Targeting SIRT4/TET2 Signaling Alleviates Human Keratinocyte Senescence by Reducing 5-hydroxymethylcytosine Loss.

Skin aging is characterized by wrinkle formation and increased frailty and laxity, leading to the risk of age-related skin diseases. Keratinocyte is an important component of the epidermis in skin structure, and keratinocyte senescence has been identified as a pivotal factor in skin aging development. Because epigenetic pathways play a vital role in the regulation of skin aging, we evaluated human skin samples for DNA hydroxymethylation (5-hydroxymethylcytosine; 5-hmC) and SIRT4 expressions. Results found that both 5-hmC and SIRT4 showed a significant decrease in aged human skin samples. To test the results in vitro, human keratinocytes were cultured in H2O2, which modulates skin aging in vivo. However, H2O2-induced keratinocytes showed senescence-associated protein expression and significant downregulation of 5-hmC and SIRT4 expressions. Moreover, 5-hmC-converting enzymes ten eleven translocation 2 (TET2) showed a decrease and enhanced TET2 acetylation level in H2O2-induced keratinocytes. However, the overexpression of SIRT4 in keratinocytes alleviates the senescence phenotype, such as senescence-associated protein expression, decreases the TET2 acetylation, but increases TET2 and 5-hmC expressions. Our results provide a novel relevant mechanism whereby the epigenetic regulation of keratinocytes in skin aging may be correlated with SIRT4 expression and TET2 acetylation in 5-hmC alteration. Our study may provide a potential strategy for antiskin aging, which targets the SIRT4/TET2 axis involving epigenetic modification in keratinocyte senescence.

Endothelial cell-derived extracellular vesicles modulate the therapeutic efficacy of mesenchymal stem cells through IDH2/TET pathway in ARDS.

BACKGROUND: Acute respiratory distress syndrome (ARDS) is a severe and fatal disease. Although mesenchymal stem cell (MSC)-based therapy has shown remarkable efficacy in treating ARDS in animal experiments, clinical outcomes have been unsatisfactory, which may be attributed to the influence of the lung microenvironment during MSC administration. Extracellular vesicles (EVs) derived from endothelial cells (EC-EVs) are important components of the lung microenvironment and play a crucial role in ARDS. However, the effect of EC-EVs on MSC therapy is still unclear. In this study, we established lipopolysaccharide (LPS) - induced acute lung injury model to evaluate the impact of EC-EVs on the reparative effects of bone marrow-derived MSC (BM-MSC) transplantation on lung injury and to unravel the underlying mechanisms. METHODS: EVs were isolated from bronchoalveolar lavage fluid of mice with LPS - induced acute lung injury and patients with ARDS using ultracentrifugation. and the changes of EC-EVs were analysed using nanoflow cytometry analysis. In vitro assays were performed to establish the impact of EC-EVs on MSC functions, including cell viability and migration, while in vivo studies were performed to validate the therapeutic effect of EC-EVs on MSCs. RNA-Seq analysis, small interfering RNA (siRNA), and a recombinant lentivirus were used to investigate the underlying mechanisms. RESULTS: Compared with that in non-ARDS patients, the quantity of EC-EVs in the lung microenvironment was significantly greater in patients with ARDS. EVs derived from lipopolysaccharide-stimulated endothelial cells (LPS-EVs) significantly decreased the viability and migration of BM-MSCs. Furthermore, engrafting BM-MSCs pretreated with LPS-EVs promoted the release of inflammatory cytokines and increased pulmonary microvascular permeability, aggravating lung injury. Mechanistically, LPS-EVs reduced the expression level of isocitrate dehydrogenase 2 (IDH2), which catalyses the formation of α-ketoglutarate (α-KG), an intermediate product of the tricarboxylic acid (TCA) cycle, in BM-MSCs. α-KG is a cofactor for ten-eleven translocation (TET) enzymes, which catalyse DNA hydroxymethylation in BM-MSCs. CONCLUSIONS: This study revealed that EC-EVs in the lung microenvironment during ARDS can affect the therapeutic efficacy of BM-MSCs through the IDH2/TET pathway, providing potential strategies for improving the therapeutic efficacy of MSC-based therapy in the clinic.

