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• New label-free electrochemical method with high sensitivity was designed for TET1 detection. • Hairpin DNA was used for the formation of peroxidase-mimicking DNAzymes to amplify the signal. • Low detection limit of 0.027 ng/μL and a wide linear range of 0.7–10.5 ng/μL were obtained. • This method has been proven to be feasible for inhibitor screening of TET1. Ten-eleven translocation 1 (TET1) is a member of the TET enzyme family of dioxygenases, which plays an important role in active DNA demethylation. Therefore, the sensitive TET1 detection could help us better understand DNA methylation-demethylation in epigenetics. Here we report a detection method that consists of electrode fabrication, TET1 modification, DNA digestion, signal-on oxidoreduction, and current peak monitoring. An exquisitely designed 5′end-G-rich oligodeoxynucleotide was synthesized bearing a methylated cytosine (5-mC), which formed into hairpin dsDNA with the MspI recognition sequence (Cm C GG/GGCC). Then hairpin dsDNA was fabricated onto gold nanoparticles modified glassy carbon electrode (DNA/AuNPs/GCE) via Au-S bond. The combination uses of restriction enzyme MspI and hemin converted fabricated-dsDNA into peroxidase-mimicking DNAzyme, thereby promoting the reduction of H 2 O 2 with a current peak. However, the current peak was extremely decreased once TET1 and T4 β-GT were used in advance. We confirmed a delicately linear relationship matching between the current difference and TET1 activity from 0.7 to 10.5 ng μL−1 with a detection limit of 0.027 ng μL−1, which outcompeted the former methods at least one order of magnitudes. The TET1 activity evaluation in the existence of Bobcat339 was also tested as the proof of concept of inhibitors screening. Our strategy provides a novel, label-free, and sensitive electrochemical approach that enables us to complete both TET1 activity evaluation and potential TET1 inhibitors screening. [ABSTRACT FROM AUTHOR]

To identify underlying mechanisms involved with metastasis formation in Wilms tumors (WTs), we performed comprehensive DNA methylation and gene expression analyses of matched normal kidney (NK), WT blastemal component, and metastatic tissues (MT) from patients treated under SIOP 2001 protocol. A linear Bayesian framework model identified 497 differentially methylated positions (DMPs) between groups that discriminated NK from WT, but MT samples were divided in two groups. Accordingly, methylation variance grouped NK and three MT samples tightly together and all WT with four MT samples that showed high variability. WT were hypomethylated compared to NK, and MT had a hypermethylated pattern compared to both groups. The methylation patterns were in agreement with methylases and demethylases expression. Methylation data pointed to the existence of two groups of metastases. While hierarchical clustering analysis based on the expression of all 2569 differentially expressed genes (DEGs) discriminated WT and MT from all NK samples, the hierarchical clustering based on the expression of 44 genes with a differentially methylated region (DMR) located in their promoter region revealed two groups: one containing all NKs and three MTs and one containing all WT and four MTs. Methylation changes might be controlling expression of genes associated with WT progression. The 44 genes are candidates to be further explored as a signature for metastasis formation in WT.

Background: Both defective DNA methylation and active DNA demethylation processes are emerging as important risk factors in chronic lymphocytic leukemia (CLL). However, associations between 5-cytosine epigenetic markers and the most frequent chromosomal abnormalities detected in CLL remain to be established. Methods: CLL patients were retrospectively classified into a cytogenetic low-risk group (isolated 13q deletion), an intermediate-risk group (normal karyotype or trisomy 12) and a high-risk group (11q deletion, 17p deletion, or complex karyotype [⩾ 3 breakpoints]). The two 5-cytosine derivatives, 5-methylcytosine (5-mCyt) and 5-hydroxymethylcytosine (5-hmCyt), were tested by ELISA (n = 60), while real-time quantitative PCR was used for determining transcriptional expression levels of DNMT and TET (n = 24). Results: By using global DNA methylation/demethylation levels, in the low-risk disease group, two subgroups with significantly different clinical outcomes have been identified (median treatment-free survival [TFS] 45 versus > 120 months for 5-mCyt, p = 0.0008 and 63 versus > 120 months for 5-hmCyt, p = 0.04). A defective 5-mCyt status was further associated with a higher percentage of 13q deleted nuclei (> 80%), thus suggesting an acquired process. When considering the cytogenetic intermediate/high-risk disease groups, an association of 5-mCyt status with lymphocytosis (p = 0.0008) and the lymphocyte doubling time (p = 0.04) but not with TFS was observed, as well as a reduction of DNMT3A, TET1 and TET2 transcripts. Conclusions: Combining cytogenetic studies with 5-mCyt assessment adds accuracy to CLL patient's prognoses and particularly for those with 13q deletion as a sole cytogenetic abnormality. [ABSTRACT FROM AUTHOR]

