5-methylcytosine Content Research Articles

Global DNA Methylation and Hydroxymethylation Differ in Hepatocellular‐ and Cholangio‐Carcinoma and Relate to Survival Rate

DNA hydroxymethylation has been recently recognized as a novel epigenetic mark. Primary liver cancers, hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC), are highly prevalent but epigenetically poorly characterized. Aims were to determine by a LC/MS/MS method methylcytosine (mCyt) and hydroxymethylcytosine (hmCyt) content in HCC and CC tissues as compared with homologous non‐neoplastic tissues and to analyze the survival rate according to mCyt and hmCyt levels in peripheral blood mononuclear cells (PBMCs) DNA. Thirty‐one HCC and 16 CC patients participated to the study; mCyt content was significantly lower in HCC than CC tissues (3.97% vs 5.26% respectively, p<0.0001). HCC tissues showed a lower mCyt compared to non‐neoplastic tissues (3.97% vs 4.82% mCyt, respectively, p<0.0001). No such difference was instead detected for the comparison of CC and non‐neoplastic tissues. HmCyt was similar in HCC and CC tissues, while it was reduced in HCC and CC as compared to homologous non‐neoplastic tissues (0.044 vs 0.128, p<0.0001 for HCC and 0.030 vs 0.124, p=0.026 for CC).The survival rate at 48 months in liver cancer patients with PBMCs mCyt>5.59% was higher than in those with lower mCyt levels (p=0.034).DNA hypomethylation distinguishes HCC from CC while DNA hypo‐hydroxymethylation characterizes both HCC and CC. PBMCs DNA mCyt content >5.59% relates to a favorable outcome in primary liver cancers.

Analysis of pollutant-induced changes in mitochondrial DNA methylation.

There is increasing evidence that exposure to air pollutants is associated with human disease and may act through epigenetic modification of the nuclear genome, but there have been few publications describing their impact upon the mitochondrial genome. Mitochondrial DNA may be more susceptible to pollutant-induced changes via increased oxidative stress in the cell, and therefore this field of research is of growing interest. Many techniques employed to study DNA methylation of the nuclear genome are also applicable to mitochondrial epigenetic studies. In this chapter, we describe a protocol for the isolation of mitochondrial DNA from peripheral blood samples and the analysis of 5-methylcytosine content by bisulfite pyrosequencing.

Global DNA methylation as a possible biomarker for diabetic retinopathy.

We evaluated whether global levels of DNA methylation status were associated with retinopathy as well as providing a predictive role of DNA methylation in developing retinopathy in a case-control study of 168 patients with type 2 diabetes. The 5-methylcytosine content was assessed by reversed-phase high-pressure liquid chromatography of peripheral blood leukocytes to determine an individual's global DNA methylation status in the two groups, either with or without retinopathy. The global DNA methylation levels were significantly higher in diabetic retinopathy patients compared with those in non-retinopathy patients (4.90 ± 0.12 vs. 4.22 ± 0.13, respectively; p = 0.001). There was a significant increasing trend in global DNA methylation levels in terms of progressing retinopathy (without retinopathy, 4.22 ± 0.13; non-proliferative diabetic retinopathy, 4.62 ± 0.17; proliferative diabetic retinopathy, 5.07 ± 0.21) (p = 0.006). Additionally, global DNA methylation independent of retinopathy risk factors, which include dyslipidaemia, hypertension, hyperglycaemia and duration of diabetes, was a predictive factor for retinopathy (OR = 1.53, p = 0.015). Global DNA methylation is modulated during or possibly before the primary stage of diabetes. This observation verifies the metabolic memory effect of hyperglycaemia in early stage of an aetiological process that leads to type 2 diabetes and its associated complications.

Analysis of 5-methyl cytosine and 5-hydromethyl cytosine content and distribution in bovine placental and fetal tissues after somatic nuclear reprogramming.

