Region-specific DNA hydroxymethylation along the malignant progression of IDH-mutant gliomas.

The majority of low-grade isocitrate dehydrogenase-mutant (IDHmt) gliomas undergo malignant progression (MP), but their underlying mechanism remains unclear. IDHmt gliomas exhibit global DNA methylation, and our previous report suggested that MP could be partly attributed to passive demethylation caused by accelerated cell cycles. However, during MP, there is also active demethylation mediated by ten-eleven translocation, such as DNA hydroxymethylation. Hydroxymethylation is reported to potentially contribute to gene expression regulation, but its role in MP remains under investigation. Therefore, we conducted a comprehensive analysis of hydroxymethylation during MP of IDHmt astrocytoma. Five primary/malignantly progressed IDHmt astrocytoma pairs were analyzed with oxidative bisulfite and the Infinium EPIC methylation array, detecting 5-hydroxymethyl cytosine at over 850,000 locations for region-specific hydroxymethylation assessment. Notably, we observed significant sharing of hydroxymethylated genomic regions during MP across the samples. Hydroxymethylated CpGs were enriched in open sea and intergenic regions (p < 0.001), and genes undergoing hydroxymethylation were significantly associated with cancer-related signaling pathways. RNA sequencing data integration identified 91 genes with significant positive/negative hydroxymethylation-expression correlations. Functional analysis suggested that positively correlated genes are involved in cell-cycle promotion, while negatively correlated ones are associated with antineoplastic functions. Analyses of The Cancer Genome Atlas clinical data on glioma were in line with these findings. Motif-enrichment analysis suggested the potential involvement of the transcription factor KLF4 in hydroxymethylation-based gene regulation. Our findings shed light on the significance of region-specific DNA hydroxymethylation in glioma MP and suggest its potential role in cancer-related gene expression and IDHmt glioma malignancy.

EBS-seq: enrichment-based method for accurate analysis of 5-hydroxymethylcytosine at single-base resolution.

BACKGROUND: A growing body of research has emphasized 5-hydroxymethylcytosine (5hmC) as an important epigenetic mark. High-resolution methods to detect 5hmC require high sequencing depth and are therefore expensive. Many studies have used enrichment-based methods to detect 5hmC; however, conventional enrichment-based methods have limited resolution. To overcome these limitations, we developed EBS-seq, a cost-efficient method for 5hmC detection with single-base resolution that combines the advantages of high-resolution methods and enrichment-based methods. RESULTS: EBS-seq uses selective labeling of 5hmC, deamination of cytosine and 5-methylcytosine, pull-down of labeled 5hmC, and C-to-T conversion during DNA amplification. Using this method, we profiled 5hmC in HEK293T cells and two colorectal cancer samples. Compared with conventional enrichment-based 5hmC detection, EBS-seq improved 5hmC signals by localizing them at single-base resolution. Furthermore, EBS-seq was able to determine 5hmC levels in CpG-dense regions where distortion of signals can occur, such as CpG islands and CpG shores. Comparing EBS-seq and conventional high-resolution 5hmC detection by ACE-seq, we showed that EBS-seq is more effective at finding 5hmC sites. Using EBS-seq, we found strong associations between gene expression and gene-body 5hmC content in both HEK293T cells and colorectal cancer samples. CONCLUSIONS: EBS-seq is a reliable and cost-efficient method for 5hmC detection because it simultaneously enriches 5hmC-containing DNA fragments and localizes 5hmC signals at single-base resolution. This method is a promising choice for 5hmC detection in challenging clinical samples with low 5hmC levels, such as cancer tissues.

A plant-based diet differentially affects the global hepatic methylome in rainbow trout depending on genetic background.

Replacing fish meal and oil in trout diets with plant-derived ingredients is a contemporary challenge to move towards more sustainable aquaculture practices. However, such dietary replacement causes hepatic metabolic changes that have not yet been elucidated. Here, we aimed to decipher the effect of a 100% plant-based diet on the hepatic global DNA methylation landscape in trout and assess whether changes depend on fish genetic background. We analysed the global methylome and the expression of DNA (de)methylation-related genes of three isogenic lines that exhibit similar growth when fed a marine resource-based diet (M diet), but differ in their responses to a plant-based diet (V diet). Our results revealed that the V diet induced a decrease in 5-cytosine combined with an increase in 5-hydroxymethylcytosine in two of the three analysed lines. For one of these 2 affected lines, when fed the M diet but at the same feed intake of the V diet (MR), no methylome differences were highlighted between M and MR or between MR and V-fed trout whereas for the other affected line, M fed trout displayed a divergent methylome profile from MR and V fed fish. DNA (de)methylation-related genes were also affected by the V or MR diets. Our findings showed that the global hepatic methylome of trout is affected by a V diet, depending on genetic background. This latter effect seems to be due to either a decreased feed intake alone or combined with the effect of the dietary composition per se.

