Fructose-induced prediabetes causes persistent DNA methylation changes in white adipose tissue despite metabolic normalization.

High-fat diets (HFD) are thought to contribute to the development of metabolism-related diseases. The long-term impact of HFD may be mediated by epigenetic mechanisms, and indeed, HFD has been reported to induce DNA methylation changes in white adipose tissue (WAT) near metabolism related genes. However, previous studies were limited to a single WAT depot, a single time-point and primarily examined the pre-pubertal period. To define dynamic DNA methylation patterns specific for WAT depots, we investigated DNA methylation of Pparg2 and Leptin in gonadal adipose tissue (GAT) and subcutaneous adipose tissue (SAT), at baseline and after 6, 12 and 24 weeks of HFD exposure in adult mice. HFD induced hypermethylation of both the Leptin promoter (max. 19.6% at week 24, P = 2.6·10−3) and the Pparg2 promoter in GAT (max. 10.5% at week 12, P = 0.001). The differential methylation was independent of immune cell infiltration upon HFD exposure. In contrast, no differential methylation in the Pparg2 and Leptin promoter was observed in SAT. Leptin and Pparg2 DNA methylation were correlated with gene expression in GAT. Our study shows that prolonged exposure to HFD in adulthood is associated with a gradually increasing DNA methylation level at the Leptin and Pparg2 promoters in a depot-specific manner.

Recent evidence has indicated an important role for DNA methylation in the control of gene expression, silencing of transposons and maintenance of genome stability. The current study utilised the Amplified Methylation Polymorphism (AMP) technique to investigate the extent of DNA methylation polymorphisms and their biological importance in several plant species. DNA methylation patterns were examined in response to different environments and propagation through tissue culture, and in different tissue types of one plant species. The technique was also used to investigate the extent of DNA loss in transgenic sugarcane as a result of genetic engineering procedures. Widespread DNA methylation changes were detected in response to the environment in both species investigated, namely sugarcane (a complex monocot polyploid) and Arabidopsis thaliana (the simple dicot diploid). Sugarcane cultivar Q155 grown at three diverse locations in Queensland, Australia, showed location-specific DNA methylation patterns, and when clones were moved to common glasshouses they showed new glasshouse-specific DNA methylation patterns whilst generally retaining the older location-specific DNA methylation signatures. In addition, prolonged cold treatment (vernalisation) in Arabidopsis induced DNA methylation changes. These findings indicate that DNA methylation may be a general control mechanism for mediating phenotypic responses to environmental change. Plant genome instability with passage through tissue culture was shown to vary greatly between plant species, with wheat cultivar Hartog being particularly stable in comparison to sugarcane cultivars. It was also demonstrated that the process of producing double haploids of Hartog resulted in few, if any DNA methylation or sequence changes, thereby validating this approach for producing mapping populations for mapping important traits in wheat. In sugarcane, on the other hand, genome instability in conventionally tissue culture-propagated plants was demonstrated to be a major problem. Furthermore, gene transfer and/or selection processes increased the extent of genome instability in the form of DNA loss compared to that resulting from the conventional tissue culture-propagation process alone. While genome instability in conventionally tissue culture-propagated and genetically engineered sugarcane is currently a major problem, AMP analysis of non-transgenic sugarcane plants propagated by a new direct regeneration method indicated that the prospects of overcoming this problem of genome instability in sugarcane are encouraging. AMP profiles were shown to be distinguishable between leaf and pollen DNA in lily (Lilium longiforum), however, the extent of tissue-specific DNA methylation changes was shown to be less than that reported for mammals. Throughout the present study, the AMP technique has been proven to be a robust and efficient tool for analysing plant genome instability and assessing the DNA methylation status of specific loci throughout the genome. This study has also demonstrated the potential importance of DNA methylation in the plant response mechanism to changing environments, including responses to stresses imposed by the tissue culture process. However, more studies are necessary to elucidate the environmentally-induced DNA methylation changes. In particular, the priority would be the cloning, sequencing and mapping of the environmentally-induced DNA methylation changes. In view of the fact that the Arabidopsis genome is completely sequenced, mapping of the vernalisation-induced DNA methylation markers is currently achievable in this species and could greatly enhance our understanding of vernalisation and the control of flowering time in plants. Future applications of the AMP protocol would include efficient screening for genome integrity in tissue culture-derived and genetically engineered plants, assessing the effect of tissue culture conditions and gene transfer and selection procedures on genome instability, and further identification and characterisation of genomic regions displaying environment-specific DNA methylation.

