Search Results (106)

There is accumulating evidence that distinct mitochondrial DNA (mtDNA) methylation and hydroxymethylation patterns exist in Parkinson’s disease (PD). However, most studies have been limited to the investigation of specific target regions, rather than the entire mtDNA, and have been further hindered by other methodological discrepancies and the lack of non-CpG context investigation. Here, we provide a comprehensive profile of methylation and hydroxymethylation levels across the mitochondrial genome, at global and single-base resolution, in CpG and non-CpG (CHG, CHH) contexts in blood samples from early-onset PD (EOPD) patients (n = 39) and age- and sex-matched controls (n = 63). Bisulfite (BS) and oxidative-bisulfite (oxBS) conversions in parallel workflows followed by next-generation sequencing (NGS) using Illumina’s Novaseq 6000 sequencing system identified mitochondrial 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) in all contexts. Global mtDNA methylation was significantly higher in EOPD patients vs. matched controls in the CpG context (p = 5.63 × 10⁻³) in the BS status, and in all contexts [CpG (p = 2.67 × 10⁻⁴), CHG (p = 0.015), CHH (p = 0.012)] in the oxBS status, i.e., “true methylation”. At single-base resolution, the most statistically significant sites across the mitogenome, in the D-loop region, and CpG context, were primarily hypomethylated in EOPD patients compared to matched controls. Upon further validation, both global and base resolution mtDNA (hydroxy)methylation results could act as blood-based biomarkers for EOPD. Full article

G-quadruplex (G4) DNA structures have recently emerged as promising chiral scaffolds for enantioselective catalysis. This study investigates how thymidine loop modifications influence the catalytic performance of the telomeric G4 sequence HT21 in the asymmetric sulfoxidation of thioanisole. To this end, several singly or doubly modified HT21 derivatives were synthesized by using β-L-2′-deoxythymidine, 5-hydroxymethyl-2′-deoxyuridine, and 5-bromo-2′-deoxyuridine instead of a T residue, or β-L-2′-deoxyadonesine instead of an A residue, in specific positions within the TTA loops. The catalytic activity of these analogues was evaluated in the Cu(II)-mediated oxidation of thioanisole using hydrogen peroxide as oxidant. All modified sequences maintained complete substrate conversion, but their enantioselectivities varied markedly. Whereas the highest enantiomeric excess (84% ee) had previously been achieved with the HT21 analogue bearing a β-L-2′-deoxyadenosine in the first loop, the thymidine-based modifications, either alone or in combination, resulted in lower ee values, suggesting that loop alterations critically affect the chiral microenvironment, not all loop positions are functionally equivalent, and single substitutions within the same loop can result in different enantioselectivities. These findings highlight new insights on how individual loop residues contribute to asymmetric induction and offer further details for tuning G4-based catalytic scaffolds. Full article

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) and fibromyalgia (FM) are debilitating disorders with overlapping symptoms such as chronic pain and fatigue. Dysregulation of the endogenous opioid system, particularly µ-opioid receptor function, may contribute to their pathophysiology. This study examined whether epigenetic modifications, specifically µ-opioid receptor 1 gene (OPRM1) promoter methylation, play a role in this dysfunction. Using a repeated-measures design, 28 ME/CFS/FM patients and 26 matched healthy controls visited the hospital twice within four days. Assessments included blood sampling for epigenetic analysis, a clinical questionnaire battery, and quantitative sensory testing (QST). Global DNA (hydroxy)methylation was quantified via liquid chromatography–tandem mass spectrometry, and targeted pyrosequencing was performed on promoter regions of OPRM1, COMT, and BDNF. ME/CFS/FM patients reported significantly worse symptom outcomes. No differences in global (hydroxy)methylation were found. Patients showed significantly higher OPRM1 promoter methylation, which remained after adjusting for symptom severity and QST findings. Across timepoints, OPRM1 methylation consistently correlated with BDNF Promoter I and Exon III methylation. This is, to the best of our knowledge, the first study examining OPRM1 methylation in ME/CFS/FM. Increased OPRM1 methylation in patients, independent of symptoms or pain sensitivity measures, supports the hypothesis of dysregulated opioidergic signaling in these conditions. Full article

