Global 5mC Levels Research Articles

Methylation aberrations in partner spermatozoa and impaired expression of imprinted genes in the placentae of early-onset preeclampsia

IntroductionDisturbed paternal epigenetic status of imprinted genes has been observed in infertility and recurrent spontaneous abortions. Shallow placentation has been associated with early-onset preeclampsia. Hence, the present study aimed to investigate the methylation patterns of imprinted genes involved in placental development, in the spermatozoa of partners of women experiencing preeclampsia. MethodsThe study involved recruitment of couples into preeclampsia (n = 14) and control (n = 25) groups. Methylation analysis of imprinted gene differentially methylated regions (DMRs) and LINE1 repetitive element was carried out by pyrosequencing in the spermatozoa and placental villi. Global 5mC levels in the spermatozoa were measured through ELISA. Expression of imprinted genes was quantified in the placental villi by real time qPCR. Association of birth weight with DNA methylation and gene expression was assessed. ResultsKvDMR, PEG3 DMR, PEG10 DMR and DLK1-GTL2 IG-DMR were differentially methylated in the spermatozoa and placental villi of preeclampsia group. Global 5mC content and LINE1 methylation levels did not differ between the spermatozoa of the two groups. Increased transcript levels of PEG3, IGF2, DLK1, PHLDA2 and CDKN1C were observed in the preeclamptic placental villi. Birth weight showed significant association with KvDMR, PEG10 DMR, DLK1-GTL2 IG-DMR and LINE1 methylation levels in the spermatozoa. DLK1 expression levels showed a negative association with birth weight. DiscussionThe study highlighted the paternal contribution to early-onset preeclampsia, in the form of disrupted sperm DNA methylation patterns at imprinted gene loci. These loci, after further evaluation in future studies, could serve as sperm-based preeclampsia predictive markers, for couples planning pregnancy.

High estrogen during ovarian stimulation induced loss of maternal imprinted methylation that is essential for placental development via overexpression of TET2 in mouse oocytes.

Ovarian stimulation (OS) during assisted reproductive technology (ART) appears to be an independent factor influencing the risk of low birth weight (LBW). Previous studies identified the association between LBW and placenta deterioration, potentially resulting from disturbed genomic DNA methylation in oocytes caused by OS. However, the mechanisms by which OS leads to aberrant DNA methylation patterns in oocytes remains unclear. Mouse oocytes and mouse parthenogenetic embryonic stem cells (pESCs) were used to investigate the roles of OS in oocyte DNA methylation. Global 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) levels were evaluated using immunofluorescence or colorimetry. Genome-wide DNA methylation was quantified using an Agilent SureSelectXT mouse Methyl-Seq. The DNA methylation status of mesoderm-specific transcript homologue (Mest) promoter region was analyzed using bisulfite sequencing polymerase chain reaction (BSP). The regulatory network between estrogen receptor alpha (ERα, ESR1) and DNA methylation status of Mest promoter region was further detected following the knockdown of ERα or ten-eleven translocation 2 (Tet2). OS resulted in a significant decrease in global 5mC levels and an increase in global 5hmC levels in oocytes. Further investigation revealed that supraphysiological β-estradiol (E2) during OS induced a notable decrease in DNA 5mC and an increase in 5hmC in both oocytes and pESCs of mice, whereas inhibition of estrogen signaling abolished such induction. Moreover, Tet2 may be a direct transcriptional target gene of ERα, and through the ERα-TET2 axis, supraphysiological E2 resulted in the reduced global levels of DNA 5mC. Furthermore, we identified that MEST, a maternal imprinted gene essential for placental development, lost its imprinted methylation in parthenogenetic placentas originating from OS, and ERα and TET2 combined together to form a protein complex that may promote Mest demethylation. In this study, a possible mechanism of loss of DNA methylation in oocyte caused by OS was revealed, which may help increase safety and reduce epigenetic abnormalities in ART procedures.

Fine-grained investigation of the relationship between human nutrition and global DNA methylation patterns.

