5mC Levels Research Articles

Abstract 4431: Tandem profiling of 5-methyl and 5-hydroxymethylcytosine in glioblastoma identifies tumor-specific distribution of 5-hydroxymethylcytosine and associations with patient survival

Abstract Glioblastomas exhibit widespread molecular alterations including a highly distorted epigenome. Genome-wide assessment of epigenetic alterations to cytosines in glioblastoma has largely been limited to 5-methylcytosine (5mC). 5-hydroxymethylcytosine (5hmC) is an additional cytosine modification that is deregulated in cancer, though the extent and context of these alterations at single cytosine resolution remain unclear. Through parallel processing of DNA with bisulfite and oxidative bisulfite treatments on Illumina 450K arrays we resolved both 5mC and 5hmC in 30 glioblastomas. We developed and applied a novel technique for estimating 5mC, 5hmC, and unmethylated proportions from array data to glioblastoma tissues and compare with normal brain tissue. Genomic distribution of 5hmC was associated with features of transcription despite the glioblastoma genome being relatively depleted of 5hmC. When integrating 5mC and 5hmC data using a Gaussian finite mixture model approach, we observed significant associations between 5hmC levels and gene-sets involved in immune and RNA regulatory processes while high 5mC classes were associated with receptor-mediated processes and cancer gene-sets. Significant differences in patient survival were observed among classes of 5hmC obtained from a recursively partitioned mixture model. Gene expression across epigenetic enzymes including methyltransferases and TETs was also investigated for relation with patterns and levels of 5hmC and 5mC. Analyses of elevated 5hmC with alternative splicing events that may promote disease progression are underway. Together, our results provide a genome-wide map of 5hmC in glioblastoma, indicate that transcription patterns as well as disrupted processes are associated with 5hmC patterns, and highlight the potential clinical prognostic utility of 5hmC in cancer. Citation Format: Kevin C. Johnson, E. Andres Houseman, Jessica E. King, Camilo E. Fadul, Brock C. Christensen. Tandem profiling of 5-methyl and 5-hydroxymethylcytosine in glioblastoma identifies tumor-specific distribution of 5-hydroxymethylcytosine and associations with patient survival. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4431.

Similar kinetics for 5-methylcytosine and 5-hydroxymethylcytosine during human preimplantation development in vitro.

After fertilization, the mammalian embryo undergoes epigenetic reprogramming with genome-wide DNA demethylation and subsequent remethylation. Oxidation of 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC) was suggested to be an intermediate step in the DNA demethylation pathway. Other evidence, such as the stability of 5hmC in specific tissues, suggests that 5hmC constitutes a new epigenetic modification with its own biological function. Since few studies have been conducted on human material compared to animal models and species-specific epigenetic differences have been reported, we studied global DNA methylation and hydroxymethylation patterns in human in vitro preimplantation embryos using immunocytochemistry, comparing these patterns in good-quality and abnormally developing embryos. Our data showed that DNA methylation and hydroxymethylation modifications co-exist. 5mC and 5hmC signals were found in oocytes and in paternal and maternal pronuclei of zygotes, present in non-reciprocal patterns-which contrasts published data for the mouse. These two epigenetic modifications are present between Days 1 and 7 of in vitro development, with 5mC levels declining over cell divisions without noticeable remethylation during this period. A main decline in 5mC and 5hmC occurred as the embryo progressed from compaction to the blastocyst stage. No difference in (hydroxy)methylation was found between the inner cell mass and trophectoderm. When comparing normally and abnormally developing embryos, DNA (hydroxy)methylation reprogramming was abnormal in poor-quality embryos, especially during the first cleavages. Mol. Reprod. Dev. 83: 594-605, 2016 © 2016 Wiley Periodicals, Inc.

