Ten-eleven translocation (Tet) methylcytosine dioxygenase-dependent viral DNA demethylation mediates in vivo hepatitis B virus (HBV) biosynthesis.

Liver-specific ten-eleven translocation (Tet) methylcytosine dioxygenases 2 and 3 (Tet2 plus Tet3)-deficient hepatitis B virus (HBV) transgenic mice fail to support viral biosynthesis. The levels of viral transcription and replication intermediates are dramatically reduced. Hepatitis B core antigen is only observed in a very limited number of pericentral hepatocytes in a pattern that is similar to glutamate-ammonia ligase (Glul), a ß-catenin target gene. HBV transcript abundance in adult Tet-deficient mice resembles that observed in wild-type neonatal mice. Furthermore, the RNA levels of several ß-catenin target genes including Glul, Lhpp, Notun, Oat, Slc1a2, and Tbx3 in Tet-deficient mice were also similar to that observed in wild-type neonatal mice. As HBV transcription is regulated by ß-catenin, these findings support the suggestion that neonatal Tet deficiency might limit ß-catenin target gene expression, limiting viral biosynthesis. Additionally, HBV transgene DNA displays increased 5-methylcytosine (5mC) frequency at CpG sequences consistent with neonatal Tet deficiency being responsible for decreased developmental viral DNA demethylation mediated by 5mC oxidation to 5-hydroxymethylcytosine, a process that might be responsible for the reduction in cellular ß-catenin target gene expression and viral transcription and replication.IMPORTANCEChronic hepatitis B virus (HBV) infection causes significant worldwide morbidity and mortality. There are no curative therapies available to resolve chronic HBV infections, and the small viral genome limits molecular targets for drug development. An alternative approach to drug development is to target cellular genes essential for HBV biosynthesis. In the liver, ten-eleven translocation (Tet) genes encode cellular enzymes that are not essential for postnatal mouse development but represent essential activities for viral DNA demethylation and transcription. Consequently, Tet inhibitors may potentially be developed into therapeutic agents capable of inducing and/or maintaining HBV covalently closed circular DNA methylation, resulting in transcriptional silencing and the resolution of chronic viral infection.

Role of DNA Methylation Profiles as Potential Biomarkers and Novel Therapeutic Targets in Head and Neck Cancer.

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide and is associated with high mortality. The main reasons for treatment failure are a low rate of early diagnosis, high relapse rates, and distant metastasis with poor outcomes. These are largely due to a lack of diagnostic, prognostic, and predictive biomarkers in HNSCC. DNA methylation has been demonstrated to play an important role in the pathogenesis of HNSCC, and recent studies have also valued DNA methylation as a potential biomarker in HNSCC. This review summarizes the current knowledge on DNA methylation profiles in HPV-positive and HPV-negative HNSCC and how these may contribute to the pathogenesis of HNSCC. It also summarizes the potential value of DNA methylation as a biomarker in the diagnosis, prognosis, and prediction of the response to therapy. With the recent immunotherapy era in head and neck treatment, new strategies to improve immune responses by modulating TIMEs have been intensely investigated in early-phase trials. Therefore, this study additionally summarizes the role of DNA methylation in the regulation of TIMEs and potential predictive immunotherapy response biomarkers. Finally, this study reviews ongoing clinical trials using DNA methylation inhibitors in HNSCC.

Pharmacologic Ascorbate and DNMT Inhibitors Increase DUOX Expression and Peroxide-Mediated Toxicity in Pancreatic Cancer.