Nutrition is an important, modifiable, environmental factor affecting human health by modulating epigenetic processes, including DNA methylation (5mC). Numerous studies investigated the association of nutrition with global and gene-specific DNA methylation and evidences on animal models highlighted a role in DNA hydroxymethylation (5hmC) regulation. However, a more comprehensive analysis of different layers of nutrition in association with global levels of 5mC and 5hmC is lacking. We investigated the association between global levels of 5mC and 5hmC and human nutrition, through the stratification and analysis of dietary patterns into different nutritional layers: adherence to Mediterranean diet (MD), main food groups, macronutrients and micronutrients intake. ELISA technique was used to measure global 5mC and 5hmC levels in 1080 subjects from the Moli-sani cohort. Food intake during the 12months before enrolment was assessed using the semi-quantitative EPIC food frequency questionnaire. Complementary approaches involving both classical statistics and supervised machine learning analyses were used to investigate the associations between global 5mC and 5hmC levels and adherence to Mediterranean diet, main food groups, macronutrients and micronutrients intake. We found that global DNA methylation, but not hydroxymethylation, was associated with daily intake of zinc and vitamin B3. Random Forests algorithms predicting 5mC and 5hmC through intakes of food groups, macronutrients and micronutrients revealed a significant contribution of zinc, while vitamin B3 was reported among the most influential features. We found that nutrition may affect global DNA methylation, suggesting a contribution of micronutrients previously implicated as cofactors in methylation pathways.

SUN-LB009 Epigenetic Changes in Response to Metabolic Modifiers in Late Life: Exercise, High Fat Diet, and Angiotenin1-7 Effects on Metabolic Health and DNA Methylation in Frail Old Mice

Background: Reduced muscle and bone-mass and quality as well as impaired energy homeostasis are seen in both aging and the metabolic syndrome (MetS). We previously showed that exercise and Angiotensin1-7 (A1-7) ameliorate this burden in young rats, but their effect vis-a-vis aging is unknown. Aims: To assess the effects of exercise under normal and high-fat diet (HFD) with or without A1-7 on bone architecture and muscle quality as well as on musculoskeletal and fat epigenetic signatures in aged mice. Methods: 19-months-old-mice receiving HFD or normal-chow (NC) with or without 3-months exercise (treadmill, 6 days/week, 20 min/day or A1-7 (0.6 mcg/kg/d). Bone was evaluated by microCT. Global quantification of the epigenetic modification 5-methyl-or hydroxyl-methyl cytosine (5mC; 5hmc) by LC-MS/MS in white/brown adipose tissue (WAT; BAT) and Gastrocnemius muscle. Results: Old, sedentary mice were metabolically "starved" with low serum glucose and triglycerides. (A) Neither exercise nor high fat diet elicited sustained changes in body weight in old mice. B) Exercise per se rehabilitated glucose, triglycerides levels and muscle quality (enhanced muscle-fiber sectional area), accompanied by reduction in muscle 5mC and in BAT 5hmc (p<0.05). (C) Combination of exercise with A1-7 improved bone geometry and density in NC mice but not during HFD. Combination of A1-7 and exercise led to a reduction in global 5mC levels both in the gastrocnemius muscle and in WAT (p<0.05), thus suggesting that some of the effects maybe induced by changes in methylation patterns. Conclusions: The beneficial effects of A1-7 and exercise in aging skeletal tissue are operative only in NC but not in the presence of HFD. These beneficial effects are accompanied, and indeed, may be partly mediated by reversal of gene silencing as depicted by lesser DNA methylation in skeletal muscle. Finally, this is the first report that exercise can induce epigenetic changes in BAT, independent of changes in body weight. Unless otherwise noted, all abstracts presented at ENDO are embargoed until the date and time of presentation. For oral presentations, the abstracts are embargoed until the session begins. s presented at a news conference are embargoed until the date and time of the news conference. The Endocrine Society reserves the right to lift the embargo on specific abstracts that are selected for promotion prior to or during ENDO.

Oral Vitamin C Supplementation to Azacitidine in Patients with Myeloid Cancer: Normalization of Plasma Vitamin C Induces Epigenetic Changes