Although the factors responsible for the recent increase in the prevalence of diabetes worldwide are not entirely known, the morbidity associated with this disease results in substantial health and economic burden on society. Epigenetic modifications, including DNA methylation have been identified as one mechanism by which the environment interacts with the genome and there is evidence that alterations in DNA methylation may contribute to the increased prevalence of both type 1 and type 2 diabetes. This review provides a summary of DNA methylation and its role in gene regulation, and includes descriptions of various techniques to measure site-specific and genome-wide DNA methylation changes. In addition, we review current literature highlighting the complex relationship between DNA methylation, gene expression, and the development of diabetes and related complications. In studies where both DNA methylation and gene expression changes were reported, DNA methylation status had a strong inverse correlation with gene expression, suggesting that this interaction may be a potential future therapeutic target. We highlight the emerging use of genome-wide DNA methylation profiles as a biomarker to predict patients at risk of developing diabetes or specific complications of diabetes. The development of a predictive model that incorporates both genetic sequencing and DNA methylation data may be an effective diagnostic approach for all types of diabetes and could lead to additional innovative therapies.

Recently, white adipose tissue has been shown to exhibit immunological activity, and may play an important role in host defense and protection against bacterial infection. Αlpha-lipoic acid (α-LA) has been demonstrated to function as an anti-inflammatory and anti-oxidant agent. However, its influence on the inflammatory response and metabolic changes in white adipose tissue remains unknown. We used male C57BL/6 mice as models to study the effect of α-LA on the inflammatory response and metabolic changes in white adipose tissue after stimulation with lipopolysaccharide (LPS). The non-esterified fatty acid content was measured by an automatic biochemical analyzer. The expression of inflammation-, lipid- and energy metabolism-related genes and proteins was determined by quantitative real-time polymerase chain reaction and western blotting. The results indicated that α-LA significantly decreased the epididymis fat weight index and the non-esterified fatty acid content in plasma compared with the control group. LPS significantly increased the expression of inflammation genes and α-LA reduced their expression. The LPS-induced expression of nuclear factor-κB protein was decreased by α-LA. Regarding lipid metabolism, α-LA significantly counteracted the inhibitory effects of LPS on the expression of hormone-sensitive lipase gene and protein. α-LA evidently increased the gene expression of fatty acid transport protein 1 and cluster of differentiation 36. Regarding energy metabolism, α-LA significantly increased the expression of most of mitochondrial DNA-encoded genes compared with the control and LPS group. Accordingly, α-LA can alleviate acute inflammatory response and this action may be related with the promotion of lipid mobilization in white adipose tissue.

Individual differentiated somatic cells and undifferentiated stem cells have common genome, although their functions or morphological characters are very different. These differences are derived from difference of gene expression pattern. DNA methylation is generally key factor of suppression of gene and its level is globally change during mammalian early development. But, in birds, whether genome-wide changes in DNA methylation occur during embryonic development is still unknown. Here, we show that genome-wide DNA methylation to assess occurrence during early chick embryonic development. We found that the methylation status at stage 1 was approximately 57%, after which it gradually decreases, reaching a minimum at stage 10 (33%). After stage 10, DNA methylation gradually increased. These results should contribute to clarify the epigenetic mechanisms in birds.

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.