An Overview of Epigenetic Changes in the Parkinson's Disease Brain.

Parkinson's disease is a progressive neurodegenerative disorder, predominantly of the motor system. Although some genetic components and cellular mechanisms of Parkinson's have been identified, much is still unknown. In recent years, emerging evidence has indicated that non-DNA-sequence variation (in particular epigenetic mechanisms) is likely to play a crucial role in the development and progression of the disease. Here, we present an up-to-date overview of epigenetic processes including DNA methylation, DNA hydroxymethylation, histone modifications and non-coding RNAs implicated in the brain of those with Parkinson's disease. We will also discuss the limitations of current epigenetic research in Parkinson's disease, the advantages of simultaneously studying genetics and epigenetics, and putative novel epigenetic therapies.

ZMIZ1 Upregulation of TET3-Mediated Hydroxymethylation Induces M2 Polarization of Kupffer Cells in Hepatocellular Carcinogenesis by Mediating Notch1/c-Myc Signaling.

Hydroxymethylation of DNA, mediated by the ten-eleven translocation (TET) family of methylcytosine dioxygenases, represents a crucial epigenetic modification that manipulates gene expression in numerous biological processes. This study focuses on the effect of TET3 on the polarization of Kupffer cells (KCs) and its connection to the development of hepatocellular carcinoma (HCC). TET3 was found to be abundant in KCs, and its knockdown induced an M2-M1 phenotype shift, resulting in the suppression of viability, migration, and invasion of cocultured HCC cells. Additionally, the TET3 knockdown inhibited the tumorigenesis of HCC cells in nude mice. Downstream targets of TET3 were predicted using bioinformatics. TET3-mediated DNA hydroxymethylation of zinc finger MIZ-type containing 1 (ZMIZ1) promoter. The ZMIZ1 protein interacted with notch receptor 1 (Notch1) protein to activate the transcription of c-Myc. Silencing of ZMIZ1 in KCs similarly suppressed M2 polarization of KCs and malignant phenotype of cocultured HCC cells. However, these changes were counteracted by the overexpression of either Notch1 or c-Myc overexpression in KCs. In summary, this study demonstrates that TET3-mediated hydroxymethylation of ZMIZ1 enhances hepatocellular carcinogenesis by promoting M2 skewing of KCs through the Notch1/c-Myc axis.

The N-terminal domain of TET1 promotes the formation of dense chromatin regions refractory to transcription.

TET (ten-eleven translocation) enzymes initiate active cytosine demethylation via the oxidation of 5-methylcytosine. TET1 is composed of a C-terminal domain, which bears the catalytic activity of the enzyme, and a N-terminal region that is less well characterized except for the CXXC domain responsible for the targeting to CpG islands. While cytosine demethylation induced by TET1 promotes transcription, this protein also interacts with chromatin-regulating factors that rather silence this process, the coordination between these two opposite functions of TET1 being unclear. In the present work, we uncover a new function of the N-terminal part of the TET1 protein in the regulation of the chromatin architecture. This domain of the protein promotes the establishment of a compact chromatin architecture displaying reduced exchange rate of core histones and partial dissociation of the histone linker. This chromatin reorganization process, which does not rely on the CXXC domain, is associated with a global shutdown of transcription and an increase in heterochromatin-associated histone epigenetic marks. Based on these findings, we propose that the dense chromatin organization generated by the N-terminal domain of TET1 could contribute to restraining the transcription enhancement induced by the DNA demethylation activity of this enzyme.

TET2 inhibits the proliferation and metastasis of lung adenocarcinoma cells via activation of the cGAS-STING signalling pathway.