Locus-Specific Enrichment Analysis of 5-Hydroxymethylcytosine Reveals Novel Genes Associated with Breast Carcinogenesis.

An imbalance in DNA methylation is a hallmark epigenetic alteration in cancer. The conversion of 5-methylcytosine (5-mC) to 5-hydroxymethyl cytosine (5-hmC), which causes the imbalance, results in aberrant gene expression. The precise functional role of 5-hydroxymethylcytosine in breast cancer remains elusive. In this study, we describe the landscape of 5-mC and 5-hmC and their association with breast cancer development. We found a distinguishable global loss of 5-hmC in the localized and invasive types of breast cancer that strongly correlate with TET expression. Genome-wide analysis revealed a unique 5-mC and 5-hmC signature in breast cancer. The differentially methylated regions (DMRs) were primarily concentrated in the proximal regulatory regions such as the promoters and UTRs, while the differentially hydroxymethylated regions (DhMRs) were densely packed in the distal regulatory regions, such as the intergenic regions (>-5 kb from TSSs). Our results indicate 4809 DMRs and 4841 DhMRs associated with breast cancer. Validation of nine 5-hmC enriched loci in a distinct set of breast cancer and normal samples positively correlated with their corresponding gene expression. The novel 5-hmC candidates such as TXNL1, and CNIH3 implicate a pro-oncogenic role in breast cancer. Overall, these results provide new insights into the loci-specific accumulation of 5-mC and 5-hmC, which are aberrantly methylated and demethylated in breast cancer.

The role of DNA methylation in epigenetics of aging.

Recent research suggests that epigenetics, especially DNA methylation, plays a mechanistic role in aging. Epigenetic clocks, which measure changes in a few hundred specific CpG sites, can accurately predict chronological age in a variety of species, including humans. These clocks are currently the best biomarkers for predicting mortality in humans. Additionally, several studies have characterized the effects of aging across the methylome in a wide variety of tissues from humans and mice. A small fraction (~2%) of the CpG sites show age-related changes, either hypermethylation or hypomethylation with aging. Evaluation of non-CpG site methylation has only been examined in a few studies, with about ~0.5% of these sites showing a change with age. Therefore, while only a small fraction of cytosines in the genome show changes in DNA methylation with age, this represents 2 to 3 million cytosines in the genome. Importantly, the only study to compare the effect of aging on DNA methylation in male and female mice and humans found that >95% of the age-related changes in DNA methylation in the hippocampus were sexually divergent, i.e., the methylation did not differ between males and females at young age but age-related changes occurred in one sex but not the other. The age-related changes in DNA methylation tend to be enriched and under-represented in specific genomic contexts, with some commonalities between tissues and species that require further investigation. The strongest evidence that the age-related changes in DNA methylation play a role in aging comes from studies of anti-aging interventions (e.g., caloric restriction, dwarfism, and rapamycin treatment) in mice. These anti-aging interventions deaccelerate the epigenetic clocks and reverse/prevent 20 to 40% of the age-related changes in DNA methylation. It will be important in the future to demonstrate that at least some of the age-related changes in DNA methylation directly lead to alterations in the transcriptome of cells/tissues that could potentially contribute to aging.

Quantification of Global DNA Methylation Levels by Mass Spectrometry.

Global DNA methylation was classically considered the relative percentage of 5-methylcysine (5mC) with respect to total cytosine (C). Early approaches were based on the use of high-performance separation technologies and UV detection. However, the recent development of protocols using mass spectrometry for the detection has increased sensibility and permitted the precise identification of peak compounds based on their molecular masses. This allows work to be conducted with much less genomic DNA starting material and also to quantify 5-hydroxymethyl-cytosine (5hmC), a recently identified form of methylated cytosine that could play an important role in active DNA demethylation. Here, we describe the protocol that we currently use in our laboratory to analyze 5mC and 5hmC by mass spectrometry. The protocol, which is based on the method originally developed by Le and colleagues using Ultra Performance Liquid Chromatography (UPLC) and mass spectrometry (triple Quadrupole (QqQ)) detection, allows for the rapid and accurate quantification of relative global 5mC and 5hmC levels starting from just 1 µg of genomic DNA, which allows for the rapid and accurate quantification of relative global 5mC and 5hmC levels.

Label free detection of 5'hydroxymethylcytosine within CpG islands using optical sensors.