High stability, the quality that made polychlorinated biphenyls (PCBs) so useful in hundreds of industrial and commercial applications during the mid-1900s, has ensured the compounds’ continuing presence in the environment despite decades of banned and restricted use.1 This class of more than 200 structurally related chemicals has been linked to both cancer and noncancer outcomes,1 and some studies suggest prenatal or early-life exposure to PCBs may adversely affect the auditory system.2,3 A new study reported in EHP specifically links postnatal—but not prenatal—PCB exposure with impaired cochlear function.2 Clusters of hair cells populate the cochlea and detect sounds transmitted to the inner ear. The cochlea, an extremely sensitive organ in the inner ear, converts soundwave vibrations to nerve impulses transmitted to the brain. Cochlear damage can impair hearing and is a well-known side effect of certain antibiotics and chemotherapeutic drugs.4 Rodent studies have shown that PCBs, too, can affect the cochlea, leading to hearing loss.5 Some human studies have reported associations between PCB exposure and hearing impairment, particularly in children,6,7 although others have found no such association.8 The current study focused on mother–infant pairs from an ongoing birth cohort study in eastern Slovakia. Study participants enrolled during hospital stays for childbirth in 2002–2004. Maternal and cord blood samples were collected for PCB and lipid measurements, and children underwent blood tests at 6, 16, and 45 months of age. At 45 months, the children also underwent otological and auditory testing, including an assessment of distortion product otoacoustic emissions (DPOAEs), a measure of cochlear function. Health records provided information on the children’s births and subsequent medical visits. Mothers completed questionnaires at study enrollment and at the 16- and 45-month followup visits, providing sociodemographic information and data on lifestyle, diet, medical history, and other factors that could affect their children’s health and hearing. A total of 351 children were included in the study. Blood tests measured 15 PCB congeners, but analyses focused on PCB-153, which was detected in nearly all samples and closely correlated with total PCB concentrations. An inverse relationship existed between decreased DPOAE amplitude (a marker of diminished cochlear function) and postnatal serum PCB concentrations, with the strongest association observed for cumulative levels of PCBs. The authors suggest this could indicate that duration of exposure, rather than timing, is most important for cochlear development. No association was observed between prenatal PCB exposure and decreased DPOAE amplitude.2 The PCB-associated decrease in DPOAE amplitude was not large.2 “Certainly, the hearing impairment would be subclinical,” says study coauthor Tomas Trnovec, a professor of environmental medicine at Slovak Medical University in Bratislava. However, the cochlea is vulnerable to any number of environmental insults, including noise, organochlorine pesticides, and drugs, among others, Trnovec says. “The combined effect of these factors is completely unknown,” he says, adding that “under different settings and other data treatment … a prenatal exposure effect cannot be excluded.” It’s also unknown whether effects persist and whether continued exposure (combined with other stressors) builds on early damage. “In my opinion, PCB ototoxicity may be involved in the pathogenesis of hearing impairment in the adult population,” says Kyoung-Bok Min, an assistant professor in occupational and environmental medicine at South Korea’s Ajou University School of Medicine, who was not involved in the study. Min and colleagues recently reported an association between PCB serum levels and increased prevalence of hearing impairment in adults who participated in the National Health and Nutrition Examination Survey.9 “Further epidemiologic studies are needed in terms of whether the impact of exposure to PCBs on cochlear function changes throughout the life span of the human,” he says. In addition, Susan Schantz, a professor of veterinary biosciences and psychology at the University of Illinois at Urbana–Champaign, raises the possibility of potential impacts beyond hearing. “One of the things that really interests me—and that I don’t know the answer to—is what does that kind of a decrease [in DPOAE amplitude] mean? Would it be enough to have subtle impacts on language acquisition in children?” asks Schantz, who was not involved in the study. Previous PCB cohort studies have shown an adverse impact on cognitive function, including verbal abilities.10 “I’ve always wondered whether subtle changes in hearing could be affecting language development, and then that could be affecting verbal abilities,” Schantz says. The new study features a particularly strong design based on multiple PCB measurements in a well-characterized population. However, it did not account for potential effects of co-contaminants, which may produce damage when combined with other exposures.11 “I think it’s very interesting that they’re seeing reductions to amplitudes related to postnatal exposure,” says Schantz. “That’s not what I would have predicted based on the animal research, but I think it’s very interesting and potentially important.”