Summary Enzymatic oxidation of 5-methylcytosine (5mC) in DNA by the Tet dioxygenases reprograms genome function in embryogenesis and postnatal development. Tet-oxidized derivatives of 5mC such as 5-hydroxymethylcytosine (5hmC) act as transient intermediates in DNA demethylation or persist as durable marks, yet how these alternative fates are specified at individual CpGs is not understood. Here, we report that the SOS response-associated peptidase (SRAP) domain protein Srap1, the mammalian ortholog of an ancient protein superfamily associated with DNA damage response operons in bacteria, binds to Tet-oxidized forms of 5mC in DNA and catalyzes turnover of these bases to unmodified cytosine by an autopeptidase-coupled nuclease. Biallelic inactivation of murine Srap1 causes embryonic sublethality associated with widespread accumulation of ectopic 5hmC. These findings establish a function for a class of DNA base modification-selective nucleases and position Srap1 as a determinant of 5mC demethylation trajectories during mammalian embryonic development. [ABSTRACT FROM AUTHOR]

DNA methylation aberrancies are hallmarks of human cancers and are characterized by global DNA hypomethylation of repetitive elements and non-CpG rich regions concomitant with locus-specific DNA hypermethylation. DNA methylation changes may result in altered gene expression profiles, most notably the silencing of tumor suppressors, microRNAs, endogenous retorviruses and tumor antigens due to promoter DNA hypermethylation, as well as oncogene upregulation due to gene-body DNA hypermethylation. Here, we review DNA methylation aberrancies in human cancers, their use in cancer surveillance and the interplay between DNA methylation and histone modifications in gene regulation. We also summarize DNA methylation inhibitors and their therapeutic effects in cancer treatment. In this context, we describe the integration of DNA methylation inhibitors with conventional chemotherapies, DNA repair inhibitors and immune-based therapies, to bring the epigenome closer to its normal state and increase sensitivity to other therapeutic agents to improve patient outcome and survival. [ABSTRACT FROM PUBLISHER]

Chronic alcohol use and abuse result in widespread changes to gene expression, some of which contribute to the development of alcohol-use disorders (AUD). Gene expression is controlled, in part, by a group of regulatory systems often referred to as epigenetic factors, which includes, among other mechanisms, chemical marks made on the histone proteins around which genomic DNA is wound to form chromatin, and on nucleotides of the DNA itself. In particular, alcohol has been shown to perturb the epigenetic machinery, leading to changes in gene expression and cellular functions characteristic of AUD and, ultimately, to altered behavior. DNA modifications in particular are seeing increasing research in the context of alcohol use and abuse. To date, studies of DNA modifications in AUD have primarily looked at global methylation profiles in human brain and blood, gene-specific methylation profiles in animal models, methylation changes associated with prenatal ethanol exposure, and the potential therapeutic abilities of DNA methyltransferase inhibitors. Future studies may be aimed at identifying changes to more recently discovered DNA modifications, utilizing new methods to discriminate methylation profiles between cell types, thus clarifying how alcohol influences the methylomes of cell-type populations and how this may affect downstream processes. These studies and more in-depth probing of DNA methylation will be key to determining whether DNA-level epigenetic regulation plays a causative role in AUD and can thus be targeted for treatment of the disorder. [ABSTRACT FROM AUTHOR]

DNA methylation plays important roles in development and disease. Yet, only recently has the dynamic nature of this epigenetic mark via oxidation and DNA repair-mediated demethylation been recognized. A major conceptual challenge to the model that DNA methylation is reversible is the risk of genomic instability, which may come with widespread DNA repair activity. Here, we focus on recent advances in mechanisms of TET-TDG mediated demethylation and cellular strategies that avoid genomic instability. We highlight the recently discovered involvement of NEIL DNA glycosylases, which cooperate with TDG in oxidative demethylation to accelerate substrate turnover and promote the organized handover of harmful repair intermediates to maintain genome stability. [ABSTRACT FROM AUTHOR]

ABSTRACT The epigenetic mark 5-hydroxymethylcytosine (5hmC) is a cytosine modification that is abundant in the central nervous system of mammals and which results from 5-methylcytosine oxidation by TET enzymes. Such a mark is suggested to play key roles in the regulation of chromatin structure and gene expression. However, its precise functions still remain poorly understood and information about its distribution in non-mammalian species is still lacking. Here, the distribution of 5hmC was investigated in the brain of adult zebrafish, African claw frog, and mouse in a comparative manner. We show that zebrafish neurons are endowed with high levels of 5hmC, whereas quiescent or proliferative neural progenitors show low to undetectable levels of the modified cytosine. In the brain of larval and juvenile Xenopus, 5hmC is also detected in neurons, while ventricular proliferative cells do not display this epigenetic mark. Similarly, 5hmC is enriched in neurons compared to neural progenitors of the ventricular zone in the mouse developing cortex. Interestingly, 5hmC colocalized with the methylated DNA binding protein MeCP2 and with the active chromatin histone modification H3K4me2 in mouse neurons. Taken together, our results show an evolutionarily conserved cerebral distribution of 5hmC between fish and tetrapods and reinforce the idea that 5hmC fulfills major functions in the control of chromatin activity in vertebrate neurons. J. Comp. Neurol. 525:478-497, 2017. © 2016 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

