Search Results (80)

DNA methylation (5-mdC) and its oxidative derivatives reflect interactions between environmental exposures and genome regulation, yet their short-term responsiveness to occupational factors remains insufficiently characterized. This exploratory pilot study evaluated whether a standard workweek is associated with measurable changes in urinary epigenetic and oxidative DNA modification profiles across distinct occupational settings. A cohort representing three exposure scenarios—cyanoacrylate-based cosmetic services, toluene diisocyanate (TDI) exposure, and polyurethane (PUR) foam processing—was examined using a paired pre-/post-exposure design. Urinary levels of 5-methyl-2′-deoxycytidine (5-mdC), 5-hydroxymethylcytosine (5-hmCyt), 5-hydroxymethyl-2′-deoxycytidine (5-hmdC), 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG), 5-hydroxymethyl-2′-deoxyuridine (5-hmdU), and 5-hydroxymethyluracil (5-hmUra) were quantified, and an epigenetic turnover ratio (5-hmdC/5-mdC) was derived. Although none of the observed shifts achieved formal statistical significance after False Discovery Rate (FDR) adjustment (p_FDR > 0.05), exposure-specific, directionally consistent non-significant trends were noted across cohorts. The isocyanate-exposed group demonstrated coordinated reductions in 5-mdC and 5-hmdC accompanied by an increase in 5-hmdU. The PUR-processing group exhibited the most pronounced response, including a marked elevation in 5-hmdU and an increased turnover ratio. In contrast, the beauty sector showed minimal median shifts but substantial inter-individual variability. These preliminary findings suggest that urinary epigenetic and oxidative DNA markers might reflect short-term, exposure-related biological trends, warranting further evaluation. Full article

Background: Long-read, single-CpG-resolution sequencing is redefining the information-to-depth ratio in epigenomics. While conventional methylome analysis often requires high coverage, we propose a scalable pipeline designed to extract high-density regulatory logic from shallow sequencing data. Methods: By utilizing the progenitor-like HepaRG cell line as a model for liver plasticity, we validated this framework across two divergent developmental trajectories: hepatic maturation and sphere-induced retrodifferentiation. Our technical approach combines CpG-centric enrichment and regional methylation aggregation to reconstruct regulatory landscapes from sparse data. Using long-read Nanopore sequencing, we mapped the dynamics of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). Results: Our pipeline revealed that these trajectories are not inverse processes but engage distinct epigenetic strategies. Hepatic maturation is characterized by the accumulation of 5hmC that partially targets repressive heterochromatin (H3K9me3, H4K20me3) and pioneer factors such as FOXA2. In contrast, retrodifferentiation increases 5mC, potentially silencing adult regulators such as HNF1A via Polycomb-associated networks. In addition, aggregation-based analysis can distinguish widespread focal perturbations from a restricted subset of transcription factors that translate epigenetic changes into regional accessibility. Conclusions: This study provides a scalable computational framework for investigating cellular fate transitions, proving that high-value epigenetic insights are attainable even at reduced sequencing depths. Full article

Small-cell lung cancer (SCLC) is an aggressive neuroendocrine carcinoma characterized by poor survival. Despite a high tumor mutation burden, biomarker discovery in SCLC remains challenging due to rapid tumor plasticity and limited tissue availability, highlighting the promise of liquid biopsy-based approaches. Epigenetic dysregulation of DNA 5-hydroxymethylcytosine (5hmC) has emerged as a cancer hallmark. However, its role in SCLC remains largely unexplored. Here, we characterized the cell-free DNA (cfDNA) 5hmC landscape in SCLC and evaluated its potential applications. We profiled the cell-free hydroxymethylomes of 107 pre-treatment SCLC patients and 53 matched controls using the 5hmC selective chemical labeling (5hmC-Seal) assay. SCLC displayed higher global 5hmC levels and distinct enrichment at neurodevelopmental and synaptic pathways, consistent with the neuroendocrine identity of SCLC. Concordance between plasma and matched circulating tumor cell patient-derived xenograft (CDX) demonstrated that cfDNA 5hmC reflects tumor epigenetic states and correlates with transcriptomic-derived molecular subtypes. Elevated SCLC-specific 5hmC levels and extensive stage (ES) disease were associated with inferior survival, with ES disease showing enrichment of pathways linked to cellular plasticity and neurodevelopment. Together, these findings indicate that cfDNA 5hmC profiling has potential as a biologically informative and clinically relevant biomarker in SCLC, with possible applications in tumor subtyping and risk stratification. Full article