Recent studies have demonstrated an important role for vitamin C in the epigenetic regulation of cancer-related genes via DNA demethylation by the ten-eleven translocation (TET) methylcytosine dioxygenase enzymes. DNA methyltransferase (DNMT) reverses this, increasing DNA methylation and decreasing gene expression. Dual oxidase (DUOX) enzymes produce hydrogen peroxide (H2O2) in normal pancreatic tissue but are silenced in pancreatic cancer (PDAC). Treatment of PDAC with pharmacologic ascorbate (P-AscH-, intravenous, high dose vitamin C) increases DUOX expression. We hypothesized that inhibiting DNMT may act synergistically with P-AscH- to further increase DUOX expression and cytotoxicity of PDAC. PDAC cells demonstrated dose-dependent increases in DUOX mRNA and protein expression when treated with DNMT inhibitors. PDAC cells treated with P-AscH- + DNMT inhibitors demonstrated increased DUOX expression, increased intracellular oxidation, and increased cytotoxicity in vitro and in vivo compared to either treatment alone. These findings suggest a potential therapeutic, epigenetic mechanism to treat PDAC.

Inhibiting Histone and DNA Methylation Improves Cancer Vaccination in an Experimental Model of Melanoma.

Immunotherapy has improved the treatment of malignant skin cancer of the melanoma type, yet overall clinical response rates remain low. Combination therapies could be key to meet this cogent medical need. Because epigenetic hallmarks represent promising combination therapy targets, we studied the immunogenic potential of a dual inhibitor of histone methyltransferase G9a and DNA methyltransferases (DNMTs) in the preclinical B16-OVA melanoma model. Making use of tumor transcriptomic and functional analyses, methylation-targeted epigenetic reprogramming was shown to induce tumor cell cycle arrest and apoptosis in vitro coinciding with transient tumor growth delay and an IFN-I response in immune-competent mice. In consideration of a potential impact on immune cells, the drug was shown not to interfere with dendritic cell maturation or T-cell activation in vitro. Notably, the drug promoted dendritic cell and, to a lesser extent, T-cell infiltration in vivo, yet failed to sensitize tumor cells to programmed cell death-1 inhibition. Instead, it increased therapeutic efficacy of TCR-redirected T cell and dendritic cell vaccination, jointly increasing overall survival of B16-OVA tumor-bearing mice. The reported data confirm the prospect of methylation-targeted epigenetic reprogramming in melanoma and sustain dual G9a and DNMT inhibition as a strategy to tip the cancer-immune set-point towards responsiveness to active and adoptive vaccination against melanoma.

DNA Methylation and Establishing Memory.

A single event can cause a life-long memory. Memories physically reside in neurons, and changes in neuronal gene expression are considered to be central to memory. Early models proposed that specific DNA methylations of cytosines in neuronal DNA encode memories in a stable biochemical form. This review describes recent research that elucidates the molecular mechanisms used by the mammalian brain to form DNA methylcytosine encoded memories. For example, neuron activation initiates cytosine demethylation by stimulating DNA topoisomerase II beta (TOP2B) protein to make a temporary DNA double-strand break (repaired within about 2 hours) at a promoter of an immediate early gene, EGR1, allowing expression of this gene. The EGR1 proteins then recruit methylcytosine dioxygenase TET1 proteins to initiate demethylation at several hundred genes, facilitating expression of those genes. Initiation of demethylation of cytosine also occurs when OGG1 localizes at oxidized guanine in a methylated CpG site and recruits TET1 for initiation of demethylation at that site. DNMT3A2 is another immediate early gene upregulated by synaptic activity. DNMT3A2 protein catalyzes de novo DNA methylations. These several mechanisms convert external experiences into DNA methylations and initiated demethylations of neuronal DNA cytosines, causing changes in gene expression that are the basis of long-term memories.

AML-Associated Mutations in DNA Methyltransferase DNMT3A.