Curcuminoids demonstrate diverse pharmacological activity as antioxidant, neuroprotective, antitumor, and anti-inflammatory drugs. Dimethoxycurcumin (DMC) is a metabolically stable analog of curcumin, and both drugs modify the activity of several epigenetic enzymes that affect DNA methylation and histone modifications. 5-hydroxymethylcytosine (5hmC) is an epigenetic mark involved in active demethylation and in gene expression regulation. The effect of curcuminoids on the activity and expression of TET enzymes involved in 5hmC oxidation and active demethylation in leukemia cells is unclear. In this study, we investigated the impact of curcumin and DMC on the activity and expression of the three isoforms of TET enzymes. We also studied their effect on global 5hmC and performed a genome-wide analysis of 5hmC distribution at the single CpG level using oxidative bisulfite sequencing, which can differentiate between 5hmC and 5-methylcytosine. Both curcumin and DMC increased the activity and the mRNA expression of the three isoforms of TET. Concordantly, they also increased the global 5hmC level in leukemia cells. Single CpG analysis showed that both drugs induced a 5hmC increase and active demethylation at gene promoters, CpG islands and shores, exons, introns, and intergenic regions. Curcumin induced a promoter 5hmC increase in 194 genes and promoter-active demethylation in 154 genes. On the other hand, DMC induced a promoter 5hmC increase in 173 genes and promoter-active demethylation in 171 genes. Our study identifies curcuminoids as active demethylators through the activation of TET enzymes and provides a rationale for testing their combination with DNA hypomethylating agents in leukemia animal models. Full article

Maternal exposure to polycyclic aromatic hydrocarbons (PAHs) during pregnancy may have effects on the offspring epigenome. And the change in onset epigenome may be associated with children’s neurodevelopment. The current study investigated the relationship between 5-hydroxymethylcytosine (5-hmC) levels in cord blood and PAH metabolites in maternal urine at delivery and children’s neurodevelopment at birth and at age 2. We enrolled 400 pregnant women and their newborns and collected their biological samples after obtaining written informed consent. Enzyme linked immunosorbent assay kits and Chromatin immunoprecipitation kits were used to assess the DNA hydroxymethylation level in cord blood. We observed that 1-hydroxypyrene (1-OHPyr) was inversely associated with gesell developmental scale scores, positively associated with global DNA 5-hmC levels, and associated with decreased 5-hmC levels of the brain-derived neurotrophic factor (BDNF) and methyl CpG binding protein 2 (MeCP2) gene promoter. In addition, the 5-hmC levels of the BDNF and MeCP2 gene promoters were associated with motor scores. The global DNA 5-hmC was inversely associated with motor scores. Mediation analysis showed mediation effects between 1-OHPyr and motor scores by 5-hmC. The global DNA 5-hmC and MeCP2 and BDNF gene promoter 5-hmC contributed 28.51%, 27.29%, and 18.98% of the effect on motor scores changes related to 1-OHPyr. The study results suggested that 5-hmC can be a potential mechanism between prenatal PAH exposure and children’s neurodevelopment at age 2 and provide a better understanding of the role of hydroxymethylation in neurodevelopment. Full article