▪IntroductionHematological cancer patients are often vitamin C deficient, and vitamin C is essential for the TET induced conversion of 5-methylcytosine (5mC) to 5-hydroxymethylsytosine (5hmC); the first step in active DNA demethylation. In tissue culture, we have shown that restoration of vitamin C to normal concentrations can potentiate the effect of DNA methyltransferase inhibitors (DNMTis) by activation of DNA demethylation and induction of genes in the viral defense pathway, so called ‘viral mimicry’ (Liu et al. PNAS 2016). Here, we investigated whether oral vitamin C supplementation can correct vitamin C deficiency, enhance the efficacy of active DNA demethylation and induce upregulation of genes in the viral defense pathway in patients with myeloid cancers treated with the DNMTi 5-azacytidine.Study Design and MethodsA randomized, placebo-controlled clinical trial of myelodysplastic syndrome (MDS; n=9), chronic myelomonocytic leukemia (CMML; n=4) and acute myeloid leukemia (AML; n=7) patients was performed during 3 cycles/12 weeks of DNMTi treatment (5-azacytidine, 100mg/m2 day 1 to 5 in a 4-week cycle) supplemented by oral dose of 500mg vitamin C (n=10) or placebo (n=10) daily during the last 8 weeks (Figure 1). Blood samples were drawn on day 1 and 5 of each treatment cycle before 5-azacytidine was administered and on day 28 of the third treatment cycle. Total plasma vitamin C was measured by HPLC in samples that had been acidified by 10% meta-phosphoric acid immediately after blood drawn. Mutational status of the 20 most commonly mutated genes in MDS were conducted by targeted next-generation sequencing. Global levels of 5mC and 5hmC were measured with LC-MS/MS in DNA extracted from MACS-sorted malignant cells and quoted relative to total levels of deoxyguanine. Total RNA-seq was performed on 20 RNA samples from 6 patients; cDNA libraries were prepared using KAPA RNA HyperPrep Kits and sequencing on a NextSeq 500 instrument (Illumina).ResultsFourteen patients were deficient in plasma vitamin C (<23 µM) and 4 of the remaining 6 patients took vitamin supplement at inclusion. Global DNA methylation was significantly higher in patients with severe vitamin C deficiency (<11.4 µM; P=0.004) and borderline significantly higher in DNMTi naïve (n=11) compared to non-naïve patients (P=0.095). At baseline, global 5hmC/5mC levels were lower in the 7 patients with TET2 mutations (n=7; P= 0.013). Oral supplementation restored plasma vitamin C levels to the normal range in all patients in the vitamin C arm (P<0.0005). We show for the first time that active DNA demethylation, estimated as the change in global 5hmC/5mC levels, was significantly increased in patients receiving vitamin C compared to placebo (P=0.041). Preliminary RNA sequencing data show increased upregulation of genes involved in the viral defense pathway, including IRF7 and IFIT1, in vitamin C supplemented patients that were DNMTi naïve at study inclusion.ConclusionsThe increase in active DNA demethylation, indicated by the elevation of 5hmC/5mC levels in myeloid cells from vitamin C supplemented patients compared to placebos, plus the increased expression of viral defense genes in vitamin C treated DNMTi naïve patients, suggest that the efficacy of 5-azacytidine might be increased by oral supplementation of vitamin C. We suggest that normalization of plasma vitamin C by oral supplementation may enhance the biological effects of DNMTis in patients and prompts the investigation of the clinical relevance of vitamin C supplementation to DNMTis in a large randomized placebo controlled trial.Figure 1. Study design. Days 1, 5, and 28: before vitamin C/placebo exposure. Day 32: after short-term vitamin C/placebo exposure. Days 56, 60, and 84: after longer-term vitamin C/placebo exposure. C = cycle, D = day in cycle. [Display omitted] DisclosuresJones:ZYMO Corporation: Consultancy. Grønbæk:Celgene: Membership on an entity's Board of Directors or advisory committees; Otsuka Pharma: Membership on an entity's Board of Directors or advisory committees; Janssen Pharma: Membership on an entity's Board of Directors or advisory committees.

Distinctive epigenomes characterize glioma stem cells and their response to differentiation cues

BackgroundGlioma stem cells (GSCs) are a subpopulation of stem-like cells that contribute to glioblastoma (GBM) aggressiveness, recurrence, and resistance to radiation and chemotherapy. Therapeutically targeting the GSC population may improve patient survival, but unique vulnerabilities need to be identified.ResultsWe isolate GSCs from well-characterized GBM patient-derived xenografts (PDX), characterize their stemness properties using immunofluorescence staining, profile their epigenome including 5mC, 5hmC, 5fC/5caC, and two enhancer marks, and define their transcriptome. Fetal brain-derived neural stem/progenitor cells are used as a comparison to define potential unique and common molecular features between these different brain-derived cells with stem properties. Our integrative study reveals that abnormal expression of ten-eleven-translocation (TET) family members correlates with global levels of 5mC and 5fC/5caC and may be responsible for the distinct levels of these marks between glioma and neural stem cells. Heterogenous transcriptome and epigenome signatures among GSCs converge on several genes and pathways, including DNA damage response and cell proliferation, which are highly correlated with TET expression. Distinct enhancer landscapes are also strongly associated with differential gene regulation between glioma and neural stem cells; they exhibit unique co-localization patterns with DNA epigenetic mark switching events. Upon differentiation, glioma and neural stem cells exhibit distinct responses with regard to TET expression and DNA mark changes in the genome and GSCs fail to properly remodel their epigenome.ConclusionsOur integrative epigenomic and transcriptomic characterization reveals fundamentally distinct yet potentially targetable biologic features of GSCs that result from their distinct epigenomic landscapes.