Analysis of 5-methyl cytosine and 5-hydromethyl cytosine content and distribution in bovine placental and fetal tissues after somatic nuclear reprogramming.

Aberrant DNA methylation patterns in diabetic nephropathy.

BackgroundThe aim of this study was to evaluate whether global levels of DNA methylation status were associated with albuminuria and progression of diabetic nephropathy in a case-control study of 123 patients with type 2 diabetes- 53 patients with albuminuria and 70 patients without albuminuria.MethodsThe 5-methyl cytosine content was assessed by reverse phase high pressure liquid chromatography (RP-HPLC) of peripheral blood mononuclear cells to determine individual global DNA methylation status in two groups.ResultsGlobal DNA methylation levels were significantly higher in patients with albuminuria compared with those in normal range of albuminuria (p = 0.01). There were significant differences in global levels of DNA methylation in relation to albuminuria (p = 0.028) and an interesting pattern of increasing global levels of DNA methylation in terms of albuminuria severity.In patients with micro- and macro albuminuria, we found no significant correlations between global DNA methylation levels and duration of diabetes (p > 0.05). In both sub groups, there were not significant differences between global DNA methylation levels with good and poor glycaemic control (p > 0.05). In addition, in patients with albuminuria, no differences in DNA methylation levels were observed between patients with and without other risk factors including age, gender, hypertension, dyslipidaemia and obesity.ConclusionsThese data may be helpful in further studies to develop novel biomarkers and new strategies for clinical care of patients at risk of diabetic nephropathy.

DNA methylation is altered in B and NK lymphocytes in obese and type 2 diabetic human

ObjectiveObesity is associated with low-grade inflammation and the infiltration of immune cells in insulin-sensitive tissues, leading to metabolic impairment. Epigenetic mechanisms control immune cell lineage determination, function and migration and are implicated in obesity and type 2 diabetes (T2D). The aim of this study was to determine the global DNA methylation profile of immune cells in obese and T2D individuals in a cell type-specific manner. Material and methodsFourteen obese subjects and 11 age-matched lean subjects, as well as 12 T2D obese subjects and 7 age-matched lean subjects were recruited. Global DNA methylation levels were measured in a cell type-specific manner by flow cytometry. We validated the assay against mass spectrometry measures of the total 5-methylcytosine content in cultured cells treated with the hypomethylation agent decitabine (r=0.97, p<0.001). ResultsGlobal DNA methylation in peripheral blood mononuclear cells, monocytes, lymphocytes or T cells was not altered in obese or T2D subjects. However, analysis of blood fractions from lean, obese, and T2D subjects showed increased methylation levels in B cells from obese and T2D subjects and in natural killer cells from T2D patients. In these cell types, DNA methylation levels were positively correlated with insulin resistance, suggesting an association between DNA methylation changes, immune function and metabolic dysfunction. ConclusionsBoth obesity and T2D are associated with an altered epigenetic signature of the immune system in a cell type-specific manner. These changes could contribute to the altered immune functions associated with obesity and insulin resistance.

Estimation of global content of 5-methylcytosine in DNA during allantoic and pulmonary respiration in the chicken embryo.

DNA methylation is an epigenetic modification that plays an important role in the proper development and functioning of an organism. The DNA methylation level is species-, tissue- and organelle-specific, and the methylation pattern is determined during embryogenesis. A correlation between methylation and age is also observed. Epigenetic phenomena are an enormously interesting research subject, not only from the perspective of pure science, but also due to their possible applications in medicine. The aim of this study was to determine the global DNA methylation level in relation to the developmental stage of the embryo. The global level of 5-methylcytosine in the DNA during pulmonary respiration was found to be higher than during allantoic respiration. The analysis shows a clear dependence between the stage of individual development and the global DNA level of 5-methylcytosine. In the future, methylation level may be a determinant of age and perhaps even a tool for predicting life expectancy. Abnormalities in the methylation process result in premature ageing at the cellular and individual level.