Pre-analytical variables of circulating cell-free nucleosomes containing 5-methylcytosine DNA or histone modification H3K9Me3

Aim: To evaluate pre-analytical variables of circulating cell-free nucleosomes containing 5-methylcytosine DNA (5mC) or histone modification H3K9Me3 (H3K9Me3).Materials and methods: Six studies were designed to assess the possible influence of pre-analytical variables. Study 1: influence of stasis and contamination with white-cells and platelets. Study 2: influence of within-day variations. Study 3: influence of day-to-day variation. Study 4: influence of temperature during handling and storage, and of neoplastic disease. Study 5: influence of colonoscopy. Study 6: influence of the surgical trauma. 5mC and H3K9Me3 measurements were performed using enzyme-linked immunosorbent assays.Results: Stasis, white-cell and platelet contamination, within-day variations, varying storage time before centrifugation, colonoscopy, and surgical trauma had no significant influence on levels of 5mC or H3K9Me3. Day-to-day variations of 12.7% and 11.5% (intra-individual) and 98.1% and 60.8% (inter-individual) were shown for 5mC and H3K9Me3, respectively. Levels of 5mC or H3K9Me3 were significantly higher in samples stored at room temperature until centrifugation compared to samples stored on ice. Patients with cancer had significantly lower levels of 5mC or H3K9Me3 compared to levels in healthy individuals.Conclusion: Levels of 5mC or H3K9Me3 appear stable in most pre-analytical settings if blood samples are stored at room temperature until centrifugation.

Bisulfite oligonucleotide-capture sequencing for targeted base- and strand-specific absolute 5-methylcytosine quantitation.

Epigenetic regulation through DNA methylation (5mC) plays an important role in development, aging, and a variety of diseases. Genome-wide studies of base- and strand-specific 5mC are limited by the extensive sequencing required. Targeting bisulfite sequencing to specific genomic regions through sequence capture with complimentary oligonucleotide probes retains the advantages of bisulfite sequencing while focusing sequencing reads on regions of interest, enables analysis of more samples by decreasing the amount of sequence required per sample, and provides base- and strand-specific absolute quantitation of CG and non-CG methylation levels. As an example, an oligonucleotide capture set to interrogate 5mC levels in all rat RefSeq gene promoter regions (18,814) and CG islands, shores, and shelves (18,411) was generated. Validation using whole-genome methylation standards and biological samples demonstrates enrichment of the targeted regions and accurate base-specific quantitation of CG and non-CG methylation for both forward and reverse genomic strands. A total of 170Mb of the rat genome is covered including 6.6 million CGs and over 67 million non-CG sites, while reducing the amount of sequencing required by ~85% as compared to existing whole-genome sequencing methods. This oligonucleotide capture targeting approach and quantitative validation workflow can also be applied to any genome of interest.

The toxic effects and possible mechanisms of Bisphenol A on oocyte maturation of porcine in vitro.

Bisphenol A (BPA) and Di-(2-ethylhexyl) phthalate (DEHP) are widely used in the plastic industry such as water bottles, containers, packaging and toys. BPA and DEHP are shown to be the endocrine disruptors which disturb the endocrine system and are linked to several diseases including infertility. In this study, we investigated the effects of BPA exposure on porcine oocyte maturation and its possible reasons. Our results showed that: (i) the rates of oocyte maturation significantly decreased with 250 μM BPA treatment in vitro, but not DEHP. This might be due to the delayed cell cycle progression of oocyte maturation. (ii) BPA treatment resulted in abnormal cytoskeletons on porcine oocytes, showing with aberrant actin distribution, spindle morphology and chromosome alignment, which was further confirmed by the reduced p-MAPK level. (iii) The fluorescence intensity of histone methylation (H3K4me2) and DNA methylation (5 mC) levels were altered after BPA treatment, indicating that epigenetic modification was disturbed. (iv) BPA-exposed oocytes had higher rates of early stage apoptosis/autophagy, and this may be resulted from the increased level of oxidative stress. Collectively, our results indicated that porcine oocytes maturation was disrupted after BPA treatment through disrupting cytoskeletal dynamics, epigenetic modifications and inducing apoptosis/autophagy.

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.

