5mC Levels Research Articles

Gestational blood levels of toxic metal and essential element mixtures and associations with global DNA methylation in pregnant women and their infants

BackgroundPregnant women and their fetuses are exposed to multiple toxic metals that together with variations in essential element levels may alter epigenetic regulation, such as DNA methylation. ObjectivesThe aim of the study was to investigate the associations between gestational levels of toxic metals and essential elements and mixtures thereof, with global DNA methylation levels in pregnant women and their newborn children. MethodsUsing 631 mother-child pairs from a prospective birth cohort (The Norwegian Mother, Father and Child Cohort Study), we measured maternal blood concentration (gestation week ~18) of five toxic metals and seven essential elements. We investigated associations as individual exposures and two-way interactions, using elastic net regression, and total mixture, using quantile g-computation, with blood levels of 5-methylcytocine (5mC) and 5-hydroxymethylcytosine (5hmC) in mothers during pregnancy and their newborn children (cord blood). Multiple testing was adjusted for using the Benjamini and Hochberg false discovery rate (FDR) approach. ResultsThe most sensitive marker of DNA methylation appeared to be 5mC levels. In pregnant mothers, elastic net regression indicated associations between 5mC and selenium and lead (non-linear), while in newborns results indicated relationships between maternal selenium, cobalt (non-linear) and mercury and 5mC, as well as copper (non-linear) and 5hmC levels. Several possible two-way interactions were identified (e.g. arsenic and mercury, and selenium and maternal smoking in newborns). None of these findings met the FDR threshold for multiple testing. No net effect was observed in the joint (mixture) exposure-approach using quantile g-computation. ConclusionWe identified few associations between gestational levels of several toxic metals and essential elements and global DNA methylation in pregnant mothers and their newborn children. As DNA methylation dysregulation might be a key mechanism in disease development and thus of high importance for public health, our results should be considered as important candidates to investigate in future studies.

Combination of azacitidine and enasidenib enhances leukemic cell differentiation and cooperatively hypomethylates DNA

Azacitidine and enasidenib are two therapies available for treatment of acute myelogenous leukemia (AML), and the mechanisms of action of these drugs involve alteration of aberrant DNA methylation. We hypothesized that a combination of these agents could have interactive effects on DNA methylation and enhance differentiation in mIDH2 cells. Combination treatment enhanced cellular differentiation in TF-1 cells overexpressing IDJ2R140Q through increased hemoglobinization and increased hemoglobin γ RNA expression compared with the effects of single agents. Furthermore, in primary AML samples (IDH2R140Q or R172K), combination treatment reduced CD34+ cells and increased CD15+ cells to a greater extent than attained with single agents. To explore the mechanism of enhanced differentiation with combination treatment, the TF-1 epigenome was analyzed by profiling 5-hydroxymethylcytosine (5hmC) and 5-methylcytosine (5mC) DNA methylation changes. Enasidenib treatment alone increased 5hmC, consistent with reactivation of ten-eleven-translocation (TET) enzyme activity. Compared with treatment with azacitidine alone, combination treatment reduced 5mC levels at greater numbers of sites and these loci were significantly enriched in regions with increased 5hMC (25.8% vs. 7.4%). Results are consistent with a model in which enasidenib-mediated reactivation of ten-eleven-translocation enzymes cooperates with azacitidine-mediated inhibition of DNA methyltransferase enzymes, leading to greater reductions in DNA methylation and enhanced erythroid differentiation.

Recent loss of the Dim2 DNA methyltransferase decreases mutation rate in repeats and changes evolutionary trajectory in a fungal pathogen.