Repetitive genomic elements and overall DNA methylation changes in acute myeloid and childhood B-cell lymphoblastic leukemia patients.

Aberrant epigenetic regulation is a hallmark of neoplastic cells. Increased DNA methylation of individual genes' promoter regions and decreases in overall DNA methylation level are both generally observed in cancer. In solid tumors, this global DNA hypomethylation is related to reduced methylation of repeated DNA elements (REs) and contributes to genome instability. The aim of the present study was to assess methylation level of LINE-1 and ALU REs and total 5-methylcytosine (5metC) content in adult acute myeloid leukemia (AML) (n = 58), childhood B-cell acute lymphoblastic leukemia (ALL) (n = 32), as the most frequent acute leukemias in two age categories and in normal adult bone marrow and children's blood samples. DNA pyrosequencing and ELISA assays were used, respectively. Global DNA hypomethylation was not observed in leukemia patients. Results revealed higher DNA methylation of LINE-1 in AML and ALL samples compared to corresponding normal controls. Elevated methylation of ALU and overall 5metC level were also observed in B-cell ALL patients. Differences of REs and global DNA methylation between AML cytogenetic-risk groups were observed, with the lowest methylation levels in intermediate-risk/cytogenetically normal patients. B-cell ALL is characterized by the highest DNA methylation level compared to AML and controls and overall DNA methylation is correlated with leukocyte count.

Coordinate Changes In RNA Expression, Transcript Splicing, and DNA Methylation In Pediatric AML

While mutations of splicing and epigenetic factors have been reported in adult AML and related myeloid disorders, relatively few such changes have been identified in pediatric AML. We previously identified a chromatin remodeling helicase, PASG (SMARCA6, HELLS, LSH), by down-regulation in AML cell lines following cytokine withdrawal and identified an alternatively spliced variant lacking a highly conserved (STRAGGLG) domain. To assess the prevalence of this splicing variant (PASGΔ75) in pediatric AML, we tested 167 diagnostic specimens from the TARGET-AML cohort for fractional PASGΔ75 expression (PASGΔ75/PASG) using a discriminatory RT-qPCR assay. These studies demonstrated a broad, continuous distribution of PASGΔ75 with right skew (mean PASGΔ75: 26%, interquartile range: 9% – 41%) that was not significantly associated with cytogenetic class (inv16, t(8;21), MLL, normal) or FAB subtype. For further comparison, specimens were quantized by PASGΔ75 quartile.Given reported associations between loss of PASG function and abnormalities of genomic methylation, we tested 48 AML specimens at the extremes of the PASGΔ75 distribution for total 5-methylcytosine (5-mC) content by liquid chromatography/tandem mass spectrometry. The mean total methylation was significantly lower in the high compared to the low PASGΔ75 groups (mean 5-mC 3.95% vs. 4.22% of cytosine, p=0.015 Mann-Whitney). To identify specific regions of altered methylation, we used high-throughput sequencing of DNA enriched by pull-down with the methyl binding domain fragment of MBD2 (MBD-Seq). Comparison of summed methylation signal across regions flanking RefSeq transcriptional start sites (TSS) showed the expected decrease in methylation just upstream of the TSS in both groups. However, methylation more distal to the TSS was proportionally lower in PASGΔ75 high than PASGΔ75 low samples (Fig 1a). To evaluate methylation at CpG islands (CGI), UCSC CGI were scaled to 500 bp and MBD-Seq data were summed across 20Kb flanking CGI. While both groups showed the anticipated increase in methylation signal on CGI, methylation in the shore regions immediately flanking CGI was proportionally lower in high PASGΔ75 compared to low PASGΔ75 samples (Fig 1b), further suggesting epigenetic differences between these sample groups. [Display omitted] Because we were unable to identify sequence variants in PASG intron 18 or flanking exons that might explain alternative splicing, we asked whether expression of PASGΔ75 was associated with global changes in transcript splicing. We evaluated gene expression patterns on the Affymetrix HuGene array, with assessment of alternative splicing using Partek software alternative splicing (altsplice) algorithm for quartile-grouped samples. In contrast to comparison of adjacent quartile groups, which showed modest changes in expression and relatively few transcripts with significant altsplice scores, comparison of the highest and lowest quartile samples showed marked changes in gene expression and a large number of alternatively spiced transcripts as assessed by significance of the altsplice score (Fig 2). In addition to splicing changes, these analyses suggested marked differences in gene expression patterns of AML specimens grouped by PASGΔ75 quartile, with clear separation of Q1 and Q4 samples by principal components analysis. Using a conservative Wilcoxon gene sets test and limiting ourselves to small, curated Biocarta pathways, we found expression patterns associated with high deletion variant expression strongly linked to overlapping pathways involving DNA repair, replication, and cell cycle progression. [Display omitted] These data suggest the existence of a previously unrecognized AML subclass characterized by widespread and coordinated changes in RNA expression, alternative transcript splicing, and epigenetic modifications. Disclosures:No relevant conflicts of interest to declare.TablePathways (Biocarta)Pathway ClassBenjamini Hochberg Corrected p-ValueATR/BRCA1/BRCA2DNA Damage Response1.3E-6RB/DNA DamageDNA Damage Response3.5E-3p27 PhosphorylationCell Cycle Progression3.5E-3G2/M CheckpointCell Cycle Progression3.5E-3G1/S CheckpointCell Cycle Progression4.1E-3PLK3Cell Cycle Progression4.1E-3MEF2DApoptosis0.01SRC/PTPaCell Cycle Progression0.02Mitochondrial Acetyl-Co ShuttleMetabolism0.02p53 SignalingDNA Damage Response0.04E2F-1Cell Cycle Progression0.04ATM SignalingDNA Damage Response0.04