Consumo, digestibilidade e balanço de nitrogênio de rações contendo diferentes teores de torta de murumuru em dietas para ovinos

This study aimed to assess the potential use of increasing levels of murumuru cake (Astrocaryum murumuru var. murumuru, M art.) (MC) in sheep diets as a replacement for Mombasa grass (Panicum maximum Jacq). Metabolic tests were performed with 20 castrated male sheep at Embrapa Amazônia Oriental, Belém, Pará, during 26 days. The experiment was arranged in a completely randomized design, with five diets and four replications. MC0: 100% grass; MC10: 10% MC and 90% grass; MC20: 20% MC and 80% grass; MC40: 40% MC and 60% grass; and MC60: 60% MC and 40% grass. The intake and the coefficient of apparent digestibility of dry matter (DMI and CDDM), organic matter (OMI and CDOM), crude protein (CPI and CDCP), neutral detergent fiber (NDFI and CDNDF), acid detergent fiber (ADFI and CDADF), ether extract (EEI and CDEE), cellulose (CELI and CDCEL), hemicellulose (HEMI and CDHEM), and nitrogen balance (NB) of experimental diets were determined. The intake of mineral material (MMI) and lignin (LIGI) were also determined. The DMI, OMI, MMI, CPI, NDFI, and ADFI displayed a decreasing linear effect with the replacement of Mombasa grass by MC in the diet. The EEI and the LIGI presented quadratic effects according to the replacement levels of MC in the diet. The CDDM, CDOM, and CDHEM displayed increasing linear effects, between MC0 and MC60. The CDEE, CDNDF, CDADF, and CDCEL displayed a quadratic effect, with optimum replacement levels of 56.65%, 41%, 31.33%, and 27.46%, respectively. The nitrogen balance presented a negative linear effect in the range of 0% to 60% of murumuru cake. One can conclude that murumuru cake is an alternative to the dietary supplementation of ruminants in replacing the Mombasa grass because it provides an increase in the digestibility of nutrients for sheep. However, a limited replacement level must be implemented, considering that from 27.46%, 31.33%, 41%, and 56.65% replacement, a decline occurs in the CDCEL, CDADF, CDNDF, and CDEE, respectively, although a negative nitrogen balance does not occur.

DNA base modifications in honey bee and fruit fly genomes suggest an active demethylation machinery with species- and tissue-specific turnover rates

Well-known epigenetic DNA modifications in mammals include the addition of a methyl group and a hydroxyl group to cytosine, resulting in 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) respectively. In contrast, the abundance and the functional implications of these modifications in invertebrate model organisms such as the honey bee (Apis mellifera) and the fruit fly (Drosophila melanogaster) are not well understood. Here we show that both adult honey bees and fruit flies contain 5mC and also 5hmC. Using a highly sensitive liquid chromatography/tandem mass spectrometry (LC/MS/MS) technique, we quantified 5mC and 5hmC in different tissues of adult honey bee worker castes and in adult fruit flies. A comparison of our data with reports from human and mouse shed light on notable differences in 5mC and 5hmC levels between tissues and species.

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.

The role of DNA methylation during anoxia tolerance in a freshwater turtle (Trachemys scripta elegans).

Oxygen deprivation is a lethal stress that only a few animals can tolerate for extended periods. This study focuses on analyzing the role of DNA methylation in aiding natural anoxia tolerance in a champion vertebrate anaerobe, the red-eared slider turtle (Trachemys scripta elegans). We examined the relative expression and total enzymatic activity of four DNA methyltransferases (DNMT1, DNMT2, DNMT3a and DNMT3b), two methyl-binding domain proteins (MBD1 and MBD2), and relative genomic levels of 5-methylcytosine under control, 5 h anoxic, and 20 h anoxic conditions in liver, heart, and white skeletal muscle (n = 4, p < 0.05). In liver, protein expression of DNMT1, DNMT2, MBD1, and MBD2 rose significantly by two- to fourfold after 5 h anoxic submergence compared to normoxic-control conditions. In heart, 5 h anoxia submergence resulted in a 1.4-fold increase in DNMT3a levels and a significant decrease in MBD1 and MBD2 levels to ~30 % of control values. In white muscle, DNMT3a and DNMT3b increased threefold and MBD1 levels increased by 50 % in response to 5 h anoxia. Total DNMT activity rose by 0.6-2.0-fold in liver and white muscle and likewise global 5mC levels significantly increased in liver and white muscle under 5 and 20 h anoxia. The results demonstrate an overall increase in DNA methylation, DNMT protein expression and enzymatic activity in response to 5 and 20 h anoxia in liver and white muscle indicating a potential downregulation of gene expression via this epigenetic mechanism during oxygen deprivation.