In mammals, DNA methylation is an essential epigenetic modification necessary for the maintenance of genome stability, regulation of gene expression, and other processes. Carcinogenesis is accompanied by multiple changes in the DNA methylation pattern and DNA methyltransferase (DNMT) genes; these changes are often associated with poor disease prognosis. Human DNA methyltransferase DNMT3A is responsible for de novo DNA methylation. Missense mutations in the DNMT3A gene occur frequently at the early stages of tumor development and are often observed in hematologic malignances, especially in acute myeloid leukemia (AML), with a prevalence of the R882H mutation. This mutation is the only one that has been extensively studied using both model DNA substrates and cancer cell lines. Biochemical characterization of other DNMT3A mutants is necessary to assess their potential effects on the DNMT3A functioning. In this review, we describe DNMT3A mutations identified in AML with special emphasis on the missense mutations in the DNMT3A catalytic domain. The impact of R882H and less common missense mutations on the DNMT3A activity toward model DNA substrates and in cancer cell lines is discussed together with the underlying molecular mechanisms. Understanding general features of these mechanisms will be useful for further development of novel approaches for early diagnostics of hematologic diseases and personalized cancer therapy.

Oxygen sensing, mitochondrial biology and experimental therapeutics for pulmonary hypertension and cancer.

The homeostatic oxygen sensing system (HOSS) optimizes systemic oxygen delivery. Specialized tissues utilize a conserved mitochondrial sensor, often involving NDUFS2 in complex I of the mitochondrial electron transport chain, as a site of pO2-responsive production of reactive oxygen species (ROS). These ROS are converted to a diffusible signaling molecule, hydrogen peroxide (H2O2), by superoxide dismutase (SOD2). H2O2 exits the mitochondria and regulates ion channels and enzymes, altering plasma membrane potential, intracellular Ca2+ and Ca2+-sensitization and controlling acute, adaptive, responses to hypoxia that involve changes in ventilation, vascular tone and neurotransmitter release. Subversion of this O2-sensing pathway creates a pseudohypoxic state that promotes disease progression in pulmonary arterial hypertension (PAH) and cancer. Pseudohypoxia is a state in which biochemical changes, normally associated with hypoxia, occur despite normal pO2. Epigenetic silencing of SOD2 by DNA methylation alters H2O2 production, activating hypoxia-inducible factor 1α, thereby disrupting mitochondrial metabolism and dynamics, accelerating cell proliferation and inhibiting apoptosis. Other epigenetic mechanisms, including dysregulation of microRNAs (miR), increase pyruvate dehydrogenase kinase and pyruvate kinase muscle isoform 2 expression in both diseases, favoring uncoupled aerobic glycolysis. This Warburg metabolic shift also accelerates cell proliferation and impairs apoptosis. Disordered mitochondrial dynamics, usually increased mitotic fission and impaired fusion, promotes disease progression in PAH and cancer. Epigenetic upregulation of dynamin-related protein 1 (Drp1) and its binding partners, MiD49 and MiD51, contributes to the pathogenesis of PAH and cancer. Finally, dysregulation of intramitochondrial Ca2+, resulting from impaired mitochondrial calcium uniporter complex (MCUC) function, links abnormal mitochondrial metabolism and dynamics. MiR-mediated decreases in MCUC function reduce intramitochondrial Ca2+, promoting Warburg metabolism, whilst increasing cytosolic Ca2+, promoting fission. Epigenetically disordered mitochondrial O2-sensing, metabolism, dynamics, and Ca2+ homeostasis offer new therapeutic targets for PAH and cancer. Promoting glucose oxidation, restoring the fission/fusion balance, and restoring mitochondrial calcium regulation are promising experimental therapeutic strategies.

Relative DNA Methylation and Demethylation Efficiencies during Postnatal Liver Development Regulate Hepatitis B Virus Biosynthesis.