Aberrant Epigenetic Modification in Murine Brain Tissues of Offspring from Preimplantation Genetic Diagnosis Blastomere Biopsies1

Preimplantation genetic diagnosis (PGD) has been prevalent in the field of assisted reproductive technology, yet the long-term risks of PGD to offspring remain unknown. In the present study, the early development of PGD embryos, postimplantation characteristics, and birth rate following PGD were determined. Moreover, the behavior of the offspring conceived from the biopsied embryos was evaluated with the Morris water maze and pole climbing tests. Finally, the epigenetic modification of the global genome and methylation patterns for the H19, Igf2, and Snrpn imprinted genes were identified. The results indicated a significant delay in the blastocoel formation of PGD embryos and a decrease in the implantation ability of these embryos, which was related to the decreased number of cells in the PGD blastocysts. The PGD mice spent more time on both the nontrained quadrant of the water maze and climbing down the pole. Furthermore, the 5-hydroxymethylcytosine content in the brain tissues of PGD mice was significantly increased, but no difference was found in 5-methylcytosine content. The differentially methylated regions of H19/Igf2 exhibited decreased methylation patterns, but that of Snrpn was normal, compared to the control group. Quantitative RT-PCR indicated that Igf2 mRNA expression was significantly decreased but that H19 and Snrpn mRNAs were expressed normally. In conclusion, blastomere biopsies in PGD procedures carry potential risks to embryo development and the behavior of resulting offspring; these risks may arise from aberrant epigenetic modification and methylation patterns in brain tissues. Further studies are needed to better understand the risks associated with PGD.