DNA methylation dynamics in mouse preimplantation embryos revealed by mass spectrometry.

Following fertilization in mammals, paternal genomic 5-methyl-2′-deoxycytidine (5 mC) content is thought to decrease via oxidation to 5-hydroxymethyl-2′-deoxycytidine (5 hmC). This reciprocal model of demethylation and hydroxymethylation is inferred from indirect, non-quantitative methods. We here report direct quantification of genomic 5 mC and 5 hmC in mouse embryos by small scale liquid chromatographic tandem mass spectrometry (SMM). Profiles of absolute 5 mC levels in embryos produced by in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) were almost identical. By 10 h after fertilization, 5 mC levels had declined by ~40%, consistent with active genomic DNA demethylation. Levels of 5 mC in androgenotes (containing only a paternal genome) and parthenogenotes (containing only a maternal genome) underwent active 5 mC loss in the first 6 h, showing that both parental genomes can undergo demethylation independently. We found no evidence for net loss of 5 mC 10–48 h after fertilization, implying that any passive ‘demethylation’ following DNA replication was balanced by active 5 mC maintenance methylation. However, levels of 5 mC declined during development after 48 h, to 1% (measured as a fraction of G-residues) in blastocysts (~96 h). 5 hmC levels were consistently low (<0.2% of G-residues) throughout development in normal diploid embryos. This work directly quantifies the dynamics of global genomic DNA modification in mouse preimplantation embryos, suggesting that SMM will be applicable to other biomedical situations with limiting sample sizes.

Both diet and gene mutation induced obesity affect oocyte quality in mice.

Obesity was shown to cause reproductive dysfunctions such as reduced conception, infertility and early pregnancy loss. However, the possible effects of obesity on oocyte quality are still not fully understood. In this study we investigated the effects of both diet and gene mutation induced obesity on impairments in mouse oocyte polarization, oxidative stress, and epigenetic modifications. Our results showed that high-fat diet induced obesity (HFD) and gene mutation induced obesity (ob/ob) could both impair oocyte meiotic maturation, disrupt spindle morphology, and reduce oocyte polarity. Oocytes from obese mice underwent oxidative stress, as shown by high DHE and ROS levels. Abnormal mitochondrial distributions and structures were observed in oocytes from obese groups of mice and early apoptosis signals were detected, which suggesting that oxidative stress had impaired mitochondrial function and resulted in oocyte apoptosis. Our results also showed that 5 mC levels and H3K9 and H3K27 methylation levels were altered in oocytes from obese mice, which indicated that DNA methylation and histone methylation had been affected. Our results showed that both HFD and ob/ob induced obesity affected oocyte maturation and that oxidative stress-induced early apoptosis and altered epigenetic modifications may be the reasons for reduced oocyte quality in obese mice.

Omega-3 Polyunsaturated Fatty Acids Inhibited Tumor Growth via Preventing the Decrease of Genomic DNA Methylation in Colorectal Cancer Rats

ABSTRACTOmge-3 polyunsaturated fatty acids (PUFAs) exhibited significant effect in inhibiting various tumors. However, the mechanisms of its anticancer role have not been fully demonstrated. The declination of 5-methylcytosine (5 mC) was closely associated with poor prognosis of tumors. To explore whether omega-3 PUFAs influences on DNA methylation level in tumors, colorectal cancer (CRC) rat model were constructed using N-methyl phosphite nitrourea and omega-3 PUFAs were fed to part of the rats during tumor induction. The PUFAs contents in the rats of 3 experimental groups were measured using gas chromatography and 5 mC level were detected by liquid chromatography tandem mass spectrometry. The results showed that tumor incidence in omega-3 treated rats was much lower than in CRC model rats, which confirmed significant antitumor role of omega-3 PUFAs. Six PUFA members categorized to omega-3 and omega-6 families were quantified and the ratio of omega-6/omega-3 PUFAs was remarkably lower in omega-3 PUFAs treatment group than in CRC model group. 5 mC content in omega-3 PUFAs treated rats was higher than in CRC model rats, suggesting omega-3 PUFAs promoted 5 mC synthesis. Therefore, omega-3 PUFAs probably inhibited tumor growth via regulating DNA methylation process, which provided a novel anticancer mechanism of omega-3 PUFAs from epigenetic view.