The incorporation of nucleoside analogs into DNA by polymerases, followed by their removal through base excision repair (BER), represents a promising strategy for cancer chemotherapy. In this study, we investigated the incorporation and cytotoxic effects of several nucleoside analogs—some of which are epigenetic reprogramming intermediates—in the U87 glioblastoma cell line. We found that two analogs, 5-hydroxymethyl-2′-deoxyuridine (5HmdU) and trifluorothymidine (TFT), are both cytotoxic and are efficiently incorporated into genomic DNA. In contrast, the 5-carboxy analogs—5-carboxy-2′-deoxyuridine (5CadU) and 5-carboxycytidine (5CadC)—showed no cytotoxicity and were not incorporated into DNA. Interestingly, 5-hydroxymethyl-2′-deoxycytidine (5HmdC) was cytotoxic but was not directly incorporated into DNA. Instead, it was deaminated into 5HmdU, which was then incorporated and likely responsible for the observed toxicity. 5HmdU is actively removed from DNA through the BER pathways. In contrast, TFT remains stably incorporated and is neither excised by BER nor does it hydrolyze into 5CadU—a known substrate for the DNA glycosylase SMUG1. We also found that N⁶-benzyladenosine (BzAdo), an inhibitor of the enzyme 2′-deoxynucleoside 5′-phosphate N-hydrolase (DNPH1), enhances the cytotoxicity of 5HmdU. However, the thymidine phosphorylase inhibitor tipiracil hydrochloride (TPI) does not increase the cytotoxic effect of TFT in U87 cells. Together, these findings highlight 5HmdU and TFT as promising chemotherapeutic agents for glioblastoma, each with distinct mechanisms of action and cellular processing. Full article

Chronic inflammation plays a central role in the pathogenesis of lung diseases including asthma, long COVID, chronic obstructive pulmonary disease (COPD), and lung cancer. Lipopolysaccharide (LPS) is a potent inflammatory agent produced by Gram-negative bacteria and also found in cigarette smoke. Our earlier study revealed that the intranasal exposure of A/J mice to LPS for 7 days altered gene expression levels in alveolar Type II epithelial cells (AECIIs), which serve as precursors to lung adenocarcinoma and are also preferentially targeted by SARS-CoV-2. In the present work, we employed a comprehensive multi-omics approach to characterize changes in DNA methylation/hydroxymethylation, gene expression, and global protein abundances in the AECIIs of A/J mice following the sub-chronic exposure to LPS and after a 4-week recovery period. Exposure to LPS led to hypermethylation at regulatory elements within the genome such as enhancer regions and expression changes in genes known to play a role in lung cancer tumorigenesis. Changes in protein abundance were consistent with an inflammatory phenotype and also included tumor suppressor proteins. Integration of the multi-omics data resulted in a model where LPS-driven inflammation in AECIIs triggers epigenetic changes that, along with genetic mutations, may contribute to lung cancer development. Full article

Microplastics (MPs), ubiquitous environmental pollutants, pose substantial threats to aquatic ecosystems and organisms, including the model species Daphnia magna. This study examined the effects of polyethylene (PE) and polystyrene (PS) MPs on D. magna, focusing on their ingestion, epigenetic alterations, and transcriptional responses. Exposure experiments revealed a concentration-dependent accumulation of MPs, with PS particles showing higher ingestion rates due to their higher density and propensity for aggregation. Epigenetic analyses demonstrated that exposure to PE MPs significantly reduced the global DNA methylation (5-mC) of Daphnia magna, suggesting hypomethylation as a potential stress response. Conversely, the DNA hydroxymethylation (5-hmC) of Daphnia magna displayed variability under PS exposure. Transcriptional analysis identified a marked downregulation of Vitellogenin 1 (v1) and upregulation of Ecdysone Receptor B (ecr-b), highlighting the occurrence of stress-related and adaptive molecular responses. These findings enhance our understanding of the molecular and epigenetic effects of MPs on aquatic organisms, offering critical insights for the development of effective environmental management and conservation strategies in the face of escalating MP pollution. Full article

Advancements in genome sequencing technologies have uncovered the multifaceted roles of long non-coding RNAs (lncRNAs) in human cells. Recent discoveries have identified lncRNAs as major players in gene regulatory pathways, highlighting their pivotal role in human cell growth and development. Their dysregulation is implicated in the onset of genetic disorders and age-related diseases, including cancer. Specifically, they have been found to orchestrate molecular mechanisms impacting epigenetics, including DNA methylation and hydroxymethylation, histone modifications, and chromatin remodeling, thereby significantly influencing gene expression. This review provides an overview of the current knowledge on lncRNA-mediated epigenetic regulation of gene expression, emphasizing the biomedical implications of lncRNAs in the development of different types of cancers and genetic diseases. Full article