Memory formation is a crucial task of the brain. It allows animals to dynamically respond to a changing environment by combining information about current and previous experiences. Thus, it promotes complex behaviours such as living in social groups and elaborate foraging tactics. The ability to form memories is present across the animal kingdom in a variety of forms. The honeybee - an eusocial insect - is capable of both ’simple’ associative and complex rule learning. Despite decades of research into the mechanisms of memory formation, however, much remains unknown. One little- understood aspect is the regulation of molecular mechanisms during memory formation. Understanding regulation of transcription is particularly important in this context, as transcription is a requirement for any stable memory. Epigenetic mechanisms regulate transcription by directly interacting with chromatin. Importantly, epigenetic mechanisms are conserved across species as distinct as humans and honeybees. This thesis aims to investigate one particular epigenetic mechanism - DNA methylation - and its role in honey bee memory formation by using behavioural, physiological and molecular assays.First, I studied the role of DNA methyltransferases (Dnmts), which catalyse DNA methylation, after olfactory reward learning. Bees were trained to associate an odour with a sugar reward. Dnmts were then blocked after conditioning using a pharmacological inhibitor. 24 hours later, bees were tested for memory retention and generalisation. Dnmt inhibition increased the generalisation to an odour that was not present during the training, thus impairing stimulus-specific memory. This effect was learning-dependent, as bees’ response to odours or sugar water alone did not change after treatment. Furthermore, this effect was robust against alterations in the training paradigm, but the directionality depended on the number of training trials.Next, I used Ca2+-imaging of the bees’ primary olfactory centre (i.e. antennal lobe, AL) to investigate whether Dnmts affect changes in AL processing established during memory formation. The AL is involved in odour discrimination and olfactory learning; both processes are also crucial for stimulus-specific memory formation. If Dnmts were inhibited after olfactory reward learning, the AL response to a new odour changed 48 hours later. This effect potentially serves as a functional explanation for the behavioural phenotype observed after Dnmt inhibition. Furthermore, this study provides the first in vivo evidence for Dnmt-mediated regulation of neuronal networks during memory formation.As DNA methylation regulates transcription, I next investigated the effect of Dnmt inhibition on gene expression. Using qPCR I studied 30 memory-associated genes. In response to Dnmt inhibition, 9 genes were upregulated after conditioning. For some of these genes I then investigated their methylation patterns using a bisulfite conversion based Mass spectrometry approach. Memory formation changed the methylation pattern compared to both unpaired (i.e. odour and sugar stimulation) and naive bees in these genes. Furthermore, I analysed the expression of Dnmts and Tet, which catalyses demethylation, after olfactory reward conditioning. Dnmt1b and Tet were upregulated after 1 hour, whereas Dnmt3 was only upregulated 5 hours after conditioning. Thus, Dnmts and Tet are likely active - in a specific temporal order - during a phase crucial for memory consolidation.Finally, I investigated genome-wide changes in DNA methylation and hydroxymethylation (i.e. intermediate of demethylation pathway) 24 hours after olfactory reward conditioning using MeDIP and hMeDIP sequencing. In order to connect and interpret the findings of my previous studies it is crucial to know which genomic regions Dnmts and Tet target during memory formation. Most memory-associated changes were located in the gene body; specifically, they were enriched in regionsflanking the transcription start and termination sites. This suggests a possible impact of DNA methylation and hydroxymethylation levels on transcription initiation and termination. Furthermore, transcription factors were enriched among the genes associated with changes in DNA methylation and hydroxymethylation. Thus, regulatory events are potentially multiplied by indirectly affecting genes targeted by these transcription factors.Overall, the results of this thesis reveal important novel aspects of DNA methylation and demethylation mediated regulation of memory formation in honey bees: (1) Dnmts are crucial for stimulusspecific memory formation and regulate cognitive, rather than perceptual, generalisation. (2) Dnmts regulate changes in odour processing in the primary olfactory centre during memory formation; likely promoting better discrimination between trained and novel odours. (3) Dnmts affect gene expression of a subgroup of memory-associated genes; but only in a few of these genes did methylation levels change as well, suggesting that Dnmts have a strong indirect effect on gene expression of memory-associated genes. This assumption is supported by the enrichment of transcription factors among genes associated with methylation or hydroxymethylation changes. (4) Dnmts and Tet are upregulated and thus likely active during a period crucial for memory consolidation. In conclusion, both DNA methylation and demethylation seem to play an important role in honey bee stimulusspecific memory formation by affecting the transcriptional landscape in neurons and consequently changing early odour processing and generalisation patterns.