MBD3L2 promotes Tet2 enzymatic activity for mediating 5-methylcytosine oxidation.

Ten-eleven translocation (Tet) proteins are key players involved in the dynamic regulation of cytosine methylation and demethylation. Inactivating mutations of Tet2 are frequently found in human malignancies, highlighting the essential role of Tet2 in cellular transformation. However, the factors that control Tet enzymatic activity remain largely unknown. Here, we found that methyl-CpG-binding domain protein 3 (MBD3) and its homolog MBD3-like 2 (MBD3L2) can specifically modulate the enzymatic activity of Tet2 protein, but not Tet1 and Tet3 proteins, in converting 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC). Moreover, MBD3L2 is more effective than MBD3 in promoting Tet2 enzymatic activity through strengthening the binding affinity between Tet2 and the methylated DNA target. Further analysis revealed pronounced decreases in 5mC levels at MBD3L2 and Tet2 co-occupied genomic regions, most of which are promoter elements associated with either cancer-related genes or genes involved in the regulation of cellular metabolic processes. Our data add new insights into the regulation of Tet2 activity by MBD3 and MBD3L2, and into how that affects Tet2-mediated modulation of its target genes in cancer development. Thus, they have important applications in understanding how dysregulation of Tet2 might contribute to human malignancy.

C9orf72 promoter hypermethylation is reduced while hydroxymethylation is acquired during reprogramming of ALS patient cells

Among several genetic mutations known to cause amyotrophic lateral sclerosis (ALS), a hexanucleotide repeat expansion in the C9orf72 gene is the most common. In approximately 30% of C9orf72-ALS cases, 5-methylcytosine (5mC) levels within the C9orf72 promoter are increased, resulting in a modestly attenuated phenotype. The developmental timing of C9orf72 promoter hypermethylation and the reason why it occurs in only a subset of patients remain unknown. In order to model the acquisition of C9orf72 hypermethylation and examine the potential role of 5-hydroxymethylcytosine (5hmC), we generated induced pluripotent stem cells (iPSCs) from an ALS patient with C9orf72 promoter hypermethylation. Our data show that 5mC levels are reduced by reprogramming and then re-acquired upon neuronal specification, while 5hmC levels increase following reprogramming and are highest in iPSCs and motor neurons. We confirmed the presence of 5hmC within the C9orf72 promoter in post-mortem brain tissues of hypermethylated patients. These findings show that iPSCs are a valuable model system for examining epigenetic perturbations caused by the C9orf72 mutation and reveal a potential role for cytosine demethylation.

Evaluation of 5-methylcytosine and 5-hydroxymethylcytosine as potential biomarkers for characterisation of chemical allergens

Epigenetic regulation of gene expression plays a pivotal role in the orchestration of immune responses. Chemical allergens form two categories: skin sensitizing chemicals associated with allergic contact dermatitis, and chemicals that cause sensitization of the respiratory tract and occupational asthma. In mice these are characterized by different T helper (Th) cell responses. Changes in DNA methylation in particular have been implicated in the in vivo responses to chemical allergy. As such it was hypothesised that differentially methylated regions (DMR) may provide candidates biomarkers of chemical allergy To examine this, mice were exposed to 2,4-dinitrochlorobenzene (DNCB; a contact allergen) or trimellitic anhydride (TMA; a respiratory allergen). DNA from draining lymph nodes was processed for methylated (5mC) and hydroxymethylated (5hmC) DNA immunoprecipitation (MeDIP/hMeDIP) then selected DMR analysed by qPCR. We describe a number of DMRs which, by combined analysis of 5mC and 5hmC, differentiate between responses induced by DNCB and those by TMA. Furthermore, these changes in methylation are specific to the draining lymph node. The Gmpr DMR is suggested as a possible biomarker for contact allergen-induced immune responses; it is characterised by divergent levels of 5mC and 5hmC DNCB-treated mice only. In contrast, the Nwc DMR was characterised by divergent 5mC and 5hmC specifically in response to TMA, highlighting its possible utility as a biomarker for responses induced by chemical respiratory allergens. These data not only represent novel analysis of 5hmC in response to chemical allergy in vivo, but with further investigation, may also provide a possible basis for differentiation between classes of chemical allergens.