Background/Objectives: Children and teenagers display a distinct metabolic dysfunction-associated steatohepatitis (MASH) phenotype, yet studies of childhood MASH are scarce and validated animal models lacking, limiting the development of treatments. Poor vitamin C (VitC) status may affect MASH progression and often co-occurs with high-fat diets and related metabolic imbalances. As a regulator of DNA methylation, poor VitC status may further contribute to MASH by regulating gene expression This study investigated guinea pigs—a species that, like humans, depends on vitC in the diet—as a model of pediatric MASH, examining the effects of poor VitC status on MASH hallmarks and global DNA methylation levels. Methods: Sixty-two juvenile guinea pigs were exposed to a high-fat diet for 16 weeks. Results: Juvenile guinea pigs exhibited hepatic histopathology representative of pediatric MASH, confirmed by portal inflammation and fibrosis. Consistent with pediatric MASH, juvenile guinea pigs displayed increased lobular and portal inflammation (p < 0.05 and p < 0.0001, respectively) but less steatosis (p < 0.001) compared to adults. Compared to the controls, the guinea pigs deprived in VitC showed lower body weight (p < 0.01), higher expression of hepatic inflammatory genes (p < 0.05), and a lower global hydroxymethylcytosine to methylcytosine ratio in the high-fat groups (p < 0.05). Conclusions: Our study validates guinea pigs as a model of pediatric MASH and suggests that VitC contributes to an altered gene expression signature through the regulation of DNA hydroxymethylation. We postulate that nutritional co-deficiencies in MASH, such as low VitC, may accelerate disease progression and deserve further attention. Full article

DNA methylation and demethylation are key epigenetic events that regulate gene expression and cell fate. DNA demethylation via oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) is typically mediated by TET (ten-eleven translocation) enzymes. The 5hmC modification is considered an intermediate state of DNA demethylation; it is particularly prevalent in the brain and is believed to play a role in the development of many cell types in the brain. Our previous studies have identified that vitamin C (Vc) and MEK inhibitor PD0325901 could significantly promote OPC (oligodendrocyte progenitor cell)-to-OL (oligodendrocyte) differentiation. Here we discovered that Vc and PD0325901 may promote OPC-to-OL differentiation by inducing DNA demethylation via hydroxymethylation. Blocking 5hmC formation almost totally blocked Vc- and PD0325901-stimulated OPC-to-OL differentiation. In addition, TET1 is not involved in Vc,- and PD0325901-promoted OL generation. We also found a synergistic effect between the two compounds in inducing OL generation, suggesting the possibility of a combination therapy for demyelination diseases in the future. Full article

Reproductive success is dependent on gamete integrity, and oxidative stress alters male nuclei, meaning that no DNA repair is possible due to chromatin compaction. The composition of sperm makes it highly sensitive to reactive oxygen species (ROS) but, at the same time, ROS are needed for sperm physiology. Over the past 30 years, much attention has been paid to the consequences of oxidative stress on sperm properties and the protective effects of antioxidant formulations to help fertility. Spermatozoa also carry epigenetic marks, critical for embryo development and the health of offspring. As DNA oxidative damage may disturb the sperm epigenome, we used an established mouse model of post-testicular sperm DNA oxidation to investigate sperm DNA methylation and hydroxymethylation. We also analyzed the potential corrective effect of oral antioxidant supplementation, proven to reduce sperm DNA oxidative damage, on sperm DNA methyl/hydroxymethyl marks. We show that sperm DNA oxidation is associated with a significant increase in overall hydroxymethylation. Oral antioxidant supplementation led to unexpected mild epigenetic changes. Antioxidant supplementation should not be proposed without proper clinical evaluation as it may alter sperm epigenetic marks, leading to a risk of paternally inherited epigenetic alterations. Full article