BACKGROUND: Effective identification and development of new molecular methods for the diagnosis, treatment and prognosis of lung adenocarcinoma (LUAD) remains an urgent clinical need. DNA methylation patterns at cytosine bases in the genome are closely related to gene expression, and abnormal DNA methylation is frequently observed in various cancers. The ten-eleven translocation (TET) enzymes oxidize 5-methylcytosine (5mC) and promote locus-specific DNA methylation reversal. This study aimed to explore the role of the TET2 protein and its downstream effector, 5-hmC/5-mC DNA modification, in LUAD progression. METHODS: The expression of TET2 was analysed by real-time PCR, Western blotting and immunohistochemistry. The 5-hmC DNA content was determined by a colorimetric kit. Activation of the cGAS-STING signalling pathway was evaluated by Western blotting. CCK-8, wound healing and Transwell assays were performed to evaluate the effect of TET2 on cell proliferation, migration and invasion abilities. A xenograft model was used to analyse the effect of TET2 on the tumorigenic ability of A549 cells. RESULTS: TET2 overexpression decreased proliferation and metastasis of A549 and H1975 cells in vitro and in vivo. However, TET2 knockdown dramatically enhanced the proliferation, migration and invasion of A549 and H1975 cells. Mechanistically, activation of the cGAS-STING signalling pathway is critical for the TET2-mediated suppression of LUAD cell tumorigenesis and metastasis. CONCLUSION: In this study, we demonstrate a tumour suppressor role of TET2 in LUAD, providing new potential molecular therapeutic targets and clinical therapies for patients with non-small cell lung cancer.

Protective effect of vitamin C against tetrachlorobenzoquinone-induced 5-hydroxymethylation-dependent apoptosis in HepG2 cells mainly via the mitochondrial apoptosis pathway.

Tetrachlorobenzoquinone (TCBQ) is an active metabolite of pentachlorophenol, and stimulates the accumulation of ROS to trigger apoptosis. The preventive effect of vitamin C (Vc) against TCBQ-induced apoptosis in HepG2 cells is unknown. And there is little known about TCBQ-triggered 5-hydromethylcytosine (5hmC)-dependent apoptosis. Here, we confirmed that Vc alleviated TCBQ-induced apoptosis. Through investigating the underlying mechanism, we found TCBQ downregulated 5hmC levels of genomic DNA in a Tet-dependent manner, with a particularly pronounced decrease in the promoter region, using UHPLC-MS-MS analysis and hydroxymethylated DNA immunoprecipitation sequencing. Notably, TCBQ exposure resulted in alterations of 5hmC abundance to ∼91% of key genes at promoters in the mitochondrial apoptosis pathway, along with changes of mRNA expression in 87% of genes. By contrast, 5hmC abundance of genes only exhibited slight changes in the death receptor/ligand pathway. Interestingly, the pretreatment with Vc, a positive stimulator of 5hmC generation, restored 5hmC in the genomic DNA to near-normal levels. More notably, Vc pretreatment further counter-regulated TCBQ-induced alteration of 5hmC abundance in the promoter with 100% of genes, accompanying the reverse modulation of mRNA expressions in 89% of genes. These data from Vc pretreatment supported the relationship between TCBQ-induced apoptosis and the altered 5hmC abundance. Additionally, Vc also suppressed TCBQ-stimulated generation of ROS, and further increased the stability of mitochondria. Our study illuminates a new mechanism of TCBQ-induced 5hmC-dependent apoptosis, and the dual mechanisms of Vc against TCBQ-stimulated apoptosis via reversely regulating 5hmC levels and scavenging ROS. The work also provided a possible strategy for the detoxification of TCBQ.

KDM2A interacts with estrogen receptor α to promote bisphenol A and S-induced breast cancer cell proliferation by repressing TET2 expression.