Association of 5-Methylcytosine and 5-Hydroxymethylcytosine with Mitochondrial DNA Content and Clinical and Biochemical Parameters in Hepatocellular Carcinoma

Increasing epidemiological evidence has indicated that inherited variations of mitochondrial DNA (mtDNA) copy number affect the genetic susceptibility of many malignancies in a tumour-specific manner and that DNA methylation also plays an important role in controlling gene expression during the differentiation and development of hepatocellular carcinoma (HCC). Our previous study demonstrated that HCC tissues showed a lower 5-hydroxymethylcytosine (5-hmC) content when compared to tumour-adjacent tissues, but the relationship among 5-hmC, 5-methylcytosine (5-mC) and mtDNA content in HCC patients is still unknown. This study aimed to clarify the correlation among mtDNA content, 5-mC and 5-hmC by quantitative real-time PCR and liquid chromatography tandem mass spectrometry analysis. We demonstrated that 5-hmC correlated with tumour size [odds ratio (OR) 0.847, 95% confidence interval (CI) 0.746–0.962, P = 0.011], and HCC patients with a tumour size ≥5.0 cm showed a lower 5-hmC content and higher levels of fasting plasma aspartate aminotransferase, the ratio of alanine amiotransferase to aspartate aminotransferase, γ-glutamyltransferase, alpha-fetoprotein than those with a tumour size <5 cm (all P<0.05). We further revealed that the mtDNA content of HCC tumour tissues was 225.97(105.42, 430.54) [median (25th Percentile, 75th Percentile)] and was negatively correlated with 5-mC content (P = 0.035), but not 5-hmC content, in genomic DNA from HCC tumour tissues.

Global DNA methylation status in laryngeal cancer

The purpose of our study was to evaluate if DNA methylation level in leukocytes may be used as a surrogate marker of genome methylation status in laryngeal cancer tissues. We evaluated global DNA methylation using an ultra performance liquid chromatography (UPLC)-based method to assess the total content of 5-methylcytosine (5 mC). The study was performed on DNA isolated from cancer tissues, adjacent normal tissues, and peripheral blood leukocytes in a group of 72 patients with laryngeal cancer. DNA hypomethylation was found in tumor tissue (56%) and normal tissue (49%). There was a significant correlation between the levels of 5 mC in these 2 types of tissue. There was no significant DNA hypomethylation in blood. A negative correlation between tumor grade and blood levels of 5 mC was found. The level of leukocyte DNA methylation measured using total 5 mC content cannot be used as a surrogate marker for genome methylation status in laryngeal cancer tissues.

A Potential Role for CHH DNA Methylation in Cotton Fiber Growth Patterns

DNA methylation controls many aspects of plant growth and development. Here, we report a novel annual growth potential change that may correlate with changes in levels of the major DNA demethylases and methyltransferases in cotton ovules harvested at different times of the year. The abundances of DNA demethylases, at both the mRNA and protein levels, increased significantly from February to August and decreased during the remainder of the 12-month period, with the opposite pattern observed for DNA methyltransferases. Over the course of one year, substantial changes in methylcytosine content was observed at certain CHH sites (H = A, C, or T) in the promoter regions of the ETHYLENE RESPONSIVE FACTOR 6 (ERF6), SUPPRESSION OF RVS 161 DELTA 4 (SUR4) and 3-KETOACYL-COA SYNTHASE 13 (KCS13), which regulate cotton fiber growth. Three independent techniques were used to confirm the annual fluctuations in DNA methylation. Furthermore, in homozygous RNAi lines specifically targeting REPRESSOR OF SILENCING 1 (ROS1, a conserved DNA demethylase domain), promotion of DNA methylation significantly reduced fiber growth during August.