DNA methylation is found throughout all domains of life, yet the extent and function of DNA methylation differ among eukaryotes. Strains of the plant pathogenic fungus Zymoseptoria tritici appeared to lack cytosine DNA methylation (5mC) because gene amplification followed by Repeat-Induced Point mutation (RIP) resulted in the inactivation of the dim2 DNA methyltransferase gene. 5mC is, however, present in closely related sister species. We demonstrate that inactivation of dim2 occurred recently as some Z. tritici isolates carry a functional dim2 gene. Moreover, we show that dim2 inactivation occurred by a different path than previously hypothesized. We mapped the genome-wide distribution of 5mC in strains with or without functional dim2 alleles. Presence of functional dim2 correlates with high levels of 5mC in transposable elements (TEs), suggesting a role in genome defense. We identified low levels of 5mC in strains carrying non-functional dim2 alleles, suggesting that 5mC is maintained over time, presumably by an active Dnmt5 DNA methyltransferase. Integration of a functional dim2 allele in strains with mutated dim2 restored normal 5mC levels, demonstrating de novo cytosine methylation activity of Dim2. To assess the importance of 5mC for genome evolution, we performed an evolution experiment, comparing genomes of strains with high levels of 5mC to genomes of strains lacking functional dim2. We found that presence of a functional dim2 allele alters nucleotide composition by promoting C to T transitions (C→T) specifically at CpA (CA) sites during mitosis, likely contributing to TE inactivation. Our results show that 5mC density at TEs is a polymorphic trait in Z. tritici populations that can impact genome evolution.

LIF maintains mouse embryonic stem cells pluripotency by modulating TET1 and JMJD2 activity in a JAK2-dependent manner.

The LIF-JAK2-STAT3 pathway is the central signal transducer that maintains undifferentiated mouse embryonic stem cells (mESCs), which is achieved by the recruitment of activated STAT3 to the master pluripotency genes and activation of the gene transcriptions. It remains unclear, however, how the epigenetic status required for the master gene transcriptions is built into LIF-treated mESC cultures. In this study, Jak2, but not Stat3, in the LIF canonical pathway, establishes an open epigenetic status in the pluripotency gene promoter regions. Upon LIF activation, cytosolic JAK2 was translocalized into the nucleus of mESCs, and reduced DNA methylation (5mC levels) along with increasing DNA hydroxymethylation (5hmC) in the pluripotent gene (Nanog/Pou5f1) promoter regions. In addition, the repressive histone codes H3K9m3/H3K27m3 were reduced by JAK2. Activated JAK2 directly interacted with the core epigenetic enzymes TET1 and JMJD2, modulating its activity and promotes the DNA and histone demethylation, respectively. The JAK2 effects were attained by tyrosine phosphorylation on the epigenetic enzymes. The effects of JAK2 phosphorylation on the enzymes were diverse, but all were merged to the epigenetic signatures associated with open DNA/chromatin structures. Taken together, these results reveal a previously unrecognized epigenetic regulatory role of JAK2 as an important mediator of mESC maintenance.

Evidence for divergence of DNA methylation maintenance and a conserved inhibitory mechanism from DNA demethylation in chickens and mammals

BackgroundDNA methylation is a significant epigenetic modification that is evolutionarily conserved in various species and often serves as a repressive mark for transcription. DNA methylation levels and patterns are regulated by a balance of opposing enzyme functions, DNA methyltransferases, DNMT1/3A/3B and methylcytosine dioxygenases, TET1/2/3. In mice, the TET enzyme converts DNA cytosine methylation (5mC) to 5-hydroxymethylcytosine (5hmC) at the beginning of fertilisation and gastrulation and initiates a global loss of 5mC, while the 5mC level is increased on the onset of cell differentiation during early embryonic development.ObjectiveGlobal loss and gain of DNA methylation may be differently regulated in diverged species.MethodsChicken B-cell lymphoma DT40 cells were used as an avian model to compare differences in the overall regulation of DNA modification with mammals.ResultsWe found that DNA methylation is maintained at high levels in DT40 cells through compact chromatin formation, which inhibits TET-mediated demethylation. Human and mouse chromosomes introduced into DT40 cells by cell fusion lost the majority of 5mC, except for human subtelomeric repeats.ConclusionOur attempt to elucidate the differences in the epigenetic regulatory mechanisms between birds and mammals explored the evidence that they share a common chromatin-based regulation of TET–DNA access, while chicken DNMT1 is involved in different target sequence recognition systems, suggesting that factors inducing DNMT–DNA association have already diverged.