Cervical cancer (CC) is an alarming global health problem, with predominantly higher incidence, lethal progression, and mortality among women of African ancestry (AA) than women of European ancestry (EA). Although persistent high-risk human papillomavirus (HPV) integration and infection are the key etiological factors, currently available evidence implicates epigenetic reprogramming as a prime contributor to ancestry-associated differences in CC pathogenesis. To address these disparities, we performed genome-wide DNA methylation profiling of HPV-positive cervical intraepithelial neoplasia (CIN) lesions from AA (n = 15) and EA (n = 15) women. Differential methylation analysis identified a distinct epigenomic landscape in AA-CIN lesions, with widespread hypermethylation and hypomethylation at promoter-associated and regulatory CpG sites. Pathway enrichment analyses highlighted dysregulation of ECM-receptor interaction, focal adhesion, PI3K-Akt, MAPK, Ras, Rap1, and RUNX-dependent transcriptional networks. Comparative analysis across CIN grades (CIN1–CIN3) revealed progressive epigenetic reprogramming affecting cell cycles, cytoskeletal dynamics, signaling, and metabolic pathways. Among hypermethylated tumor suppressor genes, SH3GL2 and ARHGAP25 showed significantly higher methylation in AA lesions, accompanied by concomitant loss of their protein expression. MBD1, a methylation-binding regulator, was upregulated in AA-CIN lesions, coinciding with global loss of 5-hydroxymethylcytosine (5hmC), suggesting enhanced transcriptional repression. In contrast, EA lesions retained protein expression and 5hmC levels. Collectively, these findings indicate that early, ancestry-specific epigenetic modifications target tumor suppressor pathways and converge on oncogenic signaling, cytoskeletal remodeling, and cell–cell adhesion. Our study provides mechanistic insight into CC health disparities, identifying SH3GL2 and ARHGAP25 hypermethylation as potential biomarkers, and highlighting epigenetic regulation as a contributor to disparate CC progression in AA women. Full article

DNA polymerase beta (Polβ) is a 39 kDa, single polypeptide enzyme that possesses both gap tailoring and nucleotidyl transferase activity and is the key polymerase involved in base excision repair (BER) and the final steps of active gene demethylation. We demonstrated that residues in the mouse Polβ protein, L301 and V303, are critical for Polβ’s interaction with the BER scaffolding protein X-ray repair cross-complementing 1 (XRCC1), and mutation of these residues impairs Polβ’s ability to bind to XRCC1, negatively impacting BER complex assembly. We developed Polβ^{L301R-V303R/L301R-V303R} knock-in mice to explore how defects with this essential protein complex impact genome stability in the mouse. We found these mice to be viable and fertile yet exhibited a modest reduction in body weight. Here, we examined the protein and mRNA levels in tissues from wild-type (WT), heterozygous (HET), and homozygous (HOM) Polβ^{L301R-V303R/L301R-V303R} mice and the derived fibroblast cell lines. We show that HOM mice have significantly diminished Polβ protein levels, as compared to WT mice, in several tissues, yet Polβ mRNA levels were not significantly different, suggesting the decreased levels of Polβ protein could not be attributed to lower gene expression. Upon examination of Polβ stability in mouse ear fibroblasts derived from WT and HOM mice, results are consistent with human cell studies that the Polβ^L301R-V303R protein is unstable and undergoes proteasome-mediated degradation. Finally, we evaluated WT, and HOM, liver and brain genomic DNA samples for 5-methylcytosine/5-hydroxymethylcytosine (5mC/5hmC) levels by nanopore sequencing to investigate the impact of suppressed Polβ protein levels on active gene demethylation. As expected, we found tissue-specific trends in methylation, when comparing the brain and liver. However, we were unable to discern substantial differences in methylation levels between WT and HOM mice, suggesting that in the absence of external stressors, low Polβ levels do not impact methylation patterns. Full article