Hepatitis B virus (HBV) transcription and replication increase progressively throughout postnatal liver development with maximal viral biosynthesis occurring at around 4 weeks of age in the HBV transgenic mouse model of chronic infection. Increasing viral biosynthesis is associated with a corresponding progressive loss of DNA methylation. The loss of DNA methylation is associated with increasing levels of 5-hydroxymethylcytosine (5hmC) residues which correlate with increased liver-enriched pioneer transcription factor Forkhead box protein A (FoxA) RNA levels, a rapid decline in postnatal liver DNA methyltransferase (Dnmt) transcripts, and a very modest reduction in ten-eleven translocation (Tet) methylcytosine dioxygenase expression. These observations are consistent with the suggestion that the balance between active HBV DNA methylation and demethylation is regulated by FoxA recruitment of Tet in the presence of declining Dnmt activity. These changes lead to demethylation of the viral genome during hepatocyte maturation with associated increases in viral biosynthesis. Consequently, manipulation of the relative activities of these two counterbalancing processes might permit the specific silencing of HBV gene expression with the loss of viral biosynthesis and the resolution of chronic HBV infections.IMPORTANCE HBV biosynthesis begins at birth and increases during early postnatal liver development in the HBV transgenic mouse model of chronic infection. The levels of viral RNA and DNA synthesis correlate with pioneer transcription factor FoxA transcript plus Tet methylcytosine dioxygenase-generated 5hmC abundance but inversely with Dnmt transcript levels and HBV DNA methylation. Together, these findings suggest that HBV DNA methylation during neonatal liver development is actively modulated by the relative contributions of FoxA-recruited Tet-mediated DNA demethylation and Dnmt-mediated DNA methylation activities. This mode of gene regulation, mediated by the loss of DNA methylation at hepatocyte-specific viral and cellular promoters, likely contributes to hepatocyte maturation during liver development in addition to the postnatal activation of HBV transcription and replication.

DNA Methylation in Nonalcoholic Fatty Liver Disease.

Nonalcoholic fatty liver disease (NAFLD) is a widespread hepatic disorder in the United States and other Westernized countries. Nonalcoholic steatohepatitis (NASH), an advanced stage of NAFLD, can progress to end-stage liver disease, including cirrhosis and liver cancer. Poor understanding of mechanisms underlying NAFLD progression from simple steatosis to NASH has limited the development of effective therapies and biomarkers. An accumulating body of studies has suggested the importance of DNA methylation, which plays pivotal roles in NAFLD pathogenesis. DNA methylation signatures that can affect gene expression are influenced by environmental and lifestyle experiences such as diet, obesity, and physical activity and are reversible. Hence, DNA methylation signatures and modifiers in NAFLD may provide the basis for developing biomarkers indicating the onset and progression of NAFLD and therapeutics for NAFLD. Herein, we review an update on the recent findings in DNA methylation signatures and their roles in the pathogenesis of NAFLD and broaden people's perspectives on potential DNA methylation-related treatments and biomarkers for NAFLD.

Characterization of stress response involved in chicken myopathy.

Myopathies (Woody Breast (WB) and White Striping (WS)) of broiler chickens have been correlated with fast growth. Recent studies reported that localized hypoxia and metabolic impairment may involve in these myopathies of birds. In order to better understand the stress response mechanisms affecting myopathies of broilers, the aim of this study was to examine effects of WB and both WB/WS on stress hormone corticosterone (CORT) levels and expressional changes of stress response genes including glucocorticoid (GC) receptor (GR), 11ß-Hydroxysteroid dehydrogenase type 1 (11ß-HSD1), DNA methylation regulators (DNMTs), and arginine vasotocin receptor 1a and 1b (V1aR, V1bR). Results of radioimmunoassay showed that CORT levels of WB and WB/WS birds were significantly higher compared to Con (p < 0.05), however, the combination of WB/WS was not significantly higher than WB birds, implying that the effects of WB and WS on CORT are not synergistic. Hepatic GR expression of both WB and WB/WS birds were significantly higher compared to Con (p < 0.05). However, GR expression levels in breast muscle of both WB and WB/WS birds were decreased compared to Con (p < 0.05). Hepatic 11ß-HSD1 expression was increased only in WB/WS birds compared to Con birds with no significant difference between Con and WB birds. 11ß-HSD1 expression was decreased and increased in WB and WB/WS birds compared to Con, respectively, in breast muscle (p < 0.05). DNMT1 expression was significantly decreased in both muscle and liver of WB birds, and in muscle of WB/WS birds, but not in liver of WB/WS birds, indicating differential effects of WS on the epigenetical stress response of muscle and liver compared to WB. V1aR expression was significantly increased in muscle of WB birds, and in liver of WB/WS birds compared to Con birds (p < 0.05). V1bR was not changed in muscle and liver of WB birds compared to Con birds. Taken together, results suggest that GC-induced myopathies occur in fast-growing broiler chickens and circulating CORT level might be a significant biochemical marker of myopathies (WB and WS) of birds. In addition, chronic stress responses in breast muscle and tissue-specific epigenetic changes of stress response genes by DNMTs may play a critical role in the occurrence of myopathies.