Loss of 5-hydroxymethylcytosine is linked to gene body hypermethylation in kidney cancer.

Both 5-methylcytosine (5mC) and its oxidized form 5-hydroxymethylcytosine (5hmC) have been proposed to be involved in tumorigenesis. Because the readout of the broadly used 5mC mapping method, bisulfite sequencing (BS-seq), is the sum of 5mC and 5hmC levels, the 5mC/5hmC patterns and relationship of these two modifications remain poorly understood. By profiling real 5mC (BS-seq corrected by Tet-assisted BS-seq, TAB-seq) and 5hmC (TAB-seq) levels simultaneously at single-nucleotide resolution, we here demonstrate that there is no global loss of 5mC in kidney tumors compared with matched normal tissues. Conversely, 5hmC was globally lost in virtually all kidney tumor tissues. The 5hmC level in tumor tissues is an independent prognostic marker for kidney cancer, with lower levels of 5hmC associated with shorter overall survival. Furthermore, we demonstrated that loss of 5hmC is linked to hypermethylation in tumors compared with matched normal tissues, particularly in gene body regions. Strikingly, gene body hypermethylation was significantly associated with silencing of the tumor-related genes. Downregulation of IDH1 was identified as a mechanism underlying 5hmC loss in kidney cancer. Restoring 5hmC levels attenuated the invasion capacity of tumor cells and suppressed tumor growth in a xenograft model. Collectively, our results demonstrate that loss of 5hmC is both a prognostic marker and an oncogenic event in kidney cancer by remodeling the DNA methylation pattern.

Human Blood Cell Level of 5-Hydroxymethylcytosine (5hmC) Declines Steadily during Aging and Is Multifactorial

Abstract BACKGROUND. The most frequently mutated genes documented in blood cells of aging individuals are known epigenetic regulator genes such as TET2 and DNMT3A, which suggests that alteration of epigenetic homeostasis could be a predisposing factor in the pathogenesis of several age-associated hematological malignancies. This study is aimed at determining if changes in hematopoietic 5-hydroxymethylcytosine (5hmC) and 5-methylcytosine (5mC) levels occur in normal individuals, and if they are independent of acquired mutations in epigenetic regulators. METHOD. The study population comprised 198 unrelated women randomly selected to form four age categories (neonates, 25-30 years, 70-75 years and &gt;90 years) from the general community. Genomic DNA from total blood cells or cord blood (12.5 ug) was hydrolysed using DNA Degradase Plus and analysed by mass spectrometry (LC-ESI-MS/MS-MRM) to quantify global 5-methyl-2'-deoxycytidine and 5-hydroxymethyl-2'-deoxycytidine levels. Statistical analysis (normality test, outlier detection, descriptive statistic, non-parametric Kruskal-Wallis and Mann Whitney tests) were performed using NCSS 07.1.21 to correlate methylation (5mC and 5hmC) levels with i) X-chromosome inactivation (XCI) patterns (evaluated by HUMARA) in polymorphonuclear (PMN) and ii) telomeres length (measured by the method of Cawthon). All individuals over 70 were sequenced in search for mutations in epigenetic regulator genes including TET2, DNMT3A, ASXL1, IDH1, IDH2 and WT1. RESULTS. 5hmC and 5mC levels: Global 5hmC levels decline steadily with age in human blood cells (30% from birth to old age, P &lt;0.000001). This reduction is progressive between 0 and 75 years of age and plateaued at a low level thereafter (Figure 1). A less severe reduction in 5mC level was also observed between newborn and elderly individuals (3%, P&lt;0.000024). Correlation with biological characteristics: Low level of 5hmC was associated with more important XCI skewing in PMN (age-adjusted, P =0.0304) as well as a reduction of telomere length (age-adjusted, P =0.0354), both surrogate markers of clonal dominance. Correlation with mutational status: Of the 100 individuals over 70 years, 16 had a somatic mutation in TET2, 10 in DNMT3A and none in IDH1, IDH2 or WT1. Individuals with TET2 gene mutation (variant allele frequency (VAF) &gt;10%) had a significant reduction in 5hmC but not the DNMT3A subjects. To evaluate if the TET2 mutated individuals were the sole drivers of the documented reduction in 5hmC, we removed these subjects from the statistical analysis and correlation was preserved. However, correlation with XCI skewing and telomere reduction was no longer significant. CONCLUSION. These results document a significant age-associated reduction in 5hmC during aging in the hematopoietic compartment. DNA hypohydroxymethylation should be considered as a new hallmark of aging hematopoiesis. We also demonstrate that acquired mutations in key epigenetic regulators such as TET2 are not the sole cause of this reduction. Epigenetic drift, which is defined as the cumulative selection of permissive stochastic epigenetic modifications, is responsible for most of the age-associated loss. These results also indicate that the impact on clonal dominance may be different according to the cause of 5hmC (mutation vs drift). Further studies are needed to evaluate the impact of 5hmC reduction on the risk of hematological cancer initiation. Figure 1. Figure 1. Disclosures Busque: Novartis: Consultancy; BMS: Consultancy; PFIZER: Consultancy.