Reduced hepatic global hydroxymethylation in mice treated with non-genotoxic carcinogens is transiently reversible with a methyl supplemented diet

Non-genotoxic carcinogens (NGCs) are known to cause perturbations in DNA methylation, which can be an early event leading to changes in gene expression and the onset of carcinogenicity. Phenobarbital (PB) has been shown to alter liver DNA methylation and hydroxymethylation patterns in mice in a time dependent manner. The goals of this study were to assess if clofibrate (CFB), a well-studied rodent NGC, would produce epigenetic changes in mice similar to PB, and if a methyl donor supplementation (MDS) would modulate epigenetic and gene expression changes induced by phenobarbital. CByB6F1 mice were treated with 0.5% clofibrate or 0.14% phenobarbital for 7 and 28 days. A subgroup of PB treated and control mice were also fed MDS diet. Liquid Chromatography-Ionization Mass Spectrometry (LC-MS) was used to quantify global liver 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) levels. Gene expression analysis was conducted using Affymetrix microarrays. A decrease in liver 5hmC but not 5mC levels was observed upon treatment with both CFB and PB with varying time of onset. We observed moderate increases in 5hmC levels in PB-treated mice when exposed to MDS diet and lower expression levels of several phenobarbital induced genes involved in cell proliferation, growth, and invasion, suggesting an early modulating effect of methyl donor supplementation. Overall, epigenetic profiling can aid in identifying early mechanism-based biomarkers of non-genotoxic carcinogenicity and increases the quality of cancer risk assessment for candidate drugs. Global DNA methylation assessment by LC-MS is an informative first step toward understanding the risk of carcinogenicity.

Molecular mechanism for vitamin C-derived C5-glyceryl-methylcytosine DNA modification catalyzed by algal TET homologue CMD1

C5-glyceryl-methylcytosine (5gmC) is a novel DNA modification catalyzed by algal TET homologue CMD1 using vitamin C (VC) as co-substrate. Here, we report the structures of CMD1 in apo form and in complexes with VC or/and dsDNA. CMD1 exhibits comparable binding affinities for DNAs of different lengths, structures, and 5mC levels, and displays a moderate substrate preference for 5mCpG-containing DNA. CMD1 adopts the typical DSBH fold of Fe2+/2-OG-dependent dioxygenases. The lactone form of VC binds to the active site and mono-coordinates the Fe2+ in a manner different from 2-OG. The dsDNA binds to a positively charged cleft of CMD1 and the 5mC/C is inserted into the active site and recognized by CMD1 in a similar manner as the TET proteins. The functions of key residues are validated by mutagenesis and activity assay. Our structural and biochemical data together reveal the molecular mechanism for the VC-derived 5gmC DNA modification by CMD1.

TET2 is a component of the estrogen receptor complex and controls 5mC to 5hmC conversion at estrogen receptor cis-regulatory regions

SummaryEstrogen receptor-α (ER) drives tumor development in ER-positive (ER+) breast cancer. The transcription factor GATA3 has been closely linked to ER function, but its precise role in this setting remains unclear. Quantitative proteomics was used to assess changes to the ER complex in response to GATA3 depletion. Unexpectedly, few proteins were lost from the ER complex in the absence of GATA3, with the only major change being depletion of the dioxygenase TET2. TET2 binding constituted a near-total subset of ER binding in multiple breast cancer models, with loss of TET2 associated with reduced activation of proliferative pathways. TET2 knockdown did not appear to change global methylated cytosine (5mC) levels; however, oxidation of 5mC to 5-hydroxymethylcytosine (5hmC) was significantly reduced, and these events occurred at ER enhancers. These findings implicate TET2 in the maintenance of 5hmC at ER sites, providing a potential mechanism for TET2-mediated regulation of ER target genes.

Single cell epigenetic visualization assay.

Characterization of the epigenetic status of individual cells remains a challenge. Current sequencing approaches have limited coverage, and it is difficult to assign an epigenetic status to the transcription state of individual gene alleles in the same cell. To address these limitations, a targeted microscopy-based epigenetic visualization assay (EVA) was developed for detection and quantification of epigenetic marks at genes of interest in single cells. The assay is based on an in situ biochemical reaction between an antibody-conjugated alkaline phosphatase bound to the epigenetic mark of interest, and a 5′-phosphorylated fluorophore-labeled DNA oligo tethered to a target gene by gene-specific oligonucleotides. When the epigenetic mark is present at the gene, phosphate group removal by the phosphatase protects the oligo from λ-exonuclease activity providing a quantitative fluorescent readout. We applied EVA to measure 5-methylcytosine (5mC) and H3K9Ac levels at different genes and the HIV-1 provirus in human cell lines. To link epigenetic marks to gene transcription, EVA was combined with RNA-FISH. Higher 5mC levels at the silenced compared to transcribed XIST gene alleles in female somatic cells validated this approach and demonstrated that EVA can be used to relate epigenetic marks to the transcription status of individual gene alleles.