Inhibition of DNA methylation in proliferating human lymphoma cells by immune cell oxidants.

Excessive generation of oxidants by immune cells results in acute tissue damage. One mechanism by which oxidant exposure could have long-term effects is modulation of epigenetic pathways. We hypothesized that methylation of newly synthesized DNA in proliferating cells can be altered by oxidants that target DNA methyltransferase activity or deplete its substrate, the methyl donor SAM. To this end, we investigated the effect of two oxidants produced by neutrophils, H2O2 and glycine chloramine, on maintenance DNA methylation in Jurkat T lymphoma cells. Using cell synchronization and MS-based analysis, we measured heavy deoxycytidine isotope incorporation into newly synthesized DNA and observed that a sublethal bolus of glycine chloramine, but not H2O2, significantly inhibited DNA methylation. Both oxidants inhibited DNA methyltransferase 1 activity, but only chloramine depleted SAM, suggesting that removal of substrate was the most effective means of inhibiting DNA methylation. These results indicate that immune cell-derived oxidants generated during inflammation have the potential to affect the epigenome of neighboring cells.

MiR-30a and miR-379 modulate retinoic acid pathway by targeting DNA methyltransferase 3B in oral cancer.

BACKGROUND: Epigenetic silencing of retinoic acid (RA) signaling-related genes have been linked with the pathogenesis and clinical outcome in oral squamous cell carcinoma (OSCC) carcinogenesis. However, the precise mechanisms underlying the abnormal silencing of RA signaling-related genes in OSCC have not been well investigated. METHODS: Using combined analysis of genome-wide gene expression and methylation profile from 40 matched normal-tumor pairs of OSCC specimens, we found a set of retinoid signaling related genes are frequently hypermethylated and downregulated in OSCC patient samples, including alcohol dehydrogenase, iron containing 1 (ADHFE1) and aldehyde dehydrogenase 1 family, member A2 (ALDH1A2), which are the important rate-limiting enzymes in synthesis of RA. The expression of ADHFE1 and ALDH1A2 in OSCC patients was determine by quantitative real-time PCR (qRT-PCR) and immunohistochemistry. The binding sites of miR-30a and miR-379 with DNA methyltransferase 3B (DNMT3B) were predicted using a series of bioinformatic tools, and validated using dual luciferase assay and Western blot analyses. The functions of miR-30a, miR-379, and DNMT3B were accessed by growth and colony formation analyses using gain- and loss-of-function approaches. Chromatin immunoprecipitation (ChIP) was performed to explore the molecular mechanisms by arecoline and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) treatment. RESULTS: We demonstrated that deregulated miR-30a and miR-379 could represent a mechanism for the silencing of ADHFE1 and ALDH1A2 in OSCC through targeting DNMT3B. Ectopic expression of miR-30a and miR-379 could induce re-expression of methylation-silenced ADHFE1 and ALDH1A2, and lead to growth inhibition in oral cancer cells. Furthermore, the dysregulation of the miRNAs and DNMT-3B may result from exposure to tobacco smoking and betel quid chewing. CONCLUSIONS: Our results demonstrate that tobacco smoking and betel quid chewing could repress miR-30a and miR-379, which upregulate the DNMT3B expression, in turn, lead to the hypermethylation of ADHFE1 and ALDH1A genes, consequently, promote the oncogenic activity. These findings highlight the potential use of retinoids in combination with epigenetic modifiers for the prevention or treatment of oral cancer.