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

Since 2022, global outbreaks of monkeypox virus (MPXV) have been repeatedly designated by the World Health Organization (WHO) as a public health emergency of international concern (PHEIC), underscoring the urgent need to elucidate the multidimensional mechanisms underlying viral evolution and transmission. Current understanding remains largely focused on genomic variation, while the critical role of epigenetic regulation has been considerably overlooked. To address this gap, this study integrates high-throughput evolutionary genomic analysis with whole-genome DNA methylation profiling. Using parallel Illumina and Nanopore sequencing platforms, we comprehensively characterized two clinically derived MPXV isolates collected locally. The results revealed that both isolates belonged to the C.1.1 ancestral lineage, diverging into distinct clades (E.3 and E.4, respectively, supporting the presence of at least two independent viral introduction events into the region, each followed by limited local transmission. They had accrued a considerable number of single-nucleotide polymorphisms (SNPs), with APOBEC3-associated substitutions constituting 84.8% and 77.6% of all observed mutations. Furthermore, both 5-hydroxymethylcytosine (5hmC) and N6-methyladenine (6mA) modifications were identified and found to be preferentially enriched within the inverted terminal repeats (ITRs) regions of MPXV genome in both viral strains; moreover, the E.4 lineage viral strain exhibits a markedly more intricate and compositionally diversified modification landscape, a pattern that indicates appreciable epigenetic heterogeneity among MPXV lineages. Our study furnishes a multi-omics framework that presents a systematic evolutionary feature of two clinical MPXV isolates and their genomic DNA 5hmC and 6mA modification topologies, and enhances our understanding of MPXV viral adaptation and diversification. 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

The red seaweed Jania rubens (J. rubens) is prevalent along the Lebanese coast and has drawn attention for its notable antineoplastic properties. Our previous data showed that its dichloromethane–methanol (DM) extract possesses antioxidant, cytotoxic, and anti-migratory effects on colon cancer cells. In the present study, a GC-MS analysis of DM extract identified a diverse profile of bioactive compounds, including flavonoids and pyrazole derivatives with antioxidant and anticancer activities. In vitro assays demonstrated that the DM extract exerts significant cytotoxic activity against various cancer cell lines, including colon, breast, and cervical types. Further investigation into the underlying molecular mechanisms revealed that the extract induces G2/M cell cycle arrest and reduces the expression of EMT (epithelial–mesenchymal transition) markers, N-cadherin and Twist. In addition, the extract showed anti-metastatic properties through its ability to decrease MMP-2 and MMP-9 activity. Mechanistically, DM caused a substantial reduction in Ten-Eleven Translocation (TET) enzymes TET-1, TET-2, and TET-3, which are essential DNA demethylation regulators, thus decreasing their enzymatic product 5-hydroxymethylcytosine (5-hmC). Interestingly, despite a significant increase in intracellular ROS (reactive oxygen species), suggesting a contribution to cytotoxicity, no substantial change in the biogenesis of promyelocytic leukemia nuclear bodies (PML-NBs) was detected. These findings demonstrate that J. rubens DM extract contains bioactive compounds with multiple anticancer effects, thus making it a promising candidate for developing new therapeutic agents. Full article