Genomic impact of transient low-dose decitabine treatment on primary AML cells

AbstractAcute myeloid leukemia (AML) is characterized by dysregulated gene expression and abnormal patterns of DNA methylation; the relationship between these events is unclear. Many AML patients are now being treated with hypomethylating agents, such as decitabine (DAC), although the mechanisms by which it induces remissions remain unknown. The goal of this study was to use a novel stromal coculture assay that can expand primary AML cells to identify the immediate changes induced by DAC with a dose (100nM) that decreases total 5-methylcytosine content and reactivates imprinted genes (without causing myeloid differentiation, which would confound downstream genomic analyses). Using array-based technologies, we found that DAC treatment caused global hypomethylation in all samples (with a preference for regions with higher levels of baseline methylation), yet there was limited correlation between changes in methylation and gene expression. Moreover, the patterns of methylation and gene expression across the samples were primarily determined by the intrinsic properties of the primary cells, rather than DAC treatment. Although DAC induces hypomethylation, we could not identify canonical target genes that are altered by DAC in primary AML cells, suggesting that the mechanism of action of DAC is more complex than previously recognized.

Increased 5-Methylcytosine and Decreased 5-Hydroxymethylcytosine Levels are Associated with Reduced Striatal A2AR Levels in Huntington’s Disease

Adenosine A2A receptor (A2AR) is a G-protein-coupled receptor highly expressed in basal ganglia. Its expression levels are severely reduced in Huntington's disease (HD), and several pharmacological therapies have shown its implication in this neurodegenerative disorder. The main goal of this study was to gain insight into the molecular mechanisms that regulate A2AR gene (ADORA2A) expression in HD. Based on previous data reported by our group, we measured the methylcytosine (5mC) and hydroxymethylcytosine (5hmC) content in the 5'UTR region of ADORA2A in the putamen of HD patients and in the striatum of R6/1 and R6/2 mice at late stages of the disease. In this genomic region, 5mC and 5hmC remained unchanged in both mice strains, although low striatal A2AR levels were associated with reduced 5mC levels in 30-week-old R6/1 mice and reduced 5hmC levels in 12-week-old R6/2 mice in exon m2. In order to analyze when this mechanism appears during the progression of the disease, a time course for A2AR protein levels was carried out in R6/1 mice striatum (8, 12, and 20weeks of age). A2AR levels were reduced from 12weeks of age onwards, and this downregulation was concomitant with reduced 5hmC levels in the 5'UTR region of ADORA2A. Interestingly, increased 5mC levels and reduced 5hmC were found in the 5'UTR region of ADORA2A in the putamen of HD patients with respect to age-matched controls. Therefore, an altered DNA methylation pattern in ADORA2A seems to play a role in the pathologically decreased A2AR expression levels found in HD.

Deciphering the Epigenetic Code: An Overview of DNA Methylation Analysis Methods

Methylation of cytosine in DNA is linked with gene regulation, and this has profound implications in development, normal biology, and disease conditions in many eukaryotic organisms. A wide range of methods and approaches exist for its identification, quantification, and mapping within the genome. While the earliest approaches were nonspecific and were at best useful for quantification of total methylated cytosines in the chunk of DNA, this field has seen considerable progress and development over the past decades. Methods for DNA methylation analysis differ in their coverage and sensitivity, and the method of choice depends on the intended application and desired level of information. Potential results include global methyl cytosine content, degree of methylation at specific loci, or genome-wide methylation maps. Introduction of more advanced approaches to DNA methylation analysis, such as microarray platforms and massively parallel sequencing, has brought us closer to unveiling the whole methylome. Sensitive quantification of DNA methylation from degraded and minute quantities of DNA and high-throughput DNA methylation mapping of single cells still remain a challenge. Developments in DNA sequencing technologies as well as the methods for identification and mapping of 5-hydroxymethylcytosine are expected to augment our current understanding of epigenomics. Here we present an overview of methodologies available for DNA methylation analysis with special focus on recent developments in genome-wide and high-throughput methods. While the application focus relates to cancer research, the methods are equally relevant to broader issues of epigenetics and redox science in this special forum.

In Vitro Decitabine Treatment Demonstrates Heterogeneous Changes in Methylation and Gene Expression in Primary AML Samples.