Effect of vitrification at different meiotic stages on epigenetic characteristics of bovine oocytes and subsequently developing embryos.

Vitrification by the Cryotop method is frequently used for bovine oocyte cryopreservation. Nevertheless, vitrified oocytes still have reduced developmental competency compared with fresh counterparts. The objective of this study was to compare the effect of vitrification either at the germinal vesicle (GV) stage or at the metaphase II (MII) stage on epigenetic characteristics of bovine oocytes and subsequently developing embryos. Our results demonstrated that vitrification of oocytes at each meiotic stage significantly reduced blastocyst development after in vitro fertilization (IVF). However, vitrification at the GV stage resulted in higher blastocyst development than did vitrification at the MII stage. Irrespective of the meiotic stage, oocyte vitrification did not affect 5-methylcytosine (5mC) immunostaining intensity in oocyte DNA. However, at both stages, it caused a similar reduction of 5mC levels in DNA of subsequently developing blastocysts. Oocyte vitrification had no effect on the intensity of H3K9me3 and acH3K9 immunostaining in oocytes and subsequent blastocysts. The results suggest that irrespective of meiotic stage, oocyte vitrification alters global methylation in resultant embryos although such alteration in the oocytes was not detected. Oocyte vitrification might not influence histone acetylation and methylation in oocytes and resultant embryos. Vitrification at the immature stage was more advantageous for blastocyst development than at the mature stage.

Cross-cultural comparisons of motor competence in southern Brazilian and Portuguese schoolchildren

Aim: This study aimed to compare the MC levels of Brazilian and Portuguese schoolchildren from 4 to 11 years. Methods: One hundred and forty-eight Brazilian children were evaluated using the Motor Competence Assessment (MCA) instrument and were later compared to the existent normative values of Portuguese children for a similar age range. Results: Our findings showed that MC increased with age, and boys outperformed girls, especially in the older age group. The distribution of the Brazilian children showed that while Brazilian boys perform above Portuguese normative values in 4 of the 6 tasks of the MCA, Brazilian girls perform below those values in all tasks, except for the standing long jump. Conclusion: The differences found, especially in girls, may be related to differences in the Physical Education curricula and how school recess periods are used.

DNA methylation defects in spermatozoa of male partners from couples experiencing recurrent pregnancy loss.

What is the sperm DNA methylation status of imprinted genes in male partners from couples experiencing recurrent pregnancy loss (RPL)? Aberrations in sperm DNA methylation status of several imprinted genes, such as insulin like growth factor-2-H19 differentially methylated region (IGF2-H19 DMR), intergenic differentially methylated region (IG-DMR), mesoderm specific transcript (MEST),zinc finger protein which regulates apoptosis and cell cycle arrest (ZAC), DMR in intron 10 of KCNQ1 gene (KvDMR), paternally expressed gene 3 (PEG3) and paternally expressed gene 10 (PEG10), as well as decreased sperm global 5-methylcytosine (5mC) levels, are associated with RPL. RPL is defined as loss of two or more pregnancies, affecting 1-2% of couples of reproductive age. Although there are several maternal and paternal aetiological factors contributing to RPL, nearly 50% of the cases remain idiopathic. Thus, there is a need to identify putative paternal factors that could be contributing towards pregnancy loss in cases of idiopathic RPL. In this case-control study, 112 couples undergoing RPL with no identifiable cause were recruited from September 2015 to May 2018. The control group comprised of 106 healthy proven fertile couples with no history of infertility or miscarriage. In this study, we investigated the paternal genetic and epigenetic factors that could be associated with RPL. We studied DNA methylation, by pyrosequencing, of selected imprinted genes implicated in embryo development, such as IGF2-H19 DMR, IG-DMR, MEST, ZAC, KvDMR, PEG3, PEG10 and small nuclear ribonucleoprotein polypeptide N (SNRPN) in sperm of men whose partners present RPL. Global DNA methylation in sperm was evaluated by studying 5mC content and long interspersed nuclear element 1 (LINE1) promoter methylation. We also studied polymorphisms by pyrosequencing in the IGF2-H19 DMR as well in the IGF2 promoter in both groups. In the RPL group, we found a significant decrease in the global sperm 5mC levels and significant decrease in DNA methylation at three CpG sites in LINE1 promoter. For IGF2-H19 DMR and IG-DMR, a significant decrease in sperm DNA methylation at specific CpG sites was observed in RPL group. For maternally imprinted genes like MEST, ZAC, KvDMR, PEG3 and PEG10 hypermethylation was noted. Polymorphism studies for IGF2-H19 DMR and IGF2 revealed significant differences in the genotypic frequencies in males. In this study, we analysed the methylation levels of selected candidate imprinted genes implicated in embryo development. Detection of methylation changes occurring at the genome-wide level may reveal further candidate genes having a better distinction between the control and study groups. Our study demonstrates that certain polymorphisms and aberrant sperm methylation status in imprinted genes are associated with RPL and could contribute to the aetiology of RPL. This study suggests that investigation of paternal genetic and epigenetic factors could be useful in identification of possible causes of idiopathic RPL. This study was funded by Department of Science and Technology-Science and Engineering Research Board (EMR/2014/000145) and National Institute for Research in Reproductive Health intramural funds (RA/872/01-2020). All authors declare no conflict of interest. N/A.