Development and Validation of a LC-ESI-MS/MS-MRM Analytical Method to Quantify 5hmC Levels in Human Blood Cells

BACKGROUND. Epigenetic dysregulation resulting from acquired mutations in the methylcytosine dioxygenase gene TET2, responsible for the conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), and the methyltransferase gene DNMT3A, responsible for the methylation (5mC) of cytosine, is emerging as an important predisposing factor for age-associated hematological cancers. This study describes an improved and validated analytical method to quantify global 5hmC and 5mC levels in DNA samples by liquid chromatography-electrospray ionization-tandem mass spectrometry-multiple reaction monitoring (LC-ESI-MS/MS-MRM).METHODS. Genomic DNA from blood cells is hydrolysed using DNA Degradase Plus and stopped by the addition of formic acid (0.1%) spiked with stable isotope labeled internal standards (2 nM of 5-hydroxymethyl-2'-deoxycytidine-d3 (2H3-5hmC), 40 nM of 5-methyl-2'-deoxycytidine-d3 (2H3-5mC) and 200 nM 2'-deoxyguanosine-13C10,15N5 (15N5-G). Global 5-methyl-2'-deoxycytidine (5mC) and 5-hydroxymethyl-2'-deoxycytidine (5hmC) levels are assessed by LC-ESI-MS/MS-MRM. 2.5 ul containing 12.5 ng of hydrolysed DNA and internal standards is injected onto a reverse phase ultra-performance liquid chromatography column (Agilent 1290 Infinity UHPLC Systems with Waters ACQUITY UPLC HSS T3 Column, 100Å, 1.8 µm, 2.1 mm X 100 mm) connected to a triple quadripole mass spectrometer (Agilent 6460 QQQ) operating in positive ion MRM mode. MRM transitions used are for 2H3-5hmC m/z 261.2>145.1, 5hmC m/z 258.2>142.1, 2H3-5mC m/z 245.2>129.1, 5mC m/z 242.2>126.1, 15N5-G m/z 283.3>162.1, G m/z 268.3>152.1. Global percentages of modified cytosine are estimated using deoxyguanosine (G) as surrogate for total cytosine (C). For the validation phase, a cohort of 472 asymptomatic aging individuals were sequenced at high coverage (95% >500x) for mutations in TET2 (Ampliseq, Ion Proton NGS) and then analysed by mass spectrometry.RESULTS. The analytical method yields linear responses across the biological range for all monitored nucleotides. For 5hmC, the limit of quantification (LOQ) is 50 amol (20 pM) with an interday accuracy of 103% and a precision of 3.3%. This sensitive and robust method was validated on a cohort of asymptomatic aging individuals (n=472) to test its capacity to identify the epigenetic phenotype associated with TET2 loss-of-function mutations: a severe reduction in 5hmC level but not in 5mC level, resulting in a reduced 5hmC/5mC ratio. In the cohort, TET2 mutations were associated with 29% reduction in 5hmC/5mC ratio compared to wild-type individuals (for mutations with VAF>20%, P<0.005). This reduction is directly function of the mutation frequency (VAF) making it possible to calculate a functional score for TET2 somatic mutations based on the severity of the associated epigenetic phenotype (Figure 1). Inversely, when using the method to screen for TET2 mutants in a population, individuals harboring a TET2 mutation above 20% VAF can be identified with 100% sensitivity and 98.43% specificity by establishing a threshold 5hmC/5mC ratio corresponding to 2 standard deviations below the mean of the wild-type population.CONCLUSION. This validated analytical method confers high sensitivity and robustness making it ideally suited to screen clinical samples for the epigenetic phenotype associated with consequential TET2 mutations. [Display omitted] DisclosuresBusque:Novartis: Consultancy; BMS: Consultancy; PFIZER: Consultancy.