Sp1 promotes ovarian cancer cell migration through repressing miR-335 expression.

Decreased miR-335 has been reported in a variety of cancers. We previously showed that miR-335 played an important role in ovarian cancer metastasis and prognosis. However, miR-335 is down-regulated in ovarian cancer by mechanisms that remain unclear. In silico analysis identified putative transcription factor specificity protein 1 (SP1) transcription factor binding sites in the miR-335 promoter. To investigate the relation between SP1 and miR-335, qRT-PCR was performed. Our results showed both Sp1 knockdown and mithramycin A increased miR-335 expression in ovarian cancer cell lines. Luciferase reporter assays indicated that Sp1 knockdown increased miR-335 transcriptional activity. ChIP experiments showed that Sp1 bound directly to miR-335 promoter. Moreover, transwell migration and wound-healing assays showed that Sp1 knockdown resulted in inhibited cell migration, which was in turn mitigated by miR-335 inhibitor. We propose that miR-335 was negatively regulated by SP1, which in turn contributes to miR-335 deregulation and tumor cells migration.

Functional Analysis of DNMT3A DNA Methyltransferase Mutations Reported in Patients with Acute Myeloid Leukemia.

In mammals, DNA methylation is necessary for the maintenance of genomic stability, gene expression regulation, and other processes. During malignant diseases progression, changes in both DNA methylation patterns and DNA methyltransferase (MTase) genes are observed. Human de novo MTase DNMT3A is most frequently mutated in acute myeloid leukemia (AML) with a striking prevalence of R882H mutation, which has been extensively studied. Here, we investigate the functional role of the missense mutations (S714C, R635W, R736H, R771L, P777R, and F752V) found in the catalytic domain of DNMT3A in AML patients. These were accordingly mutated in the murine Dnmt3a catalytic domain (S124C, R45W, R146H, R181L, P187R, and F162V) and in addition, one-site CpG-containing DNA substrates were used as a model system. The 3-15-fold decrease (S124C and P187R) or complete loss (F162V, R45W, and R146H) of Dnmt3a-CD methylation activity was observed. Remarkably, Pro 187 and Arg 146 are not located at or near the Dnmt3a functional motives. Regulatory protein Dnmt3L did not enhance the methylation activity of R45W, R146H, P187R, and F162V mutants. The key steps of the Dnmt3a-mediated methylation mechanism, including DNA binding and transient covalent intermediate formation, were examined. There was a complete loss of DNA-binding affinity for R45W located in the AdoMet binding region and for R146H. Dnmt3a mutants studied in vitro suggest functional impairment of DNMT3A during pathogenesis.

A phase 1 trial of the oral DNA methyltransferase inhibitor CC-486 and the histone deacetylase inhibitor romidepsin in advanced solid tumors.

BACKGROUND: Epigenetic abnormalities are manifold in all solid tumors and include changes in chromatin configuration and DNA methylation. The authors designed a phase 1 study to evaluate the oral DNA methyltransferase inhibitor CC-486 combined with the histone deacetylase inhibitor romidepsin in advanced solid tumors with dose expansion to further evaluate pharmacodynamics and possible clinical benefit of the recommended phase 2 dose (RP2D). METHODS: This was a phase 1 study with a 3 + 3 dose-escalation design and an expansion phase for patients with virally mediated cancers. The disease control rate (DCR) was the primary outcome for the expansion cohort. Correlative studies included long interspersed nucleotide element 1 (LINE-1) methylation and drug exposure in blood samples (clinicaltrials.gov identifier NCT01537744). RESULTS: Fourteen patients were enrolled in the dose-escalation portion at 3 dose levels. Three patients experienced dose-limiting toxicities; the RP2D was oral CC-486 300 mg daily on days 1 through 14 and romidepsin 8 mg/m2 on days 8 and 15. Because of slow accrual into the expansion phase, the trial was closed after 4 patients enrolled. Common toxicities of the combination included nausea (83.3%), anorexia (72.2%), fatigue (61.1%), and constipation (55.6%). There were 12 patients evaluable for response, 5 with stable disease, of whom 2 received >4 cycles; there were no responses. Exposure to CC-486 and romidepsin was consistent with prior data. LINE-1 methylation on C1D8 was significantly reduced (mean, -6.23; 95% CI, -12.23, -0.24; P = .04). CONCLUSIONS: Although, at the RP2D, the combination of CC-486 and romidepsin was tolerable, no significant anticancer activity was observed. Significant demethylation in post-treatment circulating tumor DNA and biopsies provided proof of target acquisition.