DNA methylation programs gene expression and is involved in numerous biological processes. Accumulating evidence supports transgenerational inheritance of DNA methylation changes in mammals via germ cells. Our aim was to determine the effect of exercise on sperm DNA methylation. Twenty-four men were recruited and assigned to an exercise intervention or control group. Clinical parameters were measured and sperm samples were donated by subjects before and after the 3-month time-period. Mature sperm global and genome-wide DNA methylation was assessed using an ELISA assay and the 450K BeadChip (Illumina). Global and genome-wide sperm DNA methylation was altered after 3 months of exercise training. DNA methylation changes occurred in genes related to numerous diseases such as schizophrenia and Parkinson's disease. Our study provides the first evidence showing exercise training reprograms the sperm methylome. Whether these DNA methylation changes are inherited to future generations warrants attention.

A mammalian cell utilizes DNA methylation to modulate gene expression in response to environmental changes during development and differentiation. Aberrant DNA methylation changes as a correlate to diseased states like cancer, neurodegenerative conditions and cardiovascular diseases have been documented. Here we show genome-wide DNA methylation changes in macrophages infected with the pathogen M. tuberculosis. Majority of the affected genomic loci were hypermethylated in M. tuberculosis infected THP1 macrophages. Hotspots of differential DNA methylation were enriched in genes involved in immune response and chromatin reorganization. Importantly, DNA methylation changes were observed predominantly for cytosines present in non-CpG dinucleotide context. This observation was consistent with our previous finding that the mycobacterial DNA methyltransferase, Rv2966c, targets non-CpG dinucleotides in the host DNA during M. tuberculosis infection and reiterates the hypothesis that pathogenic bacteria use non-canonical epigenetic strategies during infection.

The effects of ionizing radiation on DNA methylation are of importance due to the role that DNA methylation plays in maintaining genome stability, and the presence of aberrant DNA methylation in many cancers. There is limited evidence that radiation-sensitivity may influence the modulation of DNA methylation by ionizing radiation, resulting in a loss of methylation. The BALB/c, CBA and C57Bl/6 strains are the most commonly utilized mouse strains in radiation research and are classified as radiation sensitive (BALB/c and CBA) or radiation resistant (C57Bl/6). We present here the first direct comparison of changes in repeat element DNA methylation (L1, B1 and Intracisternal A Particle; IAP) over time in these three mouse strains after high-dose radiation exposure. Using a high-resolution melt assay, methylation of the spleen repeat elements was investigated between 1 and 14 days after whole-body irradiation with 1 Gy X rays. Our study demonstrated that rather than a loss of methylation at the elements, all strains exhibited an early increase in L1 methylation one day after irradiation. In the most radiosensitive strain (BALB/c) the increase was also detected at 6 days postirradiation. The radioresistant C57Bl/6 strain exhibited a loss of L1 methylation at 14 days postirradiation. Less extensive changes to the B1 and IAP elements were detected at various time points, and pyrosequencing revealed that the responses of the strains were influenced by sex, with the male BALB/c and CBA mice exhibiting a greater response to the irradiation. The results of our study do not support the hypothesis that the most radiosensitive strains exhibit the greatest loss of repeat element DNA methylation after exposure to high-dose radiation. While the exact mechanism and biological outcome of the changes in DNA methylation observed here are still to be elucidated, this study provides the first evidence that radiation exposure elicits time-dependent changes in the methylation of repeat elements that are influenced by the genetic background, gender and the type of repeat element investigated. Furthermore, it suggest that any induced changes may not be persistent.