The objective was to evaluate the effect of increased progesterone (P4) during preovulatory follicle growth during timed AI (TAI) or timed embryo transfer (TET) protocols. Lactating dairy cows with no CL and low circulating P4 (≤1.0 ng/mL) were submitted to a protocol using one or two intravaginal P4 implants (controlled intravaginal releasing device [CIDRs]), and were bred to TAI or TET. The low P4 cows for this experiment were identified on nine farms, four utilized TAI (n = 326 of 1160 cows examined), and five utilized TET (n = 445 of 1396). All cows were synchronized by insertion of P4 implant(s) (CIDR[s]) at start of protocol (Day −11) and simultaneous treatment with 2 mg of E2-benzoate. After 7 days, cows were treated with PGF (Day −4) and 2 days later treated with 1.0-mg E2-cypionate and CIDR(s) were removed (Day −2). Cows received TAI on Day 0 or TET on Day 7. Cows were randomly assigned to receive either one or two CIDRs on Day −11 until Day −2 (1CIDR vs. 2CIDR). Presence of CL was determined by ultrasound on Day −11 and Day 7 after protocol (to determine ovulation to protocol), P4 concentrations were determined on a subset of cows (Day −11, Day −4, Day 7), and ovulatory follicle diameter was evaluated on Day 0. Pregnancy success (P/AI or P/ET) was evaluated on Days 32 and 60. The 2CIDR treatment increased circulating P4 by Day −4 (1.77 ± 0.23 vs. 2.18 ± 0.24 ng/mL) but had no effect on ovulation at the end of protocol (83.6 vs. 82.6%) or ovulatory follicle diameter (15.6 ± 0.3 vs. 15.3 ± 0.3 mm). If only cows that ovulated to the protocol were included, 1CIDR tended to have lower P/AI than 2CIDR by Day 32 (42.8 vs. 52.6%; P = 0.10) and Day 60 (37.7 vs. 48.1%; P = 0.08) but there was no effect on pregnancy loss. There was an interaction (P = 0.05) between ovulatory follicle diameter and CIDR treatment on P/AI (Day 60). In cows ovulating larger follicles (≥14 mm), 2CIDR treatment increased P/AI compared with 1CIDR (53.3 vs. 34.9%; P = 0.02) but not in cows ovulating small follicles (<14 mm). There was no effect of treatment on P/ET on Day 32 (30.0% vs. 32.0%) or Day 60 (24.7% vs. 25.6%). Thus, these results add evidence to the concept that increased circulating P4 during preovulatory follicle development may improve P/AI, most likely due to improved oocyte quality in cows that ovulate larger follicles, since improvement was only in cows ovulating larger follicles and no effect of preovulatory P4 was observed in cows that received ET. [ABSTRACT FROM AUTHOR]

DNA demethylation can occur passively by 'dilution' of methylation marks by DNA replication, or actively and independently of DNA replication. Direct conversion of 5-methylcytosine (5mC) to cytosine (C), as originally proposed, does not occur. Instead, active DNA methylation involves oxidation of the methylated base by ten-eleven translocations (TETs), or deamination of the methylated or a nearby base by activation induced deaminase (AID). The modified nucleotide, possibly together with surrounding nucleotides, is then replaced by the BER pathway. Recent data clarify the roles and the regulation of well-known enzymes in this process. They identify base excision repair (BER) glycosylases that may cooperate with or replace thymine DNA glycosylase (TDG) in the base excision step, and suggest possible involvement of DNA damage repair pathways other than BER in active DNA demethylation. Here, we review these new developments. [ABSTRACT FROM AUTHOR]

Some of the best-studied neural sex differences depend on differential cell death in males and females, but other sex differences persist even if cell death is prevented. These include sex differences in neurochemical phenotype (i.e. , stable patterns of gene expression). Work in our laboratory over the last several years has tested the hypothesis that sex differences in DNA methylation early in life underlie sexual differentiation of neuronal phenotype. We have shown that 1) expression of enzymes that place or remove DNA methylation marks is greatest during the first week of life in the mouse brain and overlaps with the perinatal critical period of sexual differentiation; 2) a transient inhibition of DNA methylation during neonatal life abolishes several sex differences in cell phenotype in the mouse hypothalamus; 3) both DNA methylation and de -methylation contribute to the development of neural sex differences; and 4) the effects of DNA methylation and de-methylation are brain region- and cell type-specific. • We review evidence that sexual differentiation of hypothalamic cell types depends on DNA methylation. • DNA methylation writers/erasers are most abundant neonatally, suggesting a role in brain development. • Inhibiting DNA methylation affects ERα expression in a sex- and region-specific manner. • To our knowledge, we are the first to tie DNA de methylation to sexual differentiation. [ABSTRACT FROM AUTHOR]