Background/Objectives: The DNA methylome allows environmental signals to be converted into stable and adaptive changes in gene expression. While 5-methylcytosine (5mC) has been extensively studied, alternative epigenetic marks such as N6-methyladenine (6mA) and 5-hydroxymethylcytosine (5hmC) remain poorly understood. Comparative studies of these marks are rare, and their results are often confounded by phylogeny, tissue type, developmental stage, or methodology. Here, we aimed to disentangle the constitutive, somatic- and germline-specific, and/or age-related patterns displayed by 6mA, 5mC, and 5hmC within a single species. Methods: We generated long-read nanopore sequencing data for somatic tissues of buff-tailed bumblebee (Bombus terrestris) males and their sperm, enabling simultaneous detection of 6mA, 5mC, and 5hmC. We used a stepwise approach to successively identify (i) constitutive patterns conserved between somatic tissues and sperm, (ii) differences between the soma and the germline, and (iii) age-related changes between young and old males. Results: We found distinct constitutive, somatic and sperm, and age-related specific signatures in the genomic contexts, maintenance fidelity, and biological functions associated with 6mA, 5mC, and 5hmC. Sperm cells consistently displayed lower methylation entropy than did somatic tissues, indicating more stable methylation patterns in the germline. 5mC exhibited the greatest variation across all genomic contexts; 6mA and 5hmC displayed less dramatic differences. The influence of age was subtler but revealed context-dependent remodeling of methylation, particularly for 5hmC. Conclusions: We observed that 6mA, 5mC, and 5hmC displayed constitutive, somatic- and sperm-specific, and age-related differences that were associated with distinct genomic contexts and biological functions, supporting the complementarity of these methylation marks and their diverging epigenetic roles. 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

Epigenetic regulation, particularly DNA methylation, plays a crucial role in embryonic development by controlling gene expression patterns. The disruption of this regulation by environmental factors can have long-lasting consequences. Opioid drugs, such as morphine, are known to cross the placental barrier and affect the developing central nervous system, yet their precise epigenetic effects during early development remain unclear. This study aimed to elucidate the impact of chronic morphine exposure on the DNA methylation landscape and gene expression in mouse embryonic stem cells (mESCs). mESCs were chronically exposed to morphine (10 μM for 24 h). Genome-wide bisulfite sequencing was performed to identify DNA methylation changes, while RNA sequencing (RNA-Seq) assessed corresponding gene expression alterations. Global levels of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) were quantified using mass spectrometry. Morphine exposure induced global DNA hypomethylation and identified 16,808 differentially methylated genes (DMGs) related to development, cell signalling, metabolism, and transcriptional regulation. Integrative transcriptomic analysis with RNA-Seq data revealed 651 overlapping genes, including alterations in key epigenetic regulators involved on DNA methylation machinery. Specifically, Tet1 was upregulated with promoter hypomethylation, while Dnmt1 was downregulated, without changes in promoter methylation after morphine exposiure. Mass spectrometry results confirmed a global decrease in 5mC levels alongside increased 5hmC, indicating the involvement of both passive and active demethylation pathways. These findings demonstrate for the first time that morphine disrupts the epigenetic homeostasis of mESCs by promoting global and gene-specific DNA demethylation, which might be key to the phenotypic changes that occur in adulthood. This work provides novel mechanistic insights into how opioid exposure during early development may lead to persistent epigenetic alterations, with potential long-term implications for neurodevelopment and disease susceptibility. Full article

Impact of long-term plasma storage on biomarker analysis is critical for ensuring data reliability. Cell-free DNA (cfDNA) epigenetic markers, including 5-hydroxymethylcytosine (5hmC), have emerged for disease detection, prognosis, and treatment response. However, the effects of prolonged storage on 5hmC analysis remain unclear. We evaluated the quantity and quality of cfDNA and 5hmC sequencing analyses in 1070 plasma samples stored for up to 14 years from patients with solid tumors and acute myeloid leukemia (AML) and non-cancer individuals. In long-term stored plasma samples, cfDNA yield remained largely stable; however, uniquely mapped reads (UMRs) from 5hmC sequencing were significantly reduced in solid tumor and control samples. Notably, prolonged plasma storage independently contributed to increased genomic DNA (gDNA) contamination in solid tumor and AML samples and significantly correlated with decreased UMRs in control samples. Across all groups, samples with gDNA contamination exhibited significantly reduced UMRs. Furthermore, gDNA contamination independently compromised cfDNA fragment integrity, decreased sequencing library success in solid tumors, and reduced 5hmC sequencing UMRs across all groups. Therefore, extended plasma storage contributes to increased gDNA contamination, compromising cfDNA and 5hmC sequencing quality. Implementing measures to minimize gDNA contamination in long-term plasma storage is crucial for improving downstream cfDNA analysis reliability. Full article