Extracellular Spermine Activates DNA Methyltransferase 3A and 3B.

We first demonstrated that long-term increased polyamine (spermine, spermidine, putrescine) intake elevated blood spermine levels in mice and humans, and lifelong consumption of polyamine-rich chow inhibited aging-associated increase in aberrant DNA methylation, inhibited aging-associated pathological changes, and extend lifespan of mouse. Because gene methylation status is closely associated with aging-associated conditions and polyamine metabolism is closely associated with regulation of gene methylation, we investigated the effects of extracellular spermine supplementation on substrate concentrations and enzyme activities involved in gene methylation. Jurkat cells and human mammary epithelial cells were cultured with spermine and/or D,L-alpha-difluoromethylornithine (DFMO), an inhibitor of ornithine decarboxylase. Spermine supplementation inhibited enzymatic activities of adenosylmethionine decarboxylase in both cells. The ratio of decarboxylated S-adenosylmethionine to S-adenosyl-L-methionine increased by DFMO and decreased by spermine. In Jurkat cells cultured with DFMO, the protein levels of DNA methyltransferases (DNMTs) 1, 3A and 3B were not changed, however the activity of the three enzymes markedly decreased. The protein levels of these enzymes were not changed by addition of spermine, DNMT 3A and especially 3B were activated. We show that changes in polyamine metabolism dramatically affect substrate concentrations and activities of enzymes involved in gene methylation.

Hypermethylation of endoplasmic reticulum disulfide oxidase 1α leads to trophoblast cell apoptosis through endoplasmic reticulum stress in preeclampsia.

Abnormal trophoblast cell apoptosis is implicated in the pathogenesis of pregnancy-related disorders including preeclampsia (PE), and endoplasmic reticulum (ER) stress has been considered as a novel pathway in the regulation of cell apoptosis. In this study, we observed that both apoptosis and ER stress are triggered in trophoblast cells under hypoxia as well as in the placenta of PE rats. Quantitative polymerase chain reaction and Western blot analysis showed that the expression of endoplasmic reticulum disulfide oxidase 1α (ERO1α) is suppressed in trophoblast cells under hypoxia due to the hypermethylation of the ERO1α promoter region, and the inhibition of ERO1α expression plays an important role in ER stress and trophoblast cell apoptosis. Furthermore, we found that DNA methyltransferase 1 (DNMT1) is a key methyltransferase for DNA methylation in the regulation of ERO1α expression, and the binding level of DNMT1 to the ERO1α promoter is markedly elevated under hypoxia although DNMT1 expression is inhibited by hypoxia, suggesting that the binding level of DNMT1 to the ERO1α promoter region rather than the DNMT1 expression level contributes to the hypermethylation of ERO1α. Taken together, these results demonstrate that the hypermethylation of ERO1α mediated by increased binding of DNMT1 to the ERO1α promoter leads to trophoblast cell apoptosis through ER stress in the placenta of PE rats, which shed insight into the etiology of PE and might present a validated therapeutic target for the treatment of PE.

Iodine excess did not affect the global DNA methylation status and DNA methyltransferase expression in T and B lymphocytes from NOD.H-2h4 and Kunming mice.