As a recognized endocrine disruptor in the environment targeting estrogen receptors (ERs), Bisphenol A (BPA) and its bisphenol S (BPS) analogs are involved in the development of breast cancer. Epigenetic modifications are crucial in many biological processes, and DNA hydroxymethylation (DNAhm) coupled with histone methylation is implicated in epigenetic machinery covering cancer occurrence. Our previous study indicated that BPA/BPS induces breast cancer cell (BCC) proliferation with enhanced estrogenic transcriptional activity and causes the change of DNAhm depending on ten-eleven translocation 2 (TET2) dioxygenase. Herein, we investigated the interplay of KDM2A-mediated histone demethylation with ER-dependent estrogenic activity (EA) and identified their function in DNAhm catalyzed by TET2 for ER-positive (ER+) BCC proliferation induced by BPA/BPS. We found that BPA/BPS-treated ER+ BCCs presented increased KDM2A mRNA and protein levels but reduced TET2 and genomic DNAhm. Furthermore, KDM2A promoted H3K36me2 loss and suppressed TET2-dependent DNAhm by reducing its chromatin binding during BPA/BPS-induced cell proliferation. Results of Co-IP & ChIP assays suggested the direct interplay of KDM2A with ERα in multiple manners. KDM2A reduced the lysine methylation of ERα protein to increase its phosphorylated activation. On the other hand, ERα did not affect KDM2A expression, while KDM2A protein levels decreased after ERα deletion, indicating that ERα binding might maintain KDM2A protein stability. In conclusion, a potential feedback circuit of KDM2A/ERα-TET2-DNAhm was identified among ER+ BCCs with significant effects on regulating BPA/BPS-induced cell proliferation. These insights advanced the understanding of the relationship between histone methylation, DNAhm, and cancer cell proliferation with EA attributed to BPA/BPS exposure in the environment.

DNA methylation methods: Global DNA methylation and methylomic analyses.

DNA methylation provides a pivotal layer of epigenetic regulation in eukaryotes that has significant involvement for numerous biological processes in health and disease. The function of methylation of cytosine bases in DNA was originally proposed as a "silencing" epigenetic marker and focused on promoter regions of genes for decades. Improved technologies and accumulating studies have been extending our understanding of the roles of DNA methylation to various genomic contexts including gene bodies, repeat sequences and transcriptional start sites. The demand for comprehensively describing DNA methylation patterns spawns a diversity of DNA methylation profiling technologies that target its genomic distribution. These approaches have enabled the measurement of cytosine methylation from specific loci at restricted regions to single-base-pair resolution on a genome-scale level. In this review, we discuss the different DNA methylation analysis technologies primarily based on the initial treatments of DNA samples: bisulfite conversion, endonuclease digestion and affinity enrichment, involving methodology evolution, principles, applications, and their relative merits. This review may offer referable information for the selection of various platforms for genome-wide analysis of DNA methylation.

Arsenic exposure and human blood DNA methylation and hydroxymethylation profiles in two diverse populations from Bangladesh and Spain.

BACKGROUND: Associations of arsenic (As) with the sum of 5-mC and 5-hmC levels have been reported; however, As exposure-related differences of the separated 5-mC and 5-hmC markers have rarely been studied. METHODS: In this study, we evaluated the association of arsenic exposure biomarkers and 5-mC and 5-hmC in 30 healthy men (43-55 years) from the Aragon Workers Health Study (AWHS) (Spain) and 31 healthy men (31-50 years) from the Folic Acid and Creatinine Trial (FACT) (Bangladesh). We conducted 5-mC and 5-hmC profiling using Infinium MethylationEPIC arrays, on paired standard and modified (ox-BS in AWHS and TAB in FACT) bisulfite converted blood DNA samples. RESULTS: The median for the sum of urine inorganic and methylated As species (ΣAs) (µg/L) was 12.5 for AWHS and 89.6 for FACT. The median of blood As (µg/L) was 8.8 for AWHS and 10.2 for FACT. At a statistical significance p-value cut-off of 0.01, the differentially methylated (DMP) and hydroxymethylated (DHP) positions were mostly located in different genomic sites. Several DMPs and DHPs were consistently found in AWHS and FACT both for urine ΣAs and blood models, being of special interest those attributed to the DIP2C gene. Three DMPs (annotated to CLEC12A) for AWHS and one DHP (annotated to NPLOC4) for FACT remained statistically significant after false discovery rate (FDR) correction. Pathways related to chronic diseases including cardiovascular, cancer and neurological were enriched. CONCLUSIONS: While we identified common 5-hmC and 5-mC signatures in two populations exposed to varying levels of inorganic As, differences in As-related epigenetic sites across the study populations may additionally reflect low and high As-specific associations. This work contributes a deeper understanding of potential epigenetic dysregulations of As. However, further research is needed to confirm biological consequences associated with DIP2C epigenetic regulation and to investigate the role of 5-hmC and 5-mC separately in As-induced health disorders at different exposure levels.

