2S maintains mtDNA copy number via inhibition of Dnmt3a transcription and TFAM promoter methylation. Here, we demonstrated that deficiency of cystathionine gamma-lyase (CSE), a major H2S-producing enzyme, reduces mtDNA copy number and mitochondrial contents, and it inhibits the expressions of mitochondrial transcription factor A (TFAM) and mitochondrial marker genes in both smooth muscle cells and aorta tissues from mice. Supply of exogenous H2S stimulated mtDNA copy number and strengthened the expressions of TFAM and mitochondrial marker genes. TFAM knockdown diminished H2S-enhanced mtDNA copy number. In addition, CSE deficiency induced the expression of DNA methyltransferase 3a (Dnmt3a) and TFAM promoter DNA methylation, and H2S repressed Dnmt3a expression, resulting in TFAM promoter demethylation. We further found that H2S S-sulfhydrates transcription repressor interferon regulatory factor 1 (IRF-1) and enhances the binding of IRF-1 with Dnmt3a promoter after reduced Dnmt3a transcription. H2S had little effects on the expression of Dnmt1 and Dnmt3b as well as on ten-eleven translocation methylcytosine dioxygenase 1, 2, and 3. A sufficient level of H2S is able to inhibit TFAM promoter methylation and maintain mtDNA copy number. CSE/H2S system contributes to mtDNA replication and cellular bioenergetics and provides a novel therapeutic avenue for cardiovascular diseases.

Tet1-mediated DNA demethylation regulates neuronal cell death induced by oxidative stress.

Epigenetic regulations including DNA methylation and demethylation play critical roles in neural development. However, whether DNA methylation and demethylation may play a role in neuronal cell death remains largely unclear. Here we report that the blockade of DNA methyltransferase inhibits apoptosis of cerebellar granule cells and cortical neurons in response to oxidative stress. We found that knockdown of ten-eleven translocation methylcytosine dioxygenase (Tet1), a critical enzyme for DNA demethylation, significantly increase apoptosis of cerebellar granule cells induced by hydrogen peroxide. Moreover, cerebellar granule cells from tet1null mice appeared to be more sensitive to oxidative stress, suggesting the critical role of Tet1 in neuronal cell death. We further showed that the expression of Klotho, an antiaging protein, in cerebellar granule cells is tightly regulated by DNA methylation. Finally, we found that knockdown of Klotho diminished the rescue effects of DNA methyltransferase inhibitors and Tet1 on neuronal cell death induced by oxidative stress. Our work revealed the role of Tet1-mediated DNA demethylation on neuronal protection against oxidative stress and provided the molecular mechanisms underlying the epigenetic regulation of neuronal cell death, suggesting the role of Klotho in regulating neuronal cell death in response to oxidative stress.

The emerging nexus of active DNA demethylation and mitochondrial oxidative metabolism in post-mitotic neurons.

The variable patterns of DNA methylation in mammals have been linked to a number of physiological processes, including normal embryonic development and disease pathogenesis. Active removal of DNA methylation, which potentially regulates neuronal gene expression both globally and gene specifically, has been recently implicated in neuronal plasticity, learning and memory processes. Model pathways of active DNA demethylation involve ten-eleven translocation (TET) methylcytosine dioxygenases that are dependent on oxidative metabolites. In addition, reactive oxygen species (ROS) and oxidizing agents generate oxidative modifications of DNA bases that can be removed by base excision repair proteins. These potentially link the two processes of active DNA demethylation and mitochondrial oxidative metabolism in post-mitotic neurons. We review the current biochemical understanding of the DNA demethylation process and discuss its potential interaction with oxidative metabolism. We then summarise the emerging roles of both processes and their interaction in neural plasticity and memory formation and the pathophysiology of neurodegeneration. Finally, possible therapeutic approaches for neurodegenerative diseases are proposed, including reprogramming therapy by global DNA demethylation and mitohormesis therapy for locus-specific DNA demethylation in post-mitotic neurons.