TBIO-05. GENOME-SCALE NUCLEOTIDE-SPECIFIC CHARACTERIZATION OF 5-HYDROXYMETHYLCYTOSINE IN PEDIATRIC CENTRAL NERVOUS SYSTEM TUMORS

Though aberrant cytosine modifications are prevalent in cancer, nucleotide-specific 5-hydroxymethylcytosine (5hmC) modifications remain understudied, including in pediatric CNS tumors. Brain 5-hydroxymethylation is linked with development and differentiation. We measured genome-scale nucleotide-specific 5hmC in patients with diagnoses of glioma, ependymoma, and embryonal tumors under age 18 (n=36), and in non-tumor pediatric brain tissues (n=3). DNA was processed with tandem oxidative (OxBS) and bisulfite (BS) treatments followed by hybridization to the Illumina Methylation EPIC Array that interrogates over 860,000 CpG sites. We used the OxyBS R package to determine levels of 5hmC and 5mC. Mean 5hmC levels were lower in tumors (gliomas 4.1%, ependymomas 3.9%, and embryonal tumors 3.4%) compared to nontumor tissues (5.3%). We subset to the CpGs with the 5% highest 5hmC content for downstream analyses (37,173 CpGs). These sites were enriched among regulatory elements, including TFBS (Odds Ratio 1.14 p-value 3.57E-20) and super-enhancers (OR 1.93, p-value 1.14E-126). Linear mixed-effects models adjusted for age, sex, and cell type proportions tested the CpG-specific differences in 5hmC between tumor and nontumor samples, as well as between tumor subtypes. 5hmC levels were depleted across tumors compared with nontumor brain tissues, including at CpG islands. Model-based clustering (RPMM) results indicated that patients with low 5hmC patterns have poorer overall survival and increased risk of recurrence. Our results indicate that 5hmC localizes to sites in the DNA critical to gene regulation and is associated with patient outcomes. This study offers an opportunity to potentially contribute to classification markers for childhood brain tumors.

Global shifts in DNA methylation and DNA hydroxymethylation across multiple brain regions in transgenic models of tau and amyloid pathology

AbstractBackgroundThere is increasing evidence to support a role for altered DNA modifications in Alzheimer’s disease (AD). Global changes in both DNA methylation (5mC) and DNA hydroxymethylation (5hmC) have been robustly reported in cancer, but not widely studied in AD. We quantified global levels of 5mC and 5hmC across multiple brain regions in two transgenic models of AD pathology, relating these to the progressive changes in tau and amyloid observed in the same samples.MethodWe investigated global DNA modification levels in brain tissue from rTg4510 and J20 mice using two independent approaches. We quantified hippocampal global DNA methylation in rTg4510 and J20 transgenic (TG) and wild‐type (WT) littermate control mice using an enzymatic‐based methylation assay combined with pyrosequencing. Using immunohistochemistry, we directly quantified levels of both 5‐methylcytosine (5mC) and 5‐hydroxymethylcytosine (5hmC) in multiple brain regions in rTg4510 and J20 mice.ResultWe identified both genotype‐associated and progressive temporal changes (interaction between genotype and age) in 5mC and 5hmC in rTg4510 TG and J20 TG mice compared to WT littermate controls in several brain regions. Levels of both 5mC and 5hmc were negatively correlated with levels of tau and amyloid pathology, reflecting some evidence from human AD brain.ConclusionWe identified decreased levels of two DNA modifications (5mC and 5hmC) associated with accumulation of tau and amyloid pathology. Both genotype‐associated and progressive changes in 5mC and 5hmC measured by immunohistochemistry were observed, with these changes notably stronger in rTg4510 TG relatively to J20 TG mice, particularly in the hippocampus.