High-level dietary cadmium exposure is associated with global DNA hypermethylation in the gastropod hepatopancreas.

5-methylcytosine (5mC) is a key epigenetic mark which influences gene expression and phenotype. In vertebrates, this epigenetic mark is sensitive to Cd exposure, but there is no information linking such an event with changes in global 5mC levels in terrestrial gastropods despite their importance as excellentecotoxicological bioindicators of metal contamination. Therefore, we first evaluated total 5mC content in DNA of the hepatopancreas of adult Cantareus aspersus with the aim to determine whether this epigenetic mark is responsive to Cd exposure. The experiment was conducted under laboratory conditions and involved a continuous exposure, multiple dose- and time-point (14, 28, and 56 days) study design. Hepatopancreas cadmium levels were measured using Flame Atomic Absorption Spectrometry and the percentage of 5-mC in samples using an ELISA-based colorimetric assay. Snail death rates were also assessed. Our results, for the first time, reveal the presence of 5mC in C. aspersus and provide evidence for Cd-induced changes in global 5mC levels in DNA of gastropods and mollusks. Although less sensitive than tissue accumulation, DNA methylation levels responded in a dose- and time-dependent manner to dietary cadmium, with exposure dose having a much stronger effect than exposure duration. An obvious trend of increasing 5mC levels was observed starting at 28 days of exposure to the second highest dose and this trend persisted at the two highest treatments for close to one month, when the experiment was terminated after 56 days. Moreover, a strong association was identified between Cd concentrations in the hepatopancreas and DNA methylation levels in this organ. These data indicate an overall trend towards DNA hypermethylation with elevated Cd exposure. No consistent lethal effect was observed, irrespective of time point and Cd-dosage. Overall, our findings suggest that the total 5mC content in DNA of the hepatopancreas of land snails is responsive to sublethal Cd exposure and give new insights into invertebrate environmental epigenetics.

Effect of Inflammatory Cytokines on DNA Methylation and Demethylation

ObjectiveTo determine the effects of inflammatory cytokines and mediators on expression of DNMT and TET enzymes in human gingival fibroblasts isolated from patients with periodontitis as well as normal and transformed cell lines.MethodsExpression levels of DNA methylatransferase (DNMT1 and 3a) and demethylase (TET1) were examined by RT‐PCR in cells treated for up to 24 hours with IL‐1, IL‐6 or prostaglandin E2. The role of PGE2 in IL‐1 suppression of DNMT3a expression was assessed by treating cells with IL‐1 in the presence and absence of NS‐398. Global levels of DNA methylation following treatment with IL‐1 were measured in an ELISA‐like assay.ResultsTreatment of HGF with IL‐1 caused a three‐fold increase in levels of DNMT1 mRNA over 24 hours, but a two‐fold decrease in DNMT3a mRNA in the same samples. Global levels of 5mC were increased in HGF treated with IL‐1 for 72 hours relative to untreated controls. PGE2 caused a dose‐dependent decrease in levels of DNMT3a similar to IL‐1, but also slightly decreased levels of DNMT1.ConclusionThese data are consistent with the hypothesis that exposure to inflammatory cytokines or PGE2 alters levels and/or activity of enzymes involved in DNA methylation, resulting in global and possibly gene‐specific changes which can lead to changes in cellular characteristics over time. Further experiments will be aimed at testing the effects of these cytokines on expression of DNMT and TET enzymes in normal and transformed cell lines, and at elucidating mechanisms involved in cytokine regulation of these enzymes.Support or Funding InformationThis work has been supported by the Philadelphia College of Osteopathic Medicine Division of Research.

Alterations in global DNA methylation and hydroxymethylation are not detected in Alzheimer's disease.