BackgroundDistinct subpopulations of neoplastic cells within tumors, including hepatocellular carcinoma (HCC), display pronounced ability to initiate new tumors and induce metastasis. Recent evidence suggests that signals from transforming growth factor beta (TGF-β) may increase the survival of these so called tumor initiating cells leading to poor HCC prognosis. However, how TGF-β establishes and modifies the key features of these cell subpopulations is not fully understood.ResultsIn the present report we describe the differential DNA methylome of CD133-negative and CD133-expressing liver cancer cells. Next, we show that TGF-β is able to increase the proportion of CD133+ cells in liver cancer cell lines in a way that is stable and persistent across cell division. This process is associated with stable genome-wide changes in DNA methylation that persist through cell division. Differential methylation in response to TGF-β is under-represented at promoter CpG islands and enriched at gene bodies, including a locus in the body of the de novo DNA methyl-transferase DNMT3B gene. Moreover, phenotypic changes induced by TGF-β, including the induction of CD133, are impaired by siRNA silencing of de novo DNA methyl-transferases.ConclusionsOur study reveals a self-perpetuating crosstalk between TGF-β signaling and the DNA methylation machinery, which can be relevant in the establishment of cellular phenotypes. This is the first indication of the ability of TGF-β to induce genome-wide changes in DNA methylation, resulting in a stable change in the proportion of liver cancer cell subpopulations.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-435) contains supplementary material, which is available to authorized users.

An expanding range of genetic syndromes are characterized by genome-wide disruptions in DNA methylation profiles referred to as episignatures. Episignatures are distinct, highly sensitive, and specific biomarkers that have recently been applied in clinical diagnosis of genetic syndromes. Episignatures are contained within the broader disorder-specific genome-wide DNA methylation changes, which can share significant overlap amongdifferent conditions. In this study, we performed functional genomic assessment and comparison of disorder-specific and overlapping genome-wide DNA methylation changes related to 65 genetic syndromes with previously described episignatures. We demonstrate evidence of disorder-specific and recurring genome-wide differentially methylated probes (DMPs) and regions (DMRs). The overall distribution of DMPs and DMRs across the majority of the neurodevelopmental genetic syndromes analyzed showed substantial enrichment in gene promoters and CpG islands, and under-representation of the more variable intergenic regions. Analysis showed significant enrichment of the DMPs and DMRs in gene pathways and processes related to neurodevelopment, including neurogenesis, synaptic signaling and synaptic transmission. This study expands beyond the diagnostic utility of DNA methylation episignatures by demonstrating correlation between the function of the mutated genes and the consequent genomic DNA methylation profiles as a key functional element in the molecular etiology of genetic neurodevelopmental disorders.

Renal involvement in lupus is characterized by unique DNA methylation changes in naïve CD4+ T cells

Energy-dense diets that are high in fat are associated with a risk of metabolic diseases. The underlying molecular mechanisms could involve epigenetics, as recent data show altered DNA methylation of putative type 2 diabetes candidate genes in response to high-fat diets. We examined the effect of a short-term high-fat overfeeding (HFO) diet on genome-wide DNA methylation patterns in human skeletal muscle. Skeletal muscle biopsies were obtained from 21 healthy young men after ingestion of a short-term HFO diet and a control diet, in a randomised crossover setting. DNA methylation was measured in 27,578 CpG sites/14,475 genes using Illumina's Infinium Bead Array. Candidate gene expression was determined by quantitative real-time PCR. HFO introduced widespread DNA methylation changes affecting 6,508 genes (45%), with a maximum methylation change of 13.0 percentage points. The HFO-induced methylation changes were only partly and non-significantly reversed after 6-8weeks. Alterations in DNA methylation levels primarily affected genes involved in inflammation, the reproductive system and cancer. Few gene expression changes were observed and these had poor correlation to DNA methylation. The genome-wide DNA methylation changes induced by the short-term HFO diet could have implications for our understanding of transient epigenetic regulation in humans and its contribution to the development of metabolic diseases. The slow reversibility suggests a methylation build-up with HFO, which over time may influence gene expression levels.