Epigenetic systems are organized by different types of modifications on histones and DNA. To determine how epigenetic systems can produce variable, yet stable cellular outcomes, understanding the collaboration between these modifications is the key. A recent study by Yamagata and Kobayashi revealed the direct interplay between the regulation of two epigenetic modifications: DNA de-methylation by TET2 and histone H3-K36 methylation. Mechanistically, this finding could explain how cells are protected from oncogenesis by maintaining the integrity of active transcription. The recent identification of epigenetic modifier mutations in leukaemia suggested that it is not just the turning 'on' and 'off' of particular transcriptional events that causes disease occurrence, but rather it is the aberration in epigenetic regulation, i.e. the timing and duration of the activation/inactivation of these transcripts. Thus, a comprehensive understanding of how epigenetic interplays tune transcription will be the new perspective for disease research. [ABSTRACT FROM AUTHOR]

Epigenetic modifications include DNA methylation, DNA demethylation along with the major role fulfilled by TET protein. Epigenetic modifications refer to the regulation of gene expression without the alteration of the DNA sequence. Some of the most common epigenetic modifications include DNA methylation and demethylation, as well as the functional role of TET proteins. Epigenetic alterations are heritable traits, therefore one of the key elements to understanding the mechanisms of cancer development is to further our knowledge on the role and function of epigenetic modifications. This mini-review takes into consideration the overview of the literature on the impact of epigenetic changes in cancer development, especially in the development of CC. Researchers believe that certain compounds are capable of inhibiting the process of DNA methylation and may play an important role in future cancer therapy.

Background: Chronic lymphocytic leukemia (CLL) is the most common hematological malignancy in western countries, characterized by a heterogeneous clinical course. Although genetic studies have identified chromosomal aberrations or specific mutations, epigenetic changes have been poorly characterized in CLL. Methods: We assessed ten-eleven translocations (TET) 1, 2, and 3, isocitrate dehydrogenase (IDH) 1, and 2 messenger RNA (mRNA) expression using real-time PCR on purified leukemic B cells from 214 CLL patients (median follow-up = 75 months, range 1-380), normal peripheral blood B cells (n = 20), and umbilical cord blood B cells (n = 21). The microenvironment influence was assessed after 24 h co-culture of CLL cells with bone marrow mesenchymal stromal cells (BMSC). Finally, 5-hydroxymethylcytosine level (%5-hmC) was assessed by ELISA in CLL cells alone or with microenvironment stimuli. Results: TET 1 and 3 and IDH2 were decreased in CLL cells compared with healthy B cells (P = 0.0221, 0.0013, <0.0001, respectively), while IDH1 was overexpressed (P = 0.0037). TET2 and IDH1 were significantly correlated with treatment-free survival (TFS); patients with high TET2/IDH1 expression had a higher median TFS (111 months) than patients with low expression (78 months, P = 0.0071/0.0123). Moreover, TET1 expression decreased (P = 0.0371), while TET3 and IDH2 expression increased (P = 0.0273/0.0039) in co-cultures. However, %5-hmC was not correlated with clinical data and was unchanged following microenvironment stimuli. Conclusions: Despite a slight deregulation in CLL cells compared with normal B cells, we identified a significant association between TET/IDH gene expression and prognosis, suggesting that epigenetic changes could potentially be associated with disease progression. Moreover, despite an identical %5-hmC, TET gene expression was influenced by contact with BMSC confirming the crucial role of the microenvironment in CLL pathogenesis. [ABSTRACT FROM AUTHOR]

Epigenetic memory, in particular DNA methylation, is established during development in differentiating cells and must be erased to create naïve (induced) pluripotent stem cells. The ten-eleven translocation (TET) enzymes can catalyze the oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and further oxidized derivatives, thereby actively removing this memory. Nevertheless, the mechanism by which the TET enzymes are regulated, and the extent to which they can be manipulated, are poorly understood. Here we report that retinoic acid (RA) or retinol (vitamin A) and ascorbate (vitamin C) act as modulators of TET levels and activity. RA or retinol enhances 5hmC production in naïve embryonic stem cells by activation of TET2 and TET3 transcription, whereas ascorbate potentiates TET activity and 5hmC production through enhanced Fe2+ recycling, and not as a cofactor as reported previously. We find that both ascorbate and RA or retinol promote the derivation of induced pluripotent stem cells synergistically and enhance the erasure of epigenetic memory. This mechanistic insight has significance for the development of cell treatments for regenenerative medicine, and enhances our understanding of how intrinsic and extrinsic signals shape the epigenome. [ABSTRACT FROM AUTHOR]