AbstractAbstract 2527Acute myeloid leukemia (AML) is a hematopoietic neoplasm with high mortality that is typically treated with daunorubicin/cytarabine induction chemotherapy. Alternative therapies with cytosine analogs such as decitabine are also used in some cases with a variable clinical response that some have estimated to be as high as 25%. The mechanism of these agents is unclear, but at low doses they produce passive DNA hypomethylation by inhibiting DNMT1. Although the impact of these drugs on cell growth and DNA methylation in AML cell lines has been evaluated1, studies using primary cells are limited; importantly, most have involved extended drug treatments that may be confounded by the differentiation of the treated cells2. In addition, some evidence suggests that decitabine has a differential effect on methylation in patients who respond to treatment2, but the utility of this phenotype as an in vitro biomarker for decitabine responsiveness is unknown.In this study, we used a novel in vitro culture system for primary leukemia cells to explore the initial genomic effects of short-term low dose decitabine on primary samples from 22 AML patients. Primary bone marrow or blood samples from these patients were cultured on HS27 stromal cells in DMEM supplemented with beta-mercaptoethanol and 15% FBS along with hSCF, hIL3, hIL-6, hTPO and hFLT3L for an initial 4-day period prior to daily treatment for 3 days with either 100 nM decitabine, 100 nM cytarabine, or vehicle controls. Cells were then evaluated for growth, cell cycle effects, and differentiation (by flow cytometry and morphologic evaluation). DNA was prepared from all samples for 5-methylcytosine content measurements by mass spectrometry, and 8 samples were selected for genome-wide methylation and gene expression profiling with the Illumina Human Methylation 450 and Affymetrix Human Exon 1.0ST array platforms. Mass spectrometry revealed a mean decrease in 5-mdC of 29% (range: 13% to 62%) in the decitabine-treated samples; in comparison, cytarabine treatment resulted in a mean increase in 5-mdC of 5% (range: −10% to 37%). Methylation arrays also showed a modest shift toward lower methylation values, but unsupervised hierarchical clustering demonstrated that methylation patterns were driven by sample-specific differences and not drug treatment. Analysis of methylation changes showed the most pronounced hypomethylation at CpGs with high baseline methylation levels, irrespective of CpG island and gene-based annotation, suggesting that the initial methylation status of each CpG is responsible for preferential effects of decitabine, rather than its genomic context. Methylation at promoter-associated CpGs showed a small but statistically significant negative correlation with change in gene expression, but expression changes at individual genes were not consistent across the samples, including genes previously shown to be regulated by methylation-dependent mechanisms (eg. CDKN2B and CDx H1).In addition to these findings, we observed that a sample from a long-term decitabine responder had an exaggerated in vitro response to decitabine (58% decrease in 5-mdC after 6 days of treatment), compared to a cohort of decitabine non-responders; a sample from a second patient also showed marked hypomethylation by both mass spectrometry and methylation array, although this patient was not treated with decitabine. While more investigation is needed, this observation might suggest that extreme in vitro hypomethylation in response to decitabine could serve as a biomarker for a clinical response.In summary, our study showed that short-term low dose decitabine treatment has modest but detectable effects on DNA methylation and gene expression, but these changes did not result in activation of any canonical gene expression pathway at this early time point. We found that the baseline methylation status of a CpG appears to be the best predictor of decitabine-induced hypomethylation, with highly methylated CpGs showing the greatest change. We also observed that hypomethylation is highly variable across primary samples and at specific genes, implying that single gene approaches for measuring decitabine effect may be problematic. Finally, extreme in vitro decitabine-induced hypomethylation should be further investigated as a biomarker for decitabine responsiveness. Disclosures:No relevant conflicts of interest to declare.