Significant research has been invested in correlating genetic variations with different disease probabilities. Recently, it has become apparent that other DNA modifications, such as the addition of a methyl or hydroxymethyl group to cytosine, can also play a role. While these modifications do not change the sequence, they can negatively impact the function. Therefore, it is critical to be able to both read the genetic code and identify these modifications. Currently, the detection of hydroxymethylated cytosine (5'hmC) and the two closely related variants, cytosine (C) and 5'methylcytosine (5'mC), relies on a combination of nucleotide modification steps, followed by PCR and gene sequencing. However, this approach is not ideal because transcription errors which are inherent to the PCR process can be misinterpreted as fluctuations in the relative C:5'mC:5'hmC concentrations. As such, an alternative method which does not rely on PCR or nucleotide modification is desirable. One approach is based on label-free optical resonant cavity sensors. In the present work, toroidal resonant cavity sensors are functionalized with antibodies to enable label-free detection and discrimination between C, 5'mC, and 5'hmC in real-time without PCR. Specifically, epoxide chemistry is used to covalently attach the 5'hmC antibody to the surface of the cavity. Subsequently, to thoroughly characterize the sensor platform, detection of C, 5'mC, and 5'hmC is performed over a concentration range from pM to nM. At low (pM) concentrations, the hydroxymethylated cytosine produces a significantly larger signal than the structurally similar epigenetic markers; thus demonstrating the applicability of this platform.

Analysis of DNA Methylation and Hydroxymethylation in the Genome of Crustacean Daphnia pulex.

The aim of our study was to analyze the presence of 5-methyl-cytosine (5-mC) and 5-hydroxymethyl-cytosine (5-hmC) in the genome of crustacean Daphnia pulex. First, the presence of 5-mC and 5-hmC in genomic DNA was demonstrated by using antibodies specific to either 5-mC or 5-hmC. Then, analysis of 5-mC and 5-hmC using pairs of restriction enzymes with different sensitivity to methylation and hydroxymethylation confirmed the presence of both modifications in selected regions of three genes (Cox4, Cand2 and Ephx1). To get a detailed picture of 5-hmC distribution over the D. pulex genome, we performed 5-hmC enrichment and sequenced the enriched fraction using next generation sequencing and non-enriched library (input) as a control. Comparison of input and enriched libraries showed that 5-hmC in exons is twice as frequent as in introns. Functional analysis indicated that 5-hmC abundance is associated with genes that are involved in the adenylate cyclase-activating G-protein-coupled receptor signaling pathway, molting cycles, morphogenesis and cell fate determination. Genes that lack 5-hmC tend to be involved in the regulation of the transforming growth factor beta receptor signaling pathway and in many mRNA-related processes. Our results suggest that epigenetic modifications are present in the genome of D. pulex and most likely are involved in the regulation of gene expression of this crustacean.

Influence of ambient air pollution on global DNA methylation in healthy adults: a seasonal follow-up.

BACKGROUND: DNA methylation changes are potential pathways of environmentally induced health effects. We investigated whether exposure to ambient concentrations of NO2, PM10, PM2.5 and O3 and traffic parameters were associated with global DNA methylation in blood of healthy adults. METHODS: 48 non-smoking adults (25 males) with a median age of 39years were sampled in winter and summer. Global DNA methylation in whole blood (% 5-methyl-2'-deoxycytidine, %5mdC) was analyzed with HPLC. Exposure to air pollutants at the home address was assessed using interpolated NO2, PM10, PM2.5 and O3 concentrations for various exposure windows (60- to 1-day moving average exposures and yearly averages) and GIS-based traffic parameters. Associations between pollutants and %5mdC were tested with multiple mixed effects regression models. RESULTS: Average %5mdC (SD) was 4.30 (0.08) in winter and 4.29 (0.08) in summer. Men had higher %5mdC compared to women both in winter (4.32 vs. 4.26) and summer (4.31 vs. 4.27). When winter and summer data were analyzed together, various NO2, PM10 and PM2.5 moving average exposures were associated with changes in %5mdC (95% CI) ranging from -0.04 (-0.09 to 0.00) to -0.14 (-0.28 to 0.00) per IQR increase in pollutant. NO2, PM10, PM2.5 and O3 moving average exposures were associated with decreased %5mdC (95% CI) varying between -0.01 (-0.03 to 0.00) and -0.17 (-0.27 to -0.06) per IQR increase in pollutant in summer but not in winter. CONCLUSION: Decreased global DNA methylation in whole blood was associated with exposure to NO2, PM10, PM2.5 and O3 at the home addresses of non- adults. Most effects were observed for the 5- to 30-day moving average exposures.