Background and Objectives: The accurate discrimination between patients with and without cancer using their cell-free DNA (cfDNA) is crucial for early cancer diagnosis. The end-motifs of cfDNA serve as significant cancer biomarkers, offering compelling prospects for cancer diagnosis. This study proposes EM-DeepSD, a signal decomposition deep learning framework based on cfDNA end-motifs, which is aimed at improving the accuracy of cancer diagnosis and adapting to different sequencing modalities. Materials and Methods: This study included 146 patients diagnosed with cancer and 122 non-cancer controls. EM-DeepSD comprises three core modules. Initially, it utilizes a signal decomposition module to decompose and reconstruct the input end-motif profiles, thereby generating multiple regular subsequences that optimize the subsequent modeling process. Subsequently, both a machine learning module and a deep learning module are employed to improve the accuracy of cancer diagnosis. Furthermore, this paper compares the performance of EM-DeepSD with that of existing benchmarked methods to demonstrate its superiority. Based on the EM-DeepSD framework, we developed the EM-DeepSSA model and compared it with two benchmarked methods across different cfDNA sequencing datasets. Results: In the internal validation set, EM-DeepSSA outperformed the two benchmark methods for cancer diagnosis (area under the curve (AUC), 0.920; adjusted p value < 0.05). Meanwhile, EM-DeepSSA also exhibited the best performance on two independent external testing sets that were subjected to 5-hydroxymethylcytosine sequencing (5hmCS) and broad-range cell-free DNA sequencing (BR-cfDNA-Seq), respectively (test set-1: AUC = 0.933; test set-2: AUC = 0.956; adjusted p value < 0.05). Conclusions: In summary, we present a new framework which can achieve high classification performance in cancer diagnosis and which is applicable to different sequencing modalities. Full article

Objectives: Triple-negative breast cancer (TNBC) is an aggressive cancer subtype with limited treatment options due to the absence of estrogen, progesterone receptors, and HER2 expression. This study examined the impact of DNA methylation and demethylation markers in tumor tissues on TNBC patients’ response to neoadjuvant chemotherapy (NACT) and analyzed the correlation between 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) and clinicopathological characteristics, offering new insights into the predictive value of these epigenetic markers. Methods: The study included 53 TNBC female patients, 19 of whom received neoadjuvant chemotherapy (NACT) before surgery. Global DNA methylation and demethylation levels were quantified using an ELISA-based method to measure 5-mC and 5-hmC content in DNA isolated from pre-treatment biopsy samples (in patients undergoing NACT) and postoperative tissues (in patients without NACT). Results: In patients who received NACT, those with disease progression had significantly higher pretreatment levels of 5-hmC (p = 0.028) and a trend toward higher 5-mC levels (p = 0.054) compared to those with pathological complete response, partial response, or stable disease. Higher 5-mC and 5-hmC levels were significantly associated with higher tumor grade (p = 0.039 and p = 0.017, respectively). Additionally, a positive correlation was observed between the Ki-67 proliferation marker and both 5-mC (r_S = 0.340, p = 0.049) and 5-hmC (r_S = 0.341, p = 0.048) levels in postoperative tissues. Conclusions: Our study highlights the potential of global DNA methylation and demethylation markers as predictors of tumor aggressiveness and chemotherapy response in TNBC. Further research in larger cohorts is necessary to validate these markers’ prognostic and predictive value. Full article