Changes In 5hmC Research Articles

Proton irradiation induces persistent and tissue-specific DNA methylation changes in the left ventricle and hippocampus.

BackgroundProton irradiation poses a potential hazard to astronauts during and following a mission, with post-mitotic cells at most risk because they cannot dilute resultant epigenetic changes via cell division. Persistent epigenetic changes that result from environmental exposures include gains or losses of DNA methylation of cytosine, which can impact gene expression. In the present study, we compared the long-term epigenetic effects of whole body proton irradiation in the mouse hippocampus and left ventricle. We used an unbiased genome-wide DNA methylation study, involving ChIP-seq with antibodies to 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) to identify DNA regions in which methylation levels have changed 22 weeks after a single exposure to proton irradiation. We used DIP-Seq to profile changes in genome-wide DNA methylation and hydroxymethylation following proton irradiation. In addition, we used published RNAseq data to assess whether differentially methylated regions were linked to changes in gene expression.ResultsThe DNA methylation data showed tissue-dependent effects of proton irradiation and revealed significant major pathway changes in response to irradiation that are related to known pathophysiologic processes. Many regions affected in the ventricle mapped to genes involved in cardiovascular function pathways, whereas many regions affected in the hippocampus mapped to genes involved in neuronal functions. In the ventricle, increases in 5hmC were associated with decreases in 5mC. We also observed spatial overlap for regions where both epigenetic marks decreased in the ventricle. In hippocampus, increases in 5hmC were most significantly correlated (spatially) with regions that had increased 5mC, suggesting that deposition of hippocampal 5mC and 5hmC may be mechanistically coupled.ConclusionsThe results demonstrate long-term changes in DNA methylation patterns following a single proton irradiation, that these changes are tissue specific, and that they map to pathways consistent with tissue specific responses to proton irradiation. Further, the results suggest novel relationships between changes in 5mC and 5hmC.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2581-x) contains supplementary material, which is available to authorized users.

Reciprocal changes in DNA methylation and hydroxymethylation and a broad repressive epigenetic switch characterize FMR1 transcriptional silencing in fragile X syndrome.

BackgroundFragile X syndrome (FXS) is the most common form of inherited intellectual disability, resulting from the loss of function of the fragile X mental retardation 1 (FMR1) gene. The molecular pathways associated with FMR1 epigenetic silencing are still elusive, and their characterization may enhance the discovery of novel therapeutic targets as well as the development of novel clinical biomarkers for disease status.ResultsWe have deployed customized epigenomic profiling assays to comprehensively map the FMR1 locus chromatin landscape in peripheral mononuclear blood cells (PBMCs) from eight FXS patients and in fibroblast cell lines derived from three FXS patient. Deoxyribonucleic acid (DNA) methylation (5-methylcytosine (5mC)) and hydroxymethylation (5-hydroxymethylcytosine (5hmC)) profiling using methylated DNA immunoprecipitation (MeDIP) combined with a custom FMR1 microarray identifies novel regions of DNA (hydroxy)methylation changes within the FMR1 gene body as well as in proximal flanking regions. At the region surrounding the FMR1 transcriptional start sites, increased levels of 5mC were associated to reciprocal changes in 5hmC, representing a novel molecular feature of FXS disease. Locus-specific validation of FMR1 5mC and 5hmC changes highlighted inter-individual differences that may account for the expected DNA methylation mosaicism observed at the FMR1 locus in FXS patients. Chromatin immunoprecipitation (ChIP) profiling of FMR1 histone modifications, together with 5mC/5hmC and gene expression analyses, support a functional relationship between 5hmC levels and FMR1 transcriptional activation and reveal cell-type specific differences in FMR1 epigenetic regulation. Furthermore, whilst 5mC FMR1 levels positively correlated with FXS disease severity (clinical scores of aberrant behavior), our data reveal for the first time an inverse correlation between 5hmC FMR1 levels and FXS disease severity.ConclusionsWe identify novel, cell-type specific, regions of FMR1 epigenetic changes in FXS patient cells, providing new insights into the molecular mechanisms of FXS. We propose that the combined measurement of 5mC and 5hmC at selected regions of the FMR1 locus may significantly enhance FXS clinical diagnostics and patient stratification.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-016-0181-x) contains supplementary material, which is available to authorized users.