Abstract PR09: Developing metabolic intervention strategies to reprogram neuroblastoma epigenome and overcome tumor resistance to differentiation therapy

Abstract Patients with solid tumor usually do not respond to retinoic acid (RA)-based differentiation therapy because RA-resistance often develops. The goal of this study is to develop metabolic intervention strategies that target the cancer epigenome and overcome tumor resistance to differentiation therapy. Experimental procedures: We classified our neuroblastoma (NB) cell line collection into two categories: RA-sensitive cell lines and RA-resistant cell lines. Metabolic intervention agents were combined with RA to treat the RA-resistance cells. Chromatin markers including histone acetylations and methylations were determined using immunoblot. Global DNA methylation were accessed using dot blot. NB cell differentiation were quantified with neurite counting and immunofluorescence staining for NB differentiation marker β-tubulin III. Retinoic acid receptor (RAR) and neuron differentiation marker expression were measured using q-PCR. Summary of findings: Unlike the RA-sensitive NB cells, the RA-resistant cells did not show morphological change or proliferation arrest upon RA treatment. Previously we discovered that glycerol triacetate (GTA) supplementation restored histone acetylation and chromatin accessibility to reactivate the expression of differentiation markers and the RA-induced neuron differentiation program under hypoxia. However, the combination of GTA and RA did not induce proliferation arrest or morphologic change in RA-resistant cells. Interestingly, the combination of RA and the substrate of DNA demethylase, α-ketoglutarate (αKG), significantly induced neuronal differentiation in RA-resistant cells. RAR and differentiation markers expression were also restored by RA+αKG combination. αKG treatment rapidly reduced global 5-methylcytosine (5mC) levels within 1 hour. Although GTA alone or RA+GTA did not induce differentiation, adding GTA in combination with RA and αKG further enhanced the differentiation morphology change in resistant cells. In addition, αKG and GTA supplementation had a synergistic effect in reducing the clonogenic expansion of RA-resistant cells when combined with RA, suggesting that the RA+αKG+GTA combination inhibits self-renewal of RA-resistance NB cells. However, in RA-sensitive cells, αKG could not restore RAR repression caused by hypoxia, probably due to the elevated NADH/NAD+ ratio that favors the conversion of αKG to 2-hydroxyglutarate (2HG), which causes DNA hypermethylation. Conclusions: (1) Unlike hypoxia-induced resistance, which was mainly due to histone deacetylation, DNA methylation is the major cause of resistance in RA-resistant NB cells. (2) αKG+GTA combination effectively reduces DNA methylation and increases histone acetylation to promote RA-sensitivity in RA-resistant NB cells. Citation Format: Haowen Jiang, Yang Li, Michela Yip, Joshua Gruber, Albert Li, Jiangbin Ye. Developing metabolic intervention strategies to reprogram neuroblastoma epigenome and overcome tumor resistance to differentiation therapy [abstract]. In: Abstracts: AACR Special Virtual Conference on Epigenetics and Metabolism; October 15-16, 2020; 2020 Oct 15-16. Philadelphia (PA): AACR; Cancer Res 2020;80(23 Suppl):Abstract nr PR09.