Antidepressant-Like Effect of Sodium Butyrate is Associated with an Increase in TET1 and in 5-Hydroxymethylation Levels in the Bdnf Gene

Background:Epigenetic drugs like sodium butyrate (NaB) show antidepressant-like effects in preclinical studies, but the exact molecular mechanisms of the antidepressant effects remain unknown. While research using NaB has mainly focused on its role as a histone deacetylase inhibitor (HDACi), there is also evidence that NaB affects DNA methylation.Methods:The purpose of this study was to examine NaB’s putative antidepressant-like efficacy in relation to DNA methylation changes in the prefrontal cortex of an established genetic rat model of depression (the Flinders Sensitive Line [FSL]) and its controls (the Flinders Resistant Line).Results:The FSL rats had lower levels of ten-eleven translocation methylcytosine dioxygenase 1 (TET1), which catalyzes the conversion of DNA methylation to hydroxymethylation. As indicated by the behavioral despair test, chronic administration of NaB had antidepressant-like effects in the FSL and was accompanied by increased levels of TET1. The TET1 upregulation was also associated with an increase of hydroxymethylation and a decrease of methylation in brain-derived neurotrophic factor (Bdnf), a gene associated with neurogenesis and synaptic plasticity. These epigenetic changes were associated with a corresponding BDNF overexpression.Conclusions:Our data support the antidepressant efficacy of HDACis and suggest that their epigenetic effects may also include DNA methylation changes that are mediated by demethylation-facilitating enzymes like TET1.

Long Noncoding RNA TARID Directs Demethylation and Activation of the Tumor Suppressor TCF21 via GADD45A

DNA methylation is a dynamic and reversible process that governs gene expression during development and disease. Several examples of active DNA demethylation have been documented, involving genome-wide and gene-specific DNA demethylation. How demethylating enzymes are targeted to specific genomic loci remains largely unknown. We show that an antisense lncRNA, termed TARID (for TCF21 antisense RNA inducing demethylation), activates TCF21 expression by inducing promoter demethylation. TARID interacts with both the TCF21 promoter and GADD45A (growth arrest and DNA-damage-inducible, alpha), a regulator of DNA demethylation. GADD45A in turn recruits thymine-DNA glycosylase for base excision repair-mediated demethylation involving oxidation of 5-methylcytosine to 5-hydroxymethylcytosine in the TCF21 promoter by ten-eleven translocation methylcytosine dioxygenase proteins. The results reveal a function of lncRNAs, serving as a genomic address label for GADD45A-mediated demethylation of specific target genes.

The CpG Island Methylator Phenotype: What's in a Name?

Although the CpG island methylator phenotype (CIMP) was first identified and has been most extensively studied in colorectal cancer, the term "CIMP" has been repeatedly used over the past decade to describe CpG island promoter methylation in other tumor types, including bladder, breast, endometrial, gastric, glioblastoma (gliomas), hepatocellular, lung, ovarian, pancreatic, renal cell, and prostate cancers, as well as for leukemia, melanoma, duodenal adenocarninomas, adrenocortical carcinomas, and neuroblastomas. CIMP has been reported to be useful for predicting prognosis and response to treatment in a variety of tumor types, but it remains unclear whether or not CIMP is a universal phenomenon across human neoplasia or if there should be cancer-specific definitions of the phenotype. Recently, it was shown that somatic isocitrate dehydrogenase-1 (IDH1) mutations, frequently observed in gliomas, establish CIMP in primary human astrocytes by remodeling the methylome. Interestingly, somatic IDH1 and IDH2 mutations, and loss-of-function mutations in ten-eleven translocation (TET) methylcytosine dioxygenase-2 (TET2) associated with a hypermethylation phenotype, are also found in multiple enchondromas of patients with Ollier disease and Mafucci syndrome, and leukemia, respectively. These data provide the first clues for the elucidation of a molecular basis for CIMP. Although CIMP appears as a phenomenon that occurs in various cancer types, the definition is poorly defined and differs for each tumor. The current perspective discusses the use of the term CIMP in cancer, its significance in clinical practice, and future directions that may aid in identifying the true cause and definition of CIMP in different forms of human neoplasia.

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.