AimsGenetic factors do not seem to account fully for Alzheimer disease (AD) pathogenesis. There is evidence for the contribution of environmental factors, whose effect may be mediated by epigenetic mechanisms. Epigenetics involves the regulation of gene expression independently of DNA sequence and these epigenetic changes are influenced by age and environmental factors, with DNA methylation being one of the best characterized epigenetic mechanisms. The human genome is predominantly methylated on CpG motifs, which results in gene silencing; however methylation within the body of the gene may mark active transcription. There is evidence suggesting an involvement of environmental factors in the pathogenesis of Alzheimer's disease (AD), which prompted our study examining DNA methylation in this disorder.MethodsUsing immunohistochemistry with 5‐methylcytosine/5‐hydroxymethylcytosine antibodies we studied, in comparison with age matched controls, DNA methylation in sporadic and familial AD cases in the entorhinal cortex that exhibits substantial pathology and the cerebellum, which is relatively spared.ResultsNeuronal nuclear labelling with 5‐methylcytosine (5mC) and 5‐hydroxymethylcytosine (5hmC) was evident in all cases studied. We did not detect any significant change in the levels of nuclear staining in the AD samples compared to neurologically normal controls. In the entorhinal cortex we also examined global DNA methylation and hydroxymethylation using an enzyme‐linked immunosorbent assay (ELISA).ConclusionNo significant differences were found between AD and control cases in global levels of 5mC and 5hmC in the entorhinal cortex using immunohistochemistry and enzyme‐linked immunosorbent assays.

Altered DNA Hydroxymethylation and Methylation and High Particulate Matter Exposure in Beijing, China

Background: DNA methylation has been widely proposed as an epigenetic mediator of the effects of Particulate Matter (PM) exposure on human health. 5-hydroxymethylcytosine (5hmC), generated by oxidation of standard DNA methylation (5-methylcytosine, 5mC), has emerged as a novel epigenomic mark. 5hmC is both an intermediate for de-methylation and an active epigenetic mark that increases gene expression. Oxidant exposures such as PM may increase 5hmC, but effects in humans have not yet been evaluated. Methods: The Beijing Truck Driver Air Pollution Study included 60 office workers (OW) and 60 truck drivers (TD) examined on two workdays. Ambient PM10 was averaged from 27 Beijing monitors. Personal PM2.5 and Elemental Carbon (EC) were measured by portable monitors. We measured blood DNA global 5mC and 5hmC by ELISA. We fitted mixed-effects models adjusted for multiple confounders. Results: Ambient PM10 on the examination days was associated with increased global 5hmC in TD (β=0.012, 95% CI 0.004-0.021), and all participants (β=0.009, 95% CI 0.003- 0.015). Average ambient PM10 on days 4, 7, and 14 before the examinations was positively associated with global 5hmC in OW and all participants (p&lt;0.05). 5hmC was also positively associated with personal PM2.5 in OW (β=0.012, 95% CI 0.001-0.023), TD (β=0.012, 95% CI 0.002-0.022), and all participants (β=0.008, 95% CI 0.001-0.015). In contrast, global 5mC levels were associated among OW with personal EC (β=0.564, 95% CI 0.058-1.070), and marginally with personal PM2.5 (β=0.401, 95% CI 0.004-0.798). Conclusion: 5hmC and 5mC showed different patterns of association with PM measures and time windows. 5hmC may reflect epigenetic information not captured in 5mC.

Global changes in DNA methylation and hydroxymethylation in Alzheimer's disease human brain

DNA methylation (5-methylcytosine [5mC]) is one of several epigenetic markers altered in Alzheimer's disease (AD) brain. More recently, attention has been given to DNA hydroxymethylation (5-hydroxymethylcytosine [5hmC]), the oxidized form of 5mC. Whereas 5mC is generally associated with the inhibition of gene expression, 5hmC has been associated with increased gene expression and is involved in cellular processes such as differentiation, development, and aging. Recent findings point toward a role for 5hmC in the development of diseases including AD, potentially opening new pathways for treating AD through correcting methylation and hydroxymethylation alterations. In the present study, levels of 5mC and 5hmC were investigated in the human middle frontal gyrus (MFG) and middle temporal gyrus (MTG) by immunohistochemistry. Immunoreactivity for 5mC and 5hmC were significantly increased in AD MFG (N = 13) and MTG (N = 29) compared with age-matched controls (MFG, N = 13 and MTG, N = 29). Global levels of 5mC and 5hmC positively correlated with each other and with markers of AD including amyloid beta, tau, and ubiquitin loads. Our results showed a global hypermethylation in the AD brain and revealed that levels of 5hmC were also significantly increased in AD MFG and MTG with no apparent influence of gender, age, postmortem delay, or tissue storage time. Using double-fluorescent immunolabeling, we found that in control and AD brains, levels of 5mC and 5hmC were low in astrocytes and microglia but were elevated in neurons. In addition, our colocalization study showed that within the same nuclei, 5mC and 5hmC mostly do not coexist. The present study clearly demonstrates the involvement of 5mC and 5hmC in AD emphasizing the need for future studies determining the exact time frame of these epigenetic changes during the progression of AD pathology.