Sex-specific associations of arsenic exposure with global DNA methylation and hydroxymethylation in leukocytes: results from two studies in Bangladesh.

Depletion of global 5-hydroxymethylcytosine (5-hmC) is observed in human cancers and is strongly implicated in skin cancer development. Although arsenic (As)-a class I human carcinogen linked to skin lesion and cancer risk-is known to be associated with changes in global %5-methylcytosine (%5-mC), its influence on 5-hmC has not been widely studied. We evaluated associations of As in drinking water, urine, and blood with global %5-mC and %5-hmC in two studies of Bangladeshi adults: (i) leukocyte DNA in the Nutritional Influences on Arsenic Toxicity study (n = 196; 49% male, 19-66 years); and (ii) peripheral blood mononuclear cell DNA in the Folate and Oxidative Stress study (n = 375; 49% male, 30-63 years). Overall, As was not associated with global %5-mC or %5-hmC. Sex-specific analyses showed that associations of As exposure with global %5-hmC were positive in males and negative in females (P for interaction < 0.01). Analyses examining interactions by elevated plasma total homocysteine (tHcys), an indicator of B-vitamin deficiency, found that tHcys also modified the association between As and global %5-hmC (P for interaction < 0.10). In two samples, we observed associations between As exposure and global %5-hmC in blood DNA that were modified by sex and tHcys. Our findings suggest that As induces sex-specific changes in 5-hmC, an epigenetic mark that has been associated with cancer. Future research should explore whether altered %5-hmC is a mechanism underlying the sex-specific influences of As on skin lesion and cancer outcomes.

Drug induced changes in the mouse liver epigenome

Epigenetic reprogramming involves the erasure and re-establishment of DNA and chromatin modifications during mammalian development. Once re-established the epigenome contributes to the maintenance of tissue identity. Perturbations in the epigenome are associated with exposure to a range of drugs and toxicants, including non-genotoxic carcinogens. Application of genome-wide integrated epigenomic and transcriptomic studies reveals the extent and dynamic nature of epigenetic perturbations resulting from xenobiotic exposure. We have demonstrated that exposure to the drug phenobarbital (PB) perturbs normal 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) profiles in a dose dependent manner. The PB response gene Cyp2b10 undergoes a rapid transcriptional activation alongside a parallel active demethylation (5mC > 5hmC > C) event at its promoter region. We observe numerous changes in the two DNA modifications, which are dynamic and reciprocal in nature, with frequent losses in 5hmC observed at and around the transcriptional start sites of many genes. Dynamic changes in promoter 5hmC and 5mC can be related to a global response to PB exposure that is independent of transcriptional changes and links with cancer associated DNA modification reprogramming profiles. Our study illustrates the potential for 5mC/5hmC profiling at promoter regions to identify potential drivers of non-genotoxic carcinogenesis in the mouse liver. Drug-induced perturbations of the epigenome generate unique epigenetic signatures that can enhance our mechanistic understanding of xenobiotic exposure, and potentially lead to the identification of novel safety biomarkers.

Increased 5-hydroxymethylcytosine and decreased 5-methylcytosine are indicators of global epigenetic dysregulation in diffuse intrinsic pontine glioma