The aim of the present study was to investigate the concentration- and size-dependent effects of non-functionalized polystyrene nanoparticles (PS-NPs) of varying diameters (29 nm, 44 nm, and 72 nm) on specific epigenetic modifications and gene expression profiles related to carcinogenesis in human peripheral blood mononuclear cells (PBMCs) in vitro. This in vitro human-cell-based model is used to investigate the epigenetic effect of various environmental xenobiotics. PBMCs were exposed to PS-NPs at concentrations ranging from 0.001 to 100 µg/mL for 24 h period. The analysis encompassed epigenetic DNA modifications, including levels of 5-methyl-2′-deoxycytidine (5-mdC) and 5-(hydroxymethyl)-2′-deoxycytidine (5-hmdC), as well as the levels of 2′-deoxyuridine (dU) and 5-(hydroxymethyl)-2′-deoxyuridine (5-hmdU) by mass spectrometry methods, methylation in the promoter regions of selected tumor suppressor genes TP53 (P53), CDKN2A (P16), and CDKN1A (P21) and proto-oncogenes (CCND1, BCL2, BCL6), along with the expression profile of the indicated genes by real-time PCR assays. The results obtained revealed no significant changes in global DNA methylation/demethylation levels in PBMCs after short-term exposure to non-functionalized PS-NPs. Furthermore, there were no changes observed in the level of dU, a product of cytosine deamination. However, the level of 5-hmdU, a product of both 5-hmdC deamination and thymine oxidation, was increased at the highest concentrations of larger PS-NPs (72 nm). None of the PS-NPs caused a change in the methylation pattern of the promoter regions of the TP53, CDKN2A, CDKN1A, CCND1, BCL2 and BCL6 genes. However, gene profiling indicated that PS-NPs with a diameter of 29 nm and 44 nm altered the expression of the TP53 gene. The smallest PS-NPs with a diameter of 29 nm increased the expression of the TP53 gene at a concentration of 10 µg/mL, while PS-NPs with a diameter of 44 nm did so at a concentration of 100 µg/mL. An increase in the expression of the CDKN2A gene was also observed when PBMCs were exposed to PS-NPs with 29 nm in diameter at the highest concentration. The observed effect depended on both the concentration and the size of the PS-NPs. Full article

The chemical modifications of DNA are of pivotal importance in the epigenetic regulation of cellular processes. Although the function of 5-methylcytosine (5mC) has been extensively investigated, the significance of 5-hydroxymethylcytosine (5hmC) has only recently been acknowledged. Conventional methods for the detection of DNA methylation frequently lack the capacity to distinguish between 5mC and 5hmC, resulting in the combined reporting of both. The growing importance of 5hmC has prompted the development of a multitude of methods for the qualitative and quantitative analysis of 5hmC in recent years, thereby facilitating researchers’ understanding of the mechanisms underlying the onset and progression of numerous diseases. This review covers both established and novel methods for the detection of cytosine modifications, including 5mC, 5hmC, 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), with a particular focus on those that allow for accurate mapping and detection, particularly with third-generation sequencing. The review aims to help researchers choose the most appropriate methods based on their specific research goals and budget. Full article

In the era of precision oncology, identifying abnormal genetic and epigenetic alterations has transformed the way cancer is diagnosed, managed, and treated. 5-hydroxymethylcytosine (5hmC) is an emerging epigenetic modification formed through the oxidation of 5-methylcytosine (5mC) by ten-eleven translocase (TET) enzymes. DNA hydroxymethylation exhibits tissue- and cancer-specific patterns and is essential in DNA demethylation and gene regulation. Recent advancements in 5hmC detection methods and the discovery of 5hmC in cell-free DNA (cfDNA) have highlighted the potential for cell-free 5hmC as a cancer biomarker. This review explores the current and emerging techniques and applications of DNA hydroxymethylation in cancer, particularly in the context of cfDNA. Full article