4 SUPPRESSION OF ASH2L ALTERS DNA METHYLATION AND HISTONE PATTERNS DURING BOVINE EMBRYONIC DEVELOPMENT

Epigenetic patterns established during early bovine embryogenesis via DNA methylation and histone modification patterns are essential for proper gene expression and embryonic development. We have previously discovered that suppression of absent, small, or homeotic-like (ASH2L) with small interfering RNA (siRNA) had no significant effect during in vitro embryo development when compared with its respective control (31.3 ± 2.0% standard error of the mean, n = 466 v. 34.8 ± 1.9%, n = 418). Analysing DNA methylation and histone modifications via immunocytochemistry will further explain the role of ASH2L during embryonic development, specifically at the blastocyst stage. In this experiment, we obtained mature bovine oocytes from a commercial supplier (De Soto Biosciences, Seymour, TN) and preformed IVF following standard laboratory protocol. Eighteen hours after IVF, presumptive zygotes were divided into 3 treatments: noninjected controls, nontargeting siRNA injected controls (siNULL), and injection with siRNA targeting ASH2L (siASH2L). Each embryo was injected with ~100 pL of 20 nM siRNA previously verified to suppress expression of ASH2L by ~79%. Embryos were cultured in Bovine Evolve (Zenith Biotech, Guilford, CT) supplemented with 4 mg mL–1 of BSA (Probumin, Millipore) for 7 days. Blastocysts from each treatment (N = 601) were fixed and prepared for immunocytochemistry following standard laboratory protocol. The following primary antibodies were used to target specific DNA and histone methylation marks: 5mc mAb (Epigentek, Farmingdale, NY), 5hmc pAb, H3K4me3 pAb (Active Motif, Carlsbad, CA), H3K4me2 pAb, H3K9me2–3 mAb, and H3K27me3 mAb (Abcam, Cambridge, MA). Embryos were fluorescently labelled with the following secondary antibodies: Alexa Flour 488 Goat Anti-Rabbit, Alexa 488 Donkey Anti-Goat, and Alexa Flour 594 Goat Anti-Mouse (Invitrogen, Carlsbad, CA). The DNA was stained with Hoechst 33342 (Invitrogen). Fluorescent images were captured using the Zeiss Stallion digital imaging work station. Ratio averages (targeting mark/DNA) were calculated and statistical analysis performed using one-way ANOVA and Tukey’s honestly significant difference to assess treatment effects. The ratio of DNA methylation to total DNA increased in siASH2L as compared with control and siNULL embryos (0.35 ± 0.01, 0.26 ± 0.02, and 0.30 ± 0.01, respectively; P &lt; 0.01). The 5hmC was inversely related to 5mC levels and decreased in siASH2L embryos (0.75 ± 0.01, 0.93 ± 0.02, 0.87 ± 0.02, respectively; P &lt; 0.0001). The H3K4me3 and H3K27me3 are also inversely related with decreased H3K4me3 in siASH2L versus control and siNULL embryos (0.48 ± 0.02, 0.57 ± 0.02, 0.58 ± 0.02, respectively; P &lt; 0.001) and increased H3K27me3 (0.62 ± 0.02, 0.053 ± 0.01, 0.54 ± 0.02, respectively; P &lt; 0.001). No differences were observed in H3K9me2–3 or H3K4me2 labelling across treatments. These results indicate that ASH2L may play a role in DNA methylation by decreasing 5mc and 5hmc conversion, which is a key event during early embryonic development. Suppression of ASH2L also alters global levels of H3H4me3 and H3K27me3, which may lead to transcription aberrations. Further analysis of siASH2L embryos via RNA-seq will help define its role during early embryonic development.