IntroductionDiffuse intrinsic pontine glioma (DIPG) is a malignant pediatric brain tumor associated with dismal outcome. Recent high-throughput molecular studies have shown a high frequency of mutations in histone-encoding genes (H3F3A and HIST1B) and distinctive epigenetic alterations in these tumors. Epigenetic alterations described in DIPG include global DNA hypomethylation. In addition to the generally repressive methylcytosine DNA alteration, 5-hydroxymethylation of cytosine (5hmC) is recognized as an epigenetic mark associated with active chromatin. We hypothesized that in addition to alterations in DNA methylation, that there would be changes in 5hmC. To test this hypothesis, we performed immunohistochemical studies to compare epigenetic alterations in DIPG to extrapontine adult and pediatric glioblastoma (GBM) and normal brain. A total of 124 tumors were scored for histone 3 lysine 27 trimethylation (H3K27me3) and histone 3 lysine 9 trimethylation (H3K9me3) and 104 for 5hmC and 5-methylcytosine (5mC). An H-score was derived by multiplying intensity (0–2) by percentage of positive tumor nuclei (0-100%).ResultsWe identified decreased H3K27me3 in the DIPG cohort compared to pediatric GBM (p < 0.01), adult GBM (p < 0.0001) and normal brain (p < 0.0001). H3K9me3 was not significantly different between tumor types. Global DNA methylation as measured by 5mC levels were significantly lower in DIPG compared to pediatric GBM (p < 0.001), adult GBM (p < 0.01), and normal brain (p < 0.01). Conversely, 5hmC levels were significantly higher in DIPG compared to pediatric GBM (p < 0.0001) and adult GBM (p < 0.0001). Additionally, in an independent set of DIPG tumor samples, TET1 and TET3 mRNAs were found to be overexpressed relative to matched normal brain.ConclusionsOur findings extend the immunohistochemical study of epigenetic alterations in archival tissue to DIPG specimens. Low H3K27me3, decreased 5mC and increased 5hmC are characteristic of DIPG in comparison with extrapontine GBM. In DIPG, the relative imbalance of 5mC compared to 5hmC may represent an opportunity for therapeutic intervention.Electronic supplementary materialThe online version of this article (doi:10.1186/2051-5960-2-59) contains supplementary material, which is available to authorized users.

Transcription Factor Occupancy Can Mediate Active Turnover of DNA Methylation at Regulatory Regions

Distal regulatory elements, including enhancers, play a critical role in regulating gene activity. Transcription factor binding to these elements correlates with Low Methylated Regions (LMRs) in a process that is poorly understood. Here we ask whether and how actual occupancy of DNA-binding factors is linked to DNA methylation at the level of individual molecules. Using CTCF as an example, we observe that frequency of binding correlates with the likelihood of a demethylated state and sites of low occupancy display heterogeneous DNA methylation within the CTCF motif. In line with a dynamic model of binding and DNA methylation turnover, we find that 5-hydroxymethylcytosine (5hmC), formed as an intermediate state of active demethylation, is enriched at LMRs in stem and somatic cells. Moreover, a significant fraction of changes in 5hmC during differentiation occurs at these regions, suggesting that transcription factor activity could be a key driver for active demethylation. Since deletion of CTCF is lethal for embryonic stem cells, we used genetic deletion of REST as another DNA-binding factor implicated in LMR formation to test this hypothesis. The absence of REST leads to a decrease of hydroxymethylation and a concomitant increase of DNA methylation at its binding sites. These data support a model where DNA-binding factors can mediate turnover of DNA methylation as an integral part of maintenance and reprogramming of regulatory regions.

Vitamin C induces Tet-dependent DNA demethylation and a blastocyst-like state in ES cells