An epigenetic circuit linking oxidative stress and DNA hydroxymethylation in heart failure

Abstract Background Heart failure is a major cause of morbidity and mortality worldwide, but the underlying molecular mechanisms remain not well defined. Reactive oxygen species (ROS) in heart failure (HF) alter multitudes of mitochondrial enzymes and metabolites, such as α-ketoglutarate and its oxidised form L-2-hydroxyglutarate (L-2HG). These metabolites are cofactors for ten eleven translocation (TET) enzymes that convert DNA's 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC). Aim We hypothesize that oxidative stress during heart failure alters mitochondrial function through epigenetic remodeling, and that reduction of oxidative stress will improve cardiac function. Methods and results Targeted LC-MS/MS analysis of dilated cardiomyopathy (DCM) hearts obtained from MLP−/− and WT littermate controls showed a significant increase in the oxidized metabolite L-2HG (∼30%, p=0.004), with significant reduction of multiple TCA cycle intermediates. RNA sequencing revealed a significant reduction in mRNA levels of IDH2 in human TTN related DCM and MLP−/− HF mice. The altered activity of IDH2 contributes to ROS production in these hearts and to the production of L-2HG. No alteration in the mitochondria structures was observed. HF biopsies show decreased TET activity most likely due to increased L-2HG levels. Whole genome single base pair 5mC and 5hmC deep sequencing of gDNA from explanted human DCM heart biopsies and MLP−/− mouse model revealed significantly altered global distribution of both 5mC and 5hmC in comparison to control samples. Genes involved in hypertrophy, such as Myh7 and Fhl2 were among the top genes with differential 5mC levels. Global loss of 5hmC level was observed, especially in the intronic regions of genes involved in redox hemostasis. Reducing oxidative stress in vivo in MLP−/− using a small molecule (AZ14117925) improves heart function (EF) by 13% [EF=(Treated:47,44%, Placebo: 34,54%), n=5 (males per group), p=0.0373, unpaired t-test]. Additional bioinformatic analysis revealed that reduction of ROS most likely leads to activation of TET and activation of pro-survival pathways, anti-oxidative stress response, and significantly less activation of apoptotic pathways. Conclusion Alterations in TCA cycle metabolites may underlie changes in DNA methylation and gene expression in end-stage human DCM and mouse models and indicate a role for epigenetic regulation of mitochondrial function in HF. NRF2 activation may pose a novel therapeutic approach to treat this devastating disease. Funding Acknowledgement Type of funding source: Private company. Main funding source(s): AstraZeneca

Prenatal Lead (Pb) Exposure’s Effect on DNA Methylation (5mC) and Hydroxymethylation (5hmC) in Adolescent Whole Blood

Gestational lead (Pb) exposure may adversely impact offspring health through epigenetic alterations via DNA methylation (5mC), addition of a methyl group to the 5th carbon of cytosine, and hydroxymethylation (5hmC), an intermediate in oxidative demethylation associated with increased gene expression. Most current methods collectively measure 5mC and 5hmC without distinguishing between the two. This study aims to identify the association of prenatal Pb exposure at each trimester (T1, T2, T3) with 5mC and 5hmC levels at multiple cytosine-phosphate-guanine (CpG) sites within candidate regions of genes previously associated with prenatal Pb exposure (HCN2, NINJ2, RAB5A, and TPPP) in blood leukocytes of children ages 11-18 years. Participants from the Early Life Exposure in Mexico to Environmental Toxicants (ELEMENT) birth cohorts were selected (n=144) for pyrosequencing analysis following oxidative or standard sodium bisulfite treatment. Participants are 51% male with concurrent blood lead levels (BLL) averaging 3.29±4.44µg/dL and historical maternal BLL averaging 6.43±5.16µg/dL (T1), 5.66±5.21µg/dL (T2), and 5.86±4.34µg/dL (T3). 5mC levels were quantified in HCN2 (mean±SD, 81.3±9.63%), NINJ2 (36.7±22.5%), RAB5A (1.40±1.20%), and TPPP (92.5±8.03%). 5hmC levels were estimated in HCN2 (2.04±4.30%), NINJ2 (2.15±5.43%), RAB5A (0.64±0.80%), and TPPP (0.45±8.01%), providing evidence for 5hmC presence in blood leukocytes. Controlling for sex, current BLL, child’s age, and pyrosequencing batch, T1 maternal BLL was associated with 5mC in HCN2 (β=-1.256, p=0.019) and NINJ2 (β=0.426, p=0.004); T2 BLL with 5mC in HCN2 (β=0.368, p=0.028) and 5hmC in NINJ2 (β=0.232, p=0.011); and T3 BLL with 5mC in HCN2 (β=0.502, p=0.016) and 5hmC in NINJ2 (β=0.372, p=0.001). No associations were identified between Pb and RAB5A and TPPP. Together, these data suggest that perinatal Pb exposure results may alter 5mC and 5hmC in blood in a gene-specific manner that persists into adolescence.