The discovery of DNA methylation has provided a new perspective on how DNA may be dynamically regulated in the mammalian genome. DNA methylation is a dynamic process with a demethylation and de novo methylation from primordial germ cell to differentiation. DNA methylation and demethylation have been proposed to play important roles in somatic cell reprogramming. Some essential components were discussed, such as hydroxymethylation which has recently been confirmed as a modification of developmental importance. [ABSTRACT FROM AUTHOR]

Background: Monocyte-to-macrophage differentiation involves major biochemical and structural changes. In order to elucidate the role of gene regulatory changes during this process, we used high-throughput sequencing to analyze the complete transcriptome and epigenome of human monocytes that were differentiated in vitro by addition of colony-stimulating factor 1 in serum-free medium. Results: Numerous mRNAs and miRNAs were significantly up- or down-regulated. More than 100 discrete DNA regions, most often far away from transcription start sites, were rapidly demethylated by the ten eleven translocation enzymes, became nucleosome-free and gained histone marks indicative of active enhancers. These regions were unique for macrophages and associated with genes involved in the regulation of the actin cytoskeleton, phagocytosis and innate immune response. Conclusions: In summary, we have discovered a phagocytic gene network that is repressed by DNA methylation in monocytes and rapidly de-repressed after the onset of macrophage differentiation. [ABSTRACT FROM AUTHOR]

Background: Changes to the epigenome with aging, and DNA modifications in particular, have been proposed as a central regulator of the aging process, a predictor of mortality, and a contributor to the pathogenesis of age-related diseases. In the central nervous system, control of learning and memory, neurogenesis, and plasticity require changes in cytosine methylation and hydroxymethylation. Although genome-wide decreases in methylation with aging are often reported as scientific dogma, primary research reports describe decreases, increases, or lack of change in methylation and hydroxymethylation and their principle regulators, DNA methyltransferases and ten-eleven translocation dioxygenases in the hippocampus. Furthermore, existing data are limited to only male animals. Results: Through examination of the hippocampus in young, adult, and old male and female mice by antibodybased, pyrosequencing, and whole-genome oxidative bisulfite sequencing methods, we provide compelling evidence that contradicts the genomic hypomethylation theory of aging. We also demonstrate that expression of DNA methyltransferases and ten-eleven translocation dioxygenases is not differentially regulated with aging or between the sexes, including the proposed cognitive aging regulator DNMT3a2. Using oxidative bisulfite sequencing that discriminates methylation from hydroxymethylation and by cytosine (CG and non-CG) context, we observe sex differences in average CG methylation and hydroxymethylation of the X chromosome, and small age-related differences in hydroxymethylation of CG island shores and shelves, and methylation of promoter regions. Conclusion: These findings clarify a long-standing misconception of the epigenomic response to aging and demonstrate the need for studies of base-specific methylation and hydroxymethylation with aging in both sexes. [ABSTRACT FROM AUTHOR]

Cytosine base modifications in mammals underwent a recent expansion with the addition of several naturally occurring further modifications of methylcytosine in the last years. This expansion was accompanied by the identification of the respective enzymes and proteins reading and translating the different modifications into chromatin higher order organization as well as genome activity and stability, leading to the hypothesis of a cytosine code. Here, we summarize the current state-of-the-art on DNA modifications, the enzyme families setting the cytosine modifications and the protein families reading and translating the different modifications with emphasis on the mouse protein homologs. Throughout this review, we focus on functional and mechanistic studies performed on mammalian cells, corresponding mouse models and associated human diseases. [ABSTRACT FROM AUTHOR]

Summary Ten-eleven translocation (Tet) family proteins convert 5-methylcytosine to 5-hydroxymethylcytosine. We show that mouse embryonic stem cells (ESCs) depleted of Tet1 and/or Tet2 by RNAi exhibit short telomeres and chromosomal instability, concomitant with reduced telomere recombination. Tet1 and Tet2 double-knockout ESCs also display short telomeres but to a lesser extent. Notably, Tet1/2/3 triple-knockout ESCs show heterogeneous telomere lengths and increased frequency of telomere loss and chromosomal fusion. Mechanistically, Tets depletion or deficiency increases Dnmt3b and decreases 5hmC levels, resulting in elevated methylation levels at sub-telomeres. Consistently, knockdown of Dnmt3b or addition of 2i (MAPK and GSK3β inhibitors), which also inhibits Dnmt3b , reduces telomere shortening, partially rescuing Tet1/2 deficiency. Interestingly, Tet1/2 double or Tet1/2/3 triple knockout in ESCs consistently upregulates Zscan4 , which may counteract telomere shortening. Together, Tet enzymes play important roles in telomere maintenance and chromosomal stability of ESCs by modulating sub-telomeric methylation levels. [ABSTRACT FROM AUTHOR]