Dysregulated DNA methylation in lymphocytes has been linked to various autoimmune disorders. Excessive iodine intake leads to lymphocyte dysfunction and contributes to autoimmune thyroiditis (AIT) flares in humans and animals. However, whether excessive iodine modifies the DNA methylation status in lymphocytes is unknown. Twenty NOD.H-2h4 mice and 20 Kunming mice were randomly divided into high iodine and control groups. We scored lymphatic infiltration in the thyroid by hematoxylin and eosin (H&E) staining and assayed serum thyroglobulin antibody (TgAb) levels by an indirect enzyme-linked immunosorbent assay. CD3+ T cells and CD19+ B cells were separated by flow cytometry. Global DNA methylation levels were examined by absorptiometry. Methylation of long interspersed nucleotide element-1 (LINE-1) repeats was detected with bisulfite sequencing PCR. Expression of DNA methyltransferase (DNMT) 1, DNMT3a and DNMT3b mRNA and protein were determined by real-time PCR and Western blot, respectively. We observed evident thyroiditis in the high­iodine-treated NOD.H-2h4 mice, while mice in the other three groups did not develop thyroiditis. No differences were found in the global methylation levels and methylation status of LINE-1 repeats in T and B lymphocytes from high­iodine-treated NOD.H-2h4 mice and Kunming mice compared with those from normal­iodine-supplemented controls. We did not find obvious changes in DNMT mRNA and protein expression levels in T and B lymphocytes among the studied groups. In conclusion, we showed for the first time that excess iodine did not affect the global methylation status or DNMT expression in T and B lymphocytes in NOD.H-2h4 and Kunming mice.

The Crucial Role of DNA Methylation and MeCP2 in Neuronal Function.

A neuron is unique in its ability to dynamically modify its transcriptional output in response to synaptic activity while maintaining a core gene expression program that preserves cellular identity throughout a lifetime that is longer than almost every other cell type in the body. A contributing factor to the immense adaptability of a neuron is its unique epigenetic landscape that elicits locus-specific alterations in chromatin architecture, which in turn influences gene expression. One such epigenetic modification that is sensitive to changes in synaptic activity, as well as essential for maintaining cellular identity, is DNA methylation. The focus of this article is on the importance of DNA methylation in neuronal function, summarizing recent studies on critical players in the establishment of (the "writing"), the modification or erasure of (the "editing"), and the mediation of (the "reading") DNA methylation in neurodevelopment and neuroplasticity. One "reader" of DNA methylation in particular, methyl-CpG-binding protein 2 (MeCP2), is highlighted, given its undisputed importance in neuronal function.

DNA Methylation and Adult Neurogenesis.

The role of DNA methylation in brain development is an intense area of research because the brain has particularly high levels of CpG and mutations in many of the proteins involved in the establishment, maintenance, interpretation, and removal of DNA methylation impact brain development and/or function. These include DNA methyltransferase (DNMT), Ten-Eleven Translocation (TET), and Methyl-CpG binding proteins (MBPs). Recent advances in sequencing breadth and depth as well the detection of different forms of methylation have greatly expanded our understanding of the diversity of DNA methylation in the brain. The contributions of DNA methylation and associated proteins to embryonic and adult neurogenesis will be examined. Particular attention will be given to the impact on adult hippocampal neurogenesis (AHN), which is a key mechanism contributing to brain plasticity, learning, memory and mood regulation. DNA methylation influences multiple aspects of neurogenesis from stem cell maintenance and proliferation, fate specification, neuronal differentiation and maturation, and synaptogenesis. In addition, DNA methylation during neurogenesis has been shown to be responsive to many extrinsic signals, both under normal conditions and during disease and injury. Finally, crosstalk between DNA methylation, Methyl-DNA binding domain (MBD) proteins such as MeCP2 and MBD1 and histone modifying complexes is used as an example to illustrate the extensive interconnection between these epigenetic regulatory systems.