Low dose lead exposure induces alterations on heterochromatin hallmarks persisting through SH-SY5Y cell differentiation

Lead (Pb) is a commonly found heavy metal due to its historical applications. Recent studies have associated early-life Pb exposure with the onset of various neurodegenerative disease. The molecular mechanisms of Pb conferring long-term neurotoxicity, however, is yet to be elucidated. In this study, we explored the persistency of alteration in epigenetic marks that arise from exposure to low dose of Pb using a combination of image-based and gene expression analysis. Using SH-SY5Y as a model cell line, we observed significant alterations in global 5-methycytosine (5 mC) and histone 3 lysine 27 tri-methylation (H3K27me3) and histone 3 lysine 9 tri-methylation (H3K9me3) levels in a dose-dependent manner immediately after Pb exposure. The changes are partially associated with alterations in epigenetic enzyme expression levels. Long term culturing (14 days) after cease of exposure revealed persistent changes in 5 mC, partial recovery in H3K9me3 and overcompensation in H3K27me3 levels. The observed alterations in H3K9me3 and H3K27me3 are reversed after neuronal differentiation, while reduction in 5 mC levels are amplified with significant changes in patterns as identified via texture clustering analysis. Moreover, correlation analysis demonstrates a strong positive correlation between trends of 5 mC alteration after differentiation and neuronal morphology. Collectively, our results suggest that exposure to low dose of Pb prior to differentiation can result in persistent epigenome alterations that can potentially be responsible for the observed phenotypic changes. Our work reveals that Pb induced changes in epigenetic repressive marks can persist through neuron differentiation, which provides a plausible mechanism underlying long-term neurotoxicity associated with developmental Pb-exposure.

Ascorbic acid promotes the reproductive function of porcine immature Sertoli cells through transcriptome reprogramming

Vitamin C (ascorbic acid, AA) can regulate antioxidation and affect many cellular processes. However, the effect of AA on the reproduction of male animals remains less explored. Here, we showed that by supplementing exogenous AA to porcine immature Sertoli cells (iSCs), AA could promote the proliferation, suppress apoptosis, and decrease the global nucleic acid methylation (5 mC and m6A) levels of iSCs. After we profiled mRNA and long non-coding RNA (lncRNA) expression by transcriptome sequencing on iSCs (treated by 250 μM AA for 36 h), 1232 mRNAs and 937 lncRNAs were identified to be differentially expressed (DE). Gene enrichment analysis found multiple significantly enriched biological pathways, including oxidoreductase activity, cell proliferation and apoptosis, regulation of hormone level, regulation of catalytic activity, developmental process, ATP metabolism and reproductive process. Specifically, for the reproductive process, 49 up- and 36 down-regulated DE mRNAs (including highly expressed genes, such as Tfcp2l1, Hmgcs1, Mmp7, Fndc3a, and Zfp36l1) are involved. Moreover, AA supplementation could promote the secretion of anti-müllerian hormone, inhibin B and lactate, and enhance the activity of lactate dehydrogenase as well. Taken together, AA could promote the reproductive function of pig iSCs, potentially through reprogramming the global transcriptome, and elevating hormone secretion and metabolite production.

Integrated Analysis of DNA Methylation, Hydroxymethylation, and Gene Expression Data Using ME-Class2.

There is increasing interest in understanding the pathological role of DNA methylation changes in disease by profiling genome-wide methylation changes. This includes both 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). The typical profiling study is designed to measure 5mC and/or 5hmC levels alongside gene expression in a set of samples and controls to determine a list of candidate genes whose 5mC and/or 5hmC changes are associated with expression changes. We recently showed that ME-Class2 substantially outperforms other bioinformatic approaches at accurately identify genes with highly associated methylation and expression changes. ME-Class2 further illuminated how synergistic changes in 5mC and 5hmC potentially contribute to gene silencing and activation. Here we present a detailed protocol for using ME-Class2 to analyze genome-wide methylation (5mC and/or 5hmC) and expression data. Further, we provide advice about extending ME-Class2 to study the relationships between other epigenetic marks.