Genomic DNA hydroxymethylation reveals potential role in identification of lung injury in coal-burning arsenicosis populations.

Arsenic (As) is a toxic metalloid element that causes lung cancer and multiple non-malignant respiratory diseases. The toxicity of arsenic is mediated in part by epigenetic mechanisms, such as alterations in DNA methylation. While increasing studies have highlighted the potential importance of arsenic exposure to DNA methylation patterns and the subsequent risks for arsenic toxicity, there has been little focus on DNA hydroxymethylation-a negative regulation mechanism of DNA methylation. Therefore, this study aimed to investigate the relationship between genomic DNA methylation/hydroxymethylation and lung injury in arsenicosis populations. First, an increased risk of lung injury and exacerbation of lung function impairment in the arsenicosis population was confirmed. Levels of 5-methylcytosine/deoxycytidine (5 mC/dC), 5-hydroxymethylcytosine/deoxycytidine (5 hmC/dC) and 5 hmC/5 mC in genomic DNA of peripheral blood were decreased in the arsenicosis population compared to in the control. Additionally, multivariate logistic regression models showed an increased risk of chest digital radiography (DR) abnormalities when 5 hmC/dC and 5 hmC/5 mC levels were lower (OR = 3.12 and 3.96, all P < 0.001). For 3 years follow-up, regression analysis showed that a decline in 5 hmC/dC was significantly associated with the decline of lung function parameters [forced vital capacity (FVC), forced expiratory volume in 1 s (FEV1) and maximal mid-expiratory flow (MMEF); ß = 0.167, 0.122 and 0.073, respectively; all P < 0.05]. Using the receiver operating characteristic (ROC) curve, a combination of 5 hmC/5 dC and 5 hmC/5 mC obtained the highest value for distinguishing lung injury in all subjects (AUC = 0.82, P < 0.01). In contrast, in arsenicosis subjects, 5 hmC/dC was better at distinguishing lung injury (AUC = 0.84, P < 0.01). Together, the results revealed that a decrease in genomic DNA hydroxymethylation markers was associated with lung injury in coal-burning arsenicosis populations. Genomic DNA hydroxymethylation could be a novel biomarker for identifying the risk of lung injury caused by coal-burning arsenicosis.

MiR-3571 modulates the proliferation and migration of vascular smooth muscle cells by targeting claudin 1.

Background and aims: The miRNA-based post-transcription modification has been extensively studied in hypertension. It however remains elusive how miRNA expression is regulated in this pathological process. We hypothesize that hydroxymethylation in the promoter regions tightly controls the levels of key miRNAs, which in turn affects the development of hypertension. Methods: The levels of hydroxymethylation in the promoter regions from thoracic aortic tissues were compared between spontaneously hypertensive rats (SHRs) and normotensive Wistar-Kyoto rats (WKYs), using hydroxymethylcytosine DNA immunoprecipitation (hMeDIP) sequencing. The altered hydroxymethylation level of miR-3571 was confirmed by glucosylation-coupled hydroxymethylation-sensitive qPCR. We further identified claudin 1(CLDN1) as a key target of miR-3571 via bioinformatic prediction (targetscan) and dual-luciferase activity assays. Finally, we analyzed the contribution of miR-3571/CLDN1 axis in the proliferation and migration of vascular smooth muscle cells (VSMCs). Results: The hydroxymethylation level of miR-3571 promoter region in thoracic aortic tissue from spontaneously hypertensive rats was lower than that from normotensive Wistar-Kyoto rats. Accordingly, the expression of miR-3571 was lower during hypertension, with up-regulated CLDN1 protein levels. More importantly, we found that miR3571 overexpression led to phenotypic changes of VSMCs, and inhibited the proliferation and migration of muscle cells via suppressing CLDN1 as well. Our findings further suggested that CLDN1 up-regulation increase the activity of ERK1/2 in VSMCs. Conclusions: Our study suggested that hydroxymethylation in the promoter regions controlled the level of miR-3571 and revealed the important roles of miR-3571 and CLDN1 in VSMCs during the development of hypertension. In addition, our results also indicated that miR-3571/CLDN1 axis regulated the functions of VSMCs via the ERK1/2 pathway. Taken together, our findings support miR-3571 as a novel biomarker for the diagnosis and prevention of hypertension.