Summary Dioxygenases of the TET family impact genome functions by converting 5-methylcytosine (5mC) in DNA to 5-hydroxymethylcytosine (5hmC). Here, we identified TET2 as a crucial regulator of mast cell differentiation and proliferation. In the absence of TET2, mast cells showed disrupted gene expression and altered genome-wide 5hmC deposition, especially at enhancers and in the proximity of downregulated genes. Impaired differentiation of Tet2 -ablated cells could be relieved or further exacerbated by modulating the activity of other TET family members, and mechanistically it could be linked to the dysregulated expression of C/EBP family transcription factors. Conversely, the marked increase in proliferation induced by the loss of TET2 could be rescued exclusively by re-expression of wild-type or catalytically inactive TET2. Our data indicate that, in the absence of TET2, mast cell differentiation is under the control of compensatory mechanisms mediated by other TET family members, while proliferation is strictly dependent on TET2 expression. [ABSTRACT FROM AUTHOR]

Hebbian plasticity, including long-term potentiation and long-term depression, has long been regarded as important for local circuit refinement in the context of memory formation and stabilization. However, circuit development and stabilization additionally relies on non-Hebbian, homeostatic, forms of plasticity such as synaptic scaling. Synaptic scaling is induced by chronic increases or decreases in neuronal activity. Synaptic scaling is associated with cell-wide adjustments in postsynaptic receptor density, and can occur in a multiplicative manner resulting in preservation of relative synaptic strengths across the entire neuron's population of synapses. Both active DNA methylation and demethylation have been validated as crucial regulators of gene transcription during learning, and synaptic scaling is known to be transcriptionally dependent. However, it has been unclear whether homeostatic forms of plasticity such as synaptic scaling are regulated via epigenetic mechanisms. This review describes exciting recent work that has demonstrated a role for active changes in neuronal DNA methylation and demethylation as a controller of synaptic scaling and glutamate receptor trafficking. These findings bring together three major categories of memory-associated mechanisms that were previously largely considered separately: DNA methylation, homeostatic plasticity, and glutamate receptor trafficking. [ABSTRACT FROM AUTHOR]

The TET peptidases are large polypeptide destruction machines present among prokaryotes. They form 12-subunits hollow tetrahedral particles, and belong to the family of M42 metallo-peptidases. Structural characterization of various archaeal and bacterial complexes has revealed a unique mechanism of internal compartmentalization and peptide trafficking that distinguishes them from the other oligomeric peptidases. Different versions of the TET complex often co-exist in the cytosol of microorganisms. In depth enzymatic studies have revealed that they are non-processive cobalt-activated aminopeptidases and display contrasting substrate specificities based on the properties of the catalytic chambers. Recent studies have shed light on the assembly mechanism of homo and hetero-dodecameric TET complexes and shown that the activity of TET aminopeptidase towards polypeptides is coupled with its assembly process. These findings suggested a functional regulation based on oligomerization control in vivo . This review describes a current knowledge on M42 TET peptidases biochemistry and discuss their possible physiological roles. This article is a part of the Special Issue entitled: « A potpourri of proteases and inhibitors: from molecular toolboxes to signalling scissors » [ABSTRACT FROM AUTHOR]

Hypoxia in tumors is primarily a pathophysiologic consequence of structurally and functionally disturbed microcirculation with inadequate supply of oxygen. Tumor hypoxia is strongly associated with tumor propagation, malignant progression, and resistance to therapy. Aberrant epigenetic regulation plays a crucial role in the process of hypoxia-driven malignant progression. Convert of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) by ten-eleven translocation (TET) family enzymes plays important biological functions in embryonic stem cells, development, aging and disease. Recent reports showed that level of 5hmC and TET proteins was altered in various types of cancers. There is a strong correlation between loss of 5hmC and cancer development but research to date indicates that loss of TET activity is associated with the cancer phenotype but it is not clear whether TET proteins function as tumor suppressors or oncogenes. While loss of TET1 and TET2 expression is associated with solid cancers, implying a tumor suppressor role, TET1 exhibits a clear oncogenic role in the context of genomic rearrangements such as in MLL-fusion rearranged leukemia. Interestingly, hypoxia increases global 5hmC levels and upregulates TET1 expression in a HIF1α-dependent manner. Recently, hypoxia-induced TET1 has been demonstrated to play another important role for regulating hypoxia-responsive gene expression and epithelial-mesenchymal transition (EMT) by serving as a transcription co-activator. Furthermore, hypoxia-induced TET1 also regulates glucose metabolism and hypoxia-induced EMT through enhancing the expression of insulin induced gene 1 (INSIG1). The roles and mechanisms of action of 5hmC and TET proteins in ES cell biology and during embryonic development, as well as in cancer biology, will be the main focus in this review. [ABSTRACT FROM AUTHOR]