The correlation of genome size and DNA methylation rate in metazoans

Total DNA methylation rates are well known to vary widely between different metazoans. The phylogenetic distribution of this variation, however, has not been investigated systematically. We combine here publicly available data on methylcytosine content with the analysis of nucleotide compositions of genomes and transcriptomes of 78 metazoan species to trace the evolution of abundance and distribution of DNA methylation. The depletion of CpG and the associated enrichment of TpG and CpA dinucleotides are used to infer the intensity and localization of germline CpG methylation and to estimate its evolutionary dynamics. We observe a positive correlation of the relative methylation of CpG motifs with genome size. We tested this trend successfully by measuring total DNA methylation with LC/MS in orthopteran insects with very different genome sizes: house crickets, migratory locusts and meadow grasshoppers. We hypothesize that the observed correlation between methylation rate and genome size is due to a dependence of both variables from long-term effective population size and is driven by the accumulation of repetitive sequences that are typically methylated during periods of small population sizes. This process may result in generally methylated, large genomes such as those of jawed vertebrates. In this case, the emergence of a novel demethylation pathway and of novel reader proteins for methylcytosine may have enabled the usage of cytosine methylation for promoter-based gene regulation. On the other hand, persistently large populations may lead to a compression of the genome and to the loss of the DNA methylation machinery, as observed, e.g., in nematodes.

The Role of DNA Methylation in Aging, Rejuvenation, and Age-Related Disease

DNA methylation is a major control program that modulates gene expression in a plethora of organisms. Gene silencing through methylation occurs through the activity of DNA methyltransferases, enzymes that transfer a methyl group from S-adenosyl-L-methionine to the carbon 5 position of cytosine. DNA methylation patterns are established by the de novo DNA methyltransferases (DNMTs) DNMT3A and DNMT3B and are subsequently maintained by DNMT1. Aging and age-related diseases include defined changes in 5-methylcytosine content and are generally characterized by genome-wide hypomethylation and promoter-specific hypermethylation. These changes in the epigenetic landscape represent potential disease biomarkers and are thought to contribute to age-related pathologies, such as cancer, osteoarthritis, and neurodegeneration. Some diseases, such as a hereditary form of sensory neuropathy accompanied by dementia, are directly caused by methylomic changes. Epigenetic modifications, however, are reversible and are therefore a prime target for therapeutic intervention. Numerous drugs that specifically target DNMTs are being tested in ongoing clinical trials for a variety of cancers, and data from finished trials demonstrate that some, such as 5-azacytidine, may even be superior to standard care. DNMTs, demethylases, and associated partners are dynamically shaping the methylome and demonstrate great promise with regard to rejuvenation.

Nuclear Exclusion of TET1 Is Associated with Loss of 5-Hydroxymethylcytosine in IDH1 Wild-Type Gliomas

The recent identification of isocitrate dehydrogenase 1 (IDH1) gene mutations in gliomas stimulated various studies to explore the molecular consequences and the clinical implications of such alterations. The Cancer Genome Atlas Research Network showed evidence for a CpG island methylator phenotype in glioblastomas that was associated with IDH1 mutations. These alterations were associated with the production of the oncometabolite, 2-hydroxyglutarate, that inhibits oxygenases [ie, ten-eleven translocation (TET) enzymes involved in the oxidation of 5-methylcytosine to 5-hydroxymethylcytosine (5hmC)]. We investigated 60 gliomas for 5hmC presence, 5-methylcytosine content, TET1 expression, and IDH1 mutation to gain insight into their relationships on a histological level. Of gliomas, 61% revealed no immunoreactivity for 5hmC, and no correlation was observed between IDH1 mutations and loss of 5hmC. Interestingly, expression of TET1 showed remarkable differences regarding overall protein levels and subcellular localization. We found a highly significant (P = 0.0007) correlation between IDH1 mutations and nuclear accumulation of TET1, but not with loss of 5hmC. Of 5hmC-negative gliomas, 70% showed either exclusive or dominant cytoplasmic expression, or no detectable TET1 protein (P = 0.0122). Our data suggest that the loss of 5hmC is a frequent event in gliomas, independent of IDH1 mutation, and may be influenced by the nuclear exclusion of TET1 from the nuclei of glioma cells.