Epigenetic mapping of the somatotropic axis in Nile tilapia reveals differential DNA hydroxymethylation marks associated with growth.

In vertebrates, the somatotropic axis comprising the pituitary gland, liver and muscle plays a major role in myogenesis. Its output in terms of muscle growth is highly affected by nutritional and environmental cues, and thus likely epigenetically regulated. Hydroxymethylation is emerging as a DNA modification that modulates gene expression but a holistic characterization of the hydroxymethylome of the somatotropic axis has not been investigated to date. Using reduced representation 5-hydroxymethylcytosine profiling we demonstrate tissue-specific localization of 5-hydroxymethylcytosines at single nucleotide resolution. Their abundance within gene bodies and promoters of several growth-related genes supports their pertinent role in gene regulation. We propose that cytosine hydroxymethylation may contribute to the phenotypic plasticity of growth through epigenetic regulation of the somatotropic axis.

The mitochondrial genome and Epigenome of the Golden lion Tamarin from fecal DNA using Nanopore adaptive sequencing.

BACKGROUND: The golden lion tamarin (Leontopithecus rosalia) is an endangered Platyrrhine primate endemic to the Atlantic coastal forests of Brazil. Despite ongoing conservation efforts, genetic data on this species remains scarce. Complicating factors include limitations on sample collection and a lack of high-quality reference sequences. Here, we used nanopore adaptive sampling to resequence the L. rosalia mitogenome from feces, a sample which can be collected non-invasively. RESULTS: Adaptive sampling doubled the fraction of both host-derived and mitochondrial sequences compared to sequencing without enrichment. 258x coverage of the L. rosalia mitogenome was achieved in a single flow cell by targeting the unfinished genome of the distantly related emperor tamarin (Saguinus imperator) and the mitogenome of the closely related black lion tamarin (Leontopithecus chrysopygus). The L. rosalia mitogenome has a length of 16,597 bp, sharing 99.68% sequence identity with the L. chrysopygus mitogenome. A total of 38 SNPs between them were identified, with the majority being found in the non-coding D-loop region. DNA methylation and hydroxymethylation were directly detected using a neural network model applied to the raw signal from the MinION sequencer. In contrast to prior reports, DNA methylation was negligible in mitochondria in both CpG and non-CpG contexts. Surprisingly, a quarter of the 642 CpG sites exhibited DNA hydroxymethylation greater than 1% and 44 sites were above 5%, with concentration in the 3' side of several coding regions. CONCLUSIONS: Overall, we report a robust new mitogenome assembly for L. rosalia and direct detection of cytosine base modifications in all contexts.

TDP43 proteinopathy is associated with aberrant DNA methylation in human amyotrophic lateral sclerosis.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by motor neurone (MN) degeneration and death. ALS can be sporadic (sALS) or familial, with a number of associated gene mutations, including C9orf72 (C9ALS). DNA methylation is an epigenetic mechanism whereby a methyl group is attached to a cytosine (5mC), resulting in gene expression repression. 5mC can be further oxidized to 5-hydroxymethylcytosine (5hmC). DNA methylation has been studied in other neurodegenerative diseases, but little work has been conducted in ALS. AIMS: To assess differences in DNA methylation in individuals with ALS and the relationship between DNA methylation and TDP43 pathology. METHODS: Post mortem tissue from controls, sALS cases and C9ALS cases were assessed by immunohistochemistry for 5mC and 5hmC in spinal cord, motor cortex and prefrontal cortex. LMNs were extracted from a subset of cases using laser capture microdissection. DNA from these underwent analysis using the MethylationEPIC array to determine which molecular processes were most affected. RESULTS: There were higher levels of 5mC and 5hmC in sALS and C9ALS in the residual lower motor neurones (LMNs) of the spinal cord. Importantly, in LMNs with TDP43 pathology there was less nuclear 5mC and 5hmC compared to the majority of residual LMNs that lacked TDP43 pathology. Enrichment analysis of the array data suggested RNA metabolism was particularly affected. CONCLUSIONS: DNA methylation is a contributory factor in ALS LMN pathology. This is not so for glia or neocortical neurones.