Epigenetic regulatory mechanisms play an important role in gene silencing and genome stability; therefore, epigenetic mutations cause a variety of diseases. Analysis of the epigenome by next-generation sequencers has revealed many epigenetic mutations in various diseases such as cancer, obesity, diabetes, autism, allergies, immune diseases, and imprinting diseases. Unfortunately, it has been difficult to identify the causative epigenetic mutations because there has been no method to generate animals with target-specific epigenetic mutations. However, it has become possible to generate such animals due to the recent development of epigenome editing technology. Here, we introduce the generation of epigenome-edited mice by target-specific DNA demethylation., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Aberrations in DNA methylation patterns are observed from the early stages of carcinogenesis. However, the mechanisms that drive these changes remain elusive. The recent characterization of ten-eleven translocation (TET) enzymes as a source of newly modified cytosines (5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine) has shed new light on the DNA demethylation process. These cytosines are intermediates of an active DNA demethylation process and are epigenetic markers per se . In this review, we discuss the mechanism and function of TET proteins in biological processes as well as current knowledge regarding their expression and regulation in cancer. [ABSTRACT FROM AUTHOR]

Recent evidence has shown that deregulated expression of members of the micro RNA-29 (miR-29) family may play a critical role in human cancer, including hematological malignancies. However, the roles of miR-29 in the molecular pathophysiology of T-cell acute lymphoblastic leukemia (T- ALL) has not been investigated. Here, we show that lower levels of miR-29a were significantly associated with higher blast counts in the bone marrow and with increased disease-free survival in T- ALL patients. Furthermore, miR-29a levels are extremely reduced in T- ALL cells compared to normal T cells. Microarray analysis following introduction of synthetic miR-29a mimics into Jurkat cells revealed the downregulation of several predicted targets ( CDK6, PXDN, MCL1, PIK3R1, and CXXC6), including targets with roles in active and passive DNA demethylation (such as DNMT3a, DNMT3b, and members of the TET family and TDG). Restoring miR-29a levels in Jurkat and Molt-4 T- ALL cells led to the demethylation of many genes commonly methylated in T- ALL. Overall, our results suggest that reduced miR-29a levels may contribute to the altered epigenetic status of T- ALL, highlighting its relevance in the physiopathology of this disease. [ABSTRACT FROM AUTHOR]

Background: Global deregulation of DNA methylation is one of the crucial causes of hepato cellular carcinoma (HCC). It has been reported that the anti-cancer drug 5-azacytidine (5-AZA) mediates the activation of tumor suppressor genes through passive demethylation by inhibiting DNMT1. Recent evidence suggests that active demethylation which is mediated by ten-eleven translocation (TET) proteins may also be an important step to control global methylation. However, there exists a controversial discussion in which TET proteins are involved in the demethylation process in HCC. Therefore, we firstly wanted to identify which of the TETs are involved in demethylation and later to study whether or not 5-AZA could trigger the TET-dependent active demethylation process in HCC. HCC cell lines (Huh-7, HLE, HLF), primary human hepatocytes (hHeps), and tissues from both healthy (55 patients) and HCC patients (55 patients) were included in this study; mRNA levels of isocitrate dehydrogenase (IDH1, 2) and TETs (TET1-3) were studied via qPCR and confirmed by Western blot. The expression of 5hmC/5mC was determined by immunohistochemistry in human HCC tissues and the corresponding adjacent healthy liver. HCC cell lines were stimulated with 5-AZA (0-20 μM) and viability (Resazurin conversion), toxicity (LDH release), proliferation (PCNA), and 5hmC/5mC distribution were assessed. In addition, knockdown experiments on TET proteins in HCC cell lines using short interference RNAs (siRNAs), in the presence and absence of 5-AZA, were performed. Results: Our data applying qPCR, immunofluorescence, and Western blotting clearly show that TET2 and TET3 but not TET1 were significantly decreased in HCC tissue and different HCC cell lines compared to non-tumor liver tissues and hHeps. In addition, we show here for the first time applying knockdown experiments that 5-AZA is able to trigger an active TET2-dependent demethylation process with concomitant significant changes in 5hmC/5mC in HCC cell lines and hHeps. Conclusions: Our data clearly show that the expression and activity of TET2 and TET3 proteins but not TET1 are impaired in hepatocellular carcinoma leading to the reduction of 5hmC in HCCs. Furthermore, this study identified a novel function of 5-azacytidine in promoting a TET-mediated generation of 5hmC suggesting that the availability of 5-AZA in cancer cells will have various effects on different epigenetic targets. These findings may open new therapeutic strategies for epigenetic drugs to treat HCC. [ABSTRACT FROM AUTHOR]