DNA methylation is a heritable epigenetic modification involved in gene silencing, imprinting, and the suppression of retrotransposons1. Global DNA demethylation occurs in the early embryo and the germline2,3 and may be mediated by Tet (ten-eleven-translocation) enzymes4–6, which convert 5-methylcytosine (mC) to 5-hydroxymethylcytosine (hmC)7. Tet enzymes have been extensively studied in mouse embryonic stem cells (ESCs)8–12, which are generally cultured in the absence of Vitamin C (VitC), a potential co-factor for Fe(II) 2-oxoglutarate dioxygenase enzymes like Tets. Here we report that addition of VitC to ESCs promotes Tet activity leading to a rapid and global increase in hmC. This is followed by DNA demethylation of numerous gene promoters and up-regulation of demethylated germline genes. Tet1 binding is enriched near the transcription start site (TSS) of genes affected by VitC treatment. Importantly, VitC, but not other antioxidants, enhances the activity of recombinant Tet1 in a biochemical assay and the VitC-induced changes in hmC and mC are entirely suppressed in Tet1/2 double knockout (Tet DKO) ESCs. VitC has the strongest effects on regions that gain methylation in cultured ESCs compared to blastocysts and in vivo are methylated only after implantation. In contrast, imprinted regions and intracisternal A-particle (IAP) retroelements, which are resistant to demethylation in the early embryo2,13, are resistant to VitC-induced DNA demethylation. Collectively, this study establishes VitC as a direct regulator of Tet activity and DNA methylation fidelity in ESCs.

Age-Related Increase in Levels of 5-Hydroxymethylcytosine in Mouse Hippocampus is Prevented by Caloric Restriction

Aberrations in epigenetic marks have been associated with aging of the brain while caloric restriction (CR) and upregulation of endogenous antioxidants have been suggested as tools to attenuate the aging process. We have recently observed age-related increases in levels of 5-methylcytidine (5-mC) and DNA methyltransferase 3a (Dnmt3a) in the mouse hippocampus. Most of those age-related changes in these epigenetically relevant markers were prevented by CR but not by transgenic overexpression of the endogenous antioxidant superoxide dismutase 1 (SOD1). As recent work has suggested a distinct role for hydroxymethylation in epigenetic regulation of gene expression in the brain, the current study investigated age-related changes of 5-hydroxymethylcytosine (5-hmC) in the mouse hippocampus, and furthermore tested whether CR and transgenic upregulation of SOD1 affected any age-related changes in 5-hmC. Immunohistochemical analyses of 5-hmC in 12- and 24-month-old wild-type and transgenic mice overexpressing SOD1, which were kept under either a control or a calorie restricted diet, revealed an increase of 5-hmC immunoreactivity occurring with aging in the hippocampal dentate gyrus, CA3 and CA1-2 regions. Moreover, CR, but not overexpression of SOD1, prevented the age-related increase in the CA3 region. These findings indicate that the aging process in mice is connected with changes in epigenetic machinery in the hippocampus and suggest that CR acts by influencing epigenetic regulation.

2-Hydroxyglutarate in IDH mutant AML: Predicting Patient Responses, Minimal Residual Disease and Correlations with Methylcytosine and Hydroxymethylcytosine Levels