Experimental eye research / short communication format characterization of DNA hydroxymethylation in the ocular choroid.

DNA methylation and hydroxymethylation represent important epigenetic modifications involved in cell differentiation. DNA hydroxymethylation can be used to classify independent biological samples by tissue type. Relatively little is known regarding the genomic abundance and function of 5-hydroxymethylcytosine (5-hmC) in ocular tissues. The choroid supplies oxygen and nutrients to the outer retina through its dense network of blood vessels. This connective tissue is mainly composed of pigmented melanocytes, and stromal fibroblasts. Since DNA hydroxymethylation level is relatively high in cutaneous melanocytes, we investigated the presence of 5-hmC in choroidal melanocytes, as well as the expression of ten-eleven translocation methylcytosine dioxygenases (TETs) and isocitrate dehydrogenases (IDHs) implicated in this DNA demethylation pathway. Immunofluorescence, DNA slot blots and liquid chromatography coupled to tandem mass spectrometry performed with choroidal tissues and melanocytes within these tissues revealed that they have a relatively high level of 5-hmC. We also examined the expression of TET1/2 and IDH1/2 in choroidal melanocytes by gene expression profiling, qPCR and Western blotting. In addition, we detected decreased levels of 5-hmC when choroidal melanocytes were exposed to a lower concentration of oxygen. Our study therefore demonstrates that DNA hydroxymethylation is present in choroidal melanocytes, and that the abundance of this epigenetic mark is impacted by hypoxia.

Gestational arsenic exposure induces anxiety-like behaviors in adult offspring by reducing DNA hydroxymethylation in the developing brain.

Several studies found that reduction of 5-hydroxymethylcytosine (5hmC), a marker of DNA hydroxymethylation highly enriched in developing brain, is associated with anxiety-like behaviors. This study aimed to investigate whether gestational arsenic (As) exposure induces anxiety-like behaviors in adult offspring by reducing DNA hydroxymethylation in the developing brain. The dams drank ultrapure water containing NaAsO2 (15 mg/L) throughout pregnancy. Anxiety-like behaviors were evaluated and developing brain 5hmC was detected. Results showed that anxiety-like behaviors were observed in As-exposed adult offspring. In addition, 5hmC content was reduced in As-exposed fetal brain. Despite no difference on Tet1, Tet2 and Tet3 expression, TET activity was suppressed in As-exposed fetal brain. Mechanistically, alpha-ketoglutarate (α-KG), a cofactor for TET dioxygenases, was reduced and Idh2, a key enzymatic gene for mitochondrial α-KG synthesis, was downregulated in As-exposed fetal brain. Of interest, ascorbic acid, a cofactor for TET dioxygenases, reversed As-induced suppression of TET activity. Moreover, ascorbic acid attenuated As-induced reduction of 5hmC in fetal brain. In addition, ascorbic acid alleviated As-induced anxiety-like behaviors in adult offspring. Taken together, these results suggest that gestational As exposure induces anxiety-like behaviors in adult offspring, possibly at part, by inhibiting DNA hydroxymethylation in developing brain.

TET3-mediated accumulation of DNA hydroxymethylation contributes to the activity-dependent gene expression of Rab3a in post-mitotic neurons.