Epigenetic processes including aberrant promoter methylation of tumor suppressor gene play a key role in gastric carcinogenesis. TET proteins are involved in DNA demethylation; many cancers, haematological or solid, present loss-of-function mutations and aberrant expression/regulation of TET. In gastric cancer there are few studies reporting a decreased expression of TET and associations between these proteins and signaling pathways involved in carcinogenesis. Identifying connections between aberrant expression of TET, disruption of the balance between DNA methylation and demethylation and their association with gastric carcinogenesis might be useful for the development of novel therapeutic approaches. [ABSTRACT FROM PUBLISHER]

TET proteins have been found to play an important role in active demethylation at CpG sites in mammals. There are some reports implicating their functions in removal of DNA methylation imprint at the imprinted regions in the germline. However, it is not well established whether TET proteins can also be involved in demethylation of DNA methylation imprint in embryonic stem (ES) cells. Here we report that loss of TET proteins caused a significant increase in DNA methylation at the Igf2–H19 imprinted region in ES cells. We also observed a variable increase in DNA methylation at the Peg1 imprinted region in the ES clones devoid of TET proteins, in particular in the differentiated ES cells. By contrast, we did not observe a significant increase of DNA methylation imprint at the Peg3 , Snrpn and Dlk1–Dio3 imprinted regions in ES cells lacking TET proteins. Interestingly, loss of TET proteins did not result in a significant increase of DNA methylation imprint at the Igf2–H19 and Peg1 imprinted regions in the embryoid bodies (EB). Therefore, TET proteins seem to be differentially involved in maintaining DNA methylation imprint at a subset of imprinted regions in ES cells and EBs. [ABSTRACT FROM AUTHOR]

Emerging data have demonstrated that 5-methylcytosine (5-mC) and its oxidized products 5-hydroxymethylcytosine (5-hmC), 5-formylcytosine (5-fC), and 5-carboxylcytosine (5-CaC) play unique roles in several biological processes, including the control of gene expression and in the pathogenesis of cancer. In this review, we focus on 5-hmC and the disruption of its distribution in several cancers, including hematological malignancies and solid tumors. We present an outline of how 5-hmC is closely associated with metabolic pathways and may be the missing link connecting epigenetics with metabolism in the context of cancer cells. Finally, we discuss the diagnostic and prognostic importance of 5-mC and 5-hmC patterning, and how we may be able to establish new paradigms in cancer therapy based on these alterations. [ABSTRACT FROM AUTHOR]

The epigenetic mark 5-hydroxymethylcytosine (5hmC) has gained interest since 2009, when it was discovered that Ten-Eleven-Translocation (TET) proteins catalyze the conversion of 5-methylcytosine (5mC) into 5hmC. This conversion appears to be an intermediate step in the active DNA demethylation pathway. Factors that regulate DNA hydroxymethylation are frequently affected in cancer, leading to deregulated 5hmC levels. In this review, we will discuss the regulation of DNA hydroxymethylation, defects in this pathway in cancer, and novel therapies that may correct deregulated (hydroxy)methylation of DNA. [ABSTRACT FROM AUTHOR]

Background Prenatal stress (PRS) is considered a risk factor for several neurodevelopmental disorders including schizophrenia (SZ). An animal model involving restraint stress of pregnant mice suggests that PRS induces epigenetic changes in specific GABAergic and glutamatergic genes likely to be implicated in SZ, including the gene for brain-derived neurotrophic factor (BDNF). Methods Studying adult offspring of pregnant mice subjected to PRS, we explored the long-term effects of PRS on behavior and on the expression of key chromatin remodeling factors including DNA methyltransferase 1, ten-eleven-translocation hydroxylases, methyl CpG binding protein 2, histone deacetylases, and histone methyltransferases and demethylase in the frontal cortex and hippocampus. We also measured the expression of BDNF. Results Adult PRS offspring demonstrate behavioral abnormalities suggestive of SZ and molecular changes similar to changes seen in postmortem brains of patients with SZ. This includes a significant increase in DNA methyltransferase 1 and ten-eleven-translocation hydroxylase 1 in the frontal cortex and hippocampus but not in cerebellum; no changes in histone deacetylases, histone methyltransferases and demethylases, or methyl CpG binding protein 2, and a significant decrease in Bdnf messenger RNA variants. The decrease of the corresponding Bdnf transcript level was accompanied by an enrichment of 5-methylcytosine and 5-hydroxymethylcytosine at Bdnf gene regulatory regions. In addition, the expression of Bdnf transcripts (IV and IX) correlated positively with social approach in both PRS mice and nonstressed mice. Conclusions Because patients with psychosis and PRS mice show similar epigenetic signature, PRS mice may be a suitable model for understanding the behavioral and molecular epigenetic changes observed in patients with SZ. [ABSTRACT FROM AUTHOR]