Abstract 2509Mutations in the isocitrate dehydrogenase (IDH) 1 and 2 genes have recently been shown to be recurrent, frequent and prognostic in acute myeloid leukemia (AML) patients. IDH1 and IDH2 encode NADP+ dependent enzymes responsible for oxidative decarboxylation of isocitrate to α-ketoglutarate (AKG). Heterozygous point mutations in IDH1 and IDH2 result in the loss of this activity, concomitant with a neomorphic ability to convert AKG to 2-hydroxyglutarate (2HG), resulting in significant elevations in this oncometabolite. The ten-eleven translocation 2 (TET2) gene converts 5-methylcytosine (5MC) to 5-hydroxymethylcytosine (5HMC), leading to DNA demethylation. TET2 encodes a dioxygenase that is dependent on AKG, and therefore, increased levels of 2HG may disrupt TET2 function. We explored the potential of 2HG to serve as a clinical biomarker of response to therapy in AML patients with IDH mutations, and correlated fluctuations of 2HG with changes in 5MC and 5HMC levels in a clinical setting.In a Phase I/II clinical trial of 44 elderly, previously untreated AML patients who received sequential azacitidine and lenalidomide, we identified 12 patients with IDH1/2 mutations (27%). Five had IDH1 mutations (all R132), and seven had IDH2 mutations (R140 N=5, R172 N=2). 2HG levels were measured by mass spectrometry from plasma obtained from bone marrow aspirations. Six patients had pre- and post-treatment samples available for comparison. Clinical responses were determined per LeukemiaNet criteria.Two of the six patients achieved a complete remission (CR), one with an IDH1 R132 and the other with an IDH2 R140. A comparison of pre- and post-treatment samples from these patients revealed decreases in 2HG levels of −16.9 and −17.9 fold after three cycles of therapy. Four patients did not achieve a CR, although some did experience decreases in the percentage of blasts. These patients had substantially smaller decreases, or increases, in 2HG levels when measured after one cycle (Figure 1). A non-parametric comparison (Mann-Whitney U test) in this small sample set showed a trend toward a significant change in pre- vs. post-treatment 2HG levels between patients who experienced a CR and those who did not (p=0.067), suggesting 2HG levels may correlate with leukemic blast clearance. [Display omitted] Both patients who achieved a CR continued to have detectable 2HG levels. Furthermore, patient 8 ultimately relapsed, and at the time of this relapse, which was unexpected based on clinical indicators or routine peripheral blood laboratory findings, there was a 1.8-fold increase in 2HG levels. Therefore, for AML patients with IDH1 and IDH2 mutations, 2HG may provide a useful, non-invasive marker of minimal residual disease.We hypothesized that decreases in 2HG would allow functional TET2 activity, resulting in increased 5HMC and decreased 5MC. Therefore, we correlated changes in 2HG levels with changes in global 5MC and 5HMC levels, as measured by mass spectrometry, in the patients who achieved a CR.Patient 8 was wild type for TET2. As predicted, after one cycle of therapy, 2HG decreased 6.1 fold, 5HMC increased 7.8 fold and 5MC decreased 1.4 fold. At relapse, when 2HG rose 1.8 fold, 5HMC decreased 5.4 fold; the expected increase in 5MC was not observed (Figure 2). Although concomitant mutations in IDH and TET2 are exceedingly rare, patient 22 did have this genotype, which allowed for an instructive negative control. After one cycle of therapy, 2HG decreased 2.6 fold. However, in contrast to patient 8, in which it appeared TET2 became functional as shown by increased 5HMC after 2HG decreased, patient 22's mutant TET2 resulted in marginal changes in 5HMC and 5MC levels (1.3-fold increase and 1.1-fold decrease, respectively) (Figure 3). These observations provide in vivo evidence for the interaction between IDH and TET2, and suggest that these interactions are dynamic and can change with therapy. [Display omitted] [Display omitted] Disclosures:Off Label Use: Lenalidomide in AML. Schenkein:Agios: Employment. Fantin:Agios: Employment. Straley:Agios: Employment. Medeiros:Celgene: Honoraria, Research Funding.

Tissue-specific Distribution and Dynamic Changes of 5-Hydroxymethylcytosine in Mammalian Genomes

Cytosine residues in the vertebrate genome are enzymatically modified to 5-methylcytosine, which participates in transcriptional repression of genes during development and disease progression. 5-Methylcytosine can be further enzymatically modified to 5-hydroxymethylcytosine by the TET family of methylcytosine dioxygenases. Analysis of 5-methylcytosine and 5-hydroxymethylcytosine is confounded, as these modifications are indistinguishable by traditional sequencing methods even when supplemented by bisulfite conversion. Here we demonstrate a simple enzymatic approach that involves cloning, identification, and quantification of 5-hydroxymethylcytosine in various CCGG loci within murine and human genomes. 5-Hydroxymethylcytosine was prevalent in human and murine brain and heart genomic DNAs at several regions. The cultured cell lines NIH3T3 and HeLa both displayed very low or undetectable amounts of 5-hydroxymethylcytosine at the examined loci. Interestingly, 5-hydroxymethylcytosine levels in mouse embryonic stem cell DNA first increased then slowly decreased upon differentiation to embryoid bodies, whereas 5-methylcytosine levels increased gradually over time. Finally, using a quantitative PCR approach, we established that a portion of VANGL1 and EGFR gene body methylation in human tissue DNA samples is indeed hydroxymethylation.