Rab3a, a subtype protein in the Rab3 family amongst the small G proteins, is closely associated with the learning and memory formation process. Various neuronal stimuli can induce the expression of Rab3a; however, how DNA modification is involved in regulating its expression is not fully understood. Ten-eleven translocation (TET) proteins can oxidate methylcytosine to hydroxymethylcytosine, which can further activate gene expression. Previous studies reported that TET-mediated regulation of 5hmC induced by learning is involved in neuronal activation. However, whether Tet protein regulates Rab3a is unknown. To understand the role of TET-mediated 5hmC on Rab3a in neuronal activation, we adopted a KCl-induced depolarization protocol in cultured primary cortical neurons to mimic neuronal activity in vitro. After KCl treatment, Rab3a and Tet3 mRNA expression were induced. Moreover, we observed a decrease in the methylation level and an increase of hydroxymethylation level surrounding the CpG island near the transcription start site of Rab3a. Furthermore, recently, Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) has proven powerful in identifying open chromatin in the genome of various eukaryotes. Using FAIRE-qPCR, we observed a euchromatin state and the increased occupancy of Tet3, H3K4me3, and H3K27ac at the promoter region of Rab3a after KCl treatment. Finally, by using shRNA to knockdown Tet3 prior KCl treatment, all changes mentioned above vanished. Thus, our findings elucidated that the neuronal activity-induced accumulation of hydroxymethylation, which Tet3 mediates, can introduce an active and permissive chromatin structure at Rab3a promoter and lead to the induction of Rab3a mRNA expression.

Deregulation of imprinted genes expression and epigenetic regulators in placental tissue from intrauterine growth restriction.

PURPOSE: Intrauterine growth restriction (IUGR) is a fetal growth complication that can be caused by ineffective nutrient transfer from the mother to the fetus via the placenta. Abnormal placental development and function have been correlated with abnormal expression of imprinted genes, which are regulated by epigenetic modifications at imprinting control regions (ICRs). In this study, we analyzed the expression of imprinted genes known to be involved in fetal growth and epigenetic regulators involved in DNA methylation, as well as DNA methylation at the KvDMR1 imprinting control region and global levels of DNA hydroxymethylation, in IUGR cases. METHODS: Expression levels of imprinted genes and epigenetic regulators were analyzed in term placental samples from 21 IUGR cases and 9 non-IUGR (control) samples, by RT-qPCR. Additionally, KvDMR1 methylation was analyzed by bisulfite sequencing and combined bisulfite restriction analysis (COBRA) techniques. Moreover, global DNA methylation and hydroxymethylation levels were also measured. RESULTS: We observed increased expression of PHLDA2, CDKN1C, and PEG10 imprinted genes and of DNMT1, DNMT3A, DNMT3B, and TET3 epigenetic regulators in IUGR placentas. No differences in methylation levels at the KvDMR1 were observed between the IUGR and control groups; similarly, no differences in global DNA methylation and hydromethylation were detected. CONCLUSION: Our study shows that deregulation of epigenetic mechanisms, namely increased expression of imprinted genes and epigenetic regulators, might be associated with IUGR etiology. Therefore, this study adds knowledge to the molecular mechanisms underlying IUGR, which may contribute to novel prediction tools and future therapeutic options for the management of IUGR pregnancies.

Genome-wide characterization of 5-hydoxymethylcytosine in melanoma reveals major differences with nevus.

Melanoma demonstrates altered patterns of DNA methylation that are associated with genetic instability and transcriptional repression of numerous genes. Active DNA demethylation is mediated by TET enzymes that catalyze conversion of 5-methylcytosine (mC) to 5-hydroxymethylcytosine (hmC). Loss of hmC occurs in melanoma and correlates with disease progression. Here we analyzed the genomic distribution of hmC along with mC in nevus and melanoma using oxidative bisulfite chemistry combined with high-density arrays. HmC was enriched relative to mC at enhancers, 5'UTR regions and CpG shores in nevus and melanoma samples, pointing to specific TET enzyme activity. The proportion of interrogated CpG sites with high hmC levels was lower in melanoma (0.54%) than in nevus (2.0%). Depletion of hmC in melanoma was evident across all chromosomes and intragenic regions, being more pronounced in metastatic than in non-metastatic tumors. The patterns of hmC distribution in melanoma samples differed significantly from those in nevus samples, exceeding differences in mC patterns. We identified specific CpG sites and regions with significantly lower hmC levels in melanoma than in nevus that might serve as diagnostic markers. Differentially hydroxymethylated regions localized to cancer-related genes, including the PTEN gene promoter, suggesting that deregulated DNA hydroxymethylation may contribute to melanoma pathogenesis.