Differential Methylation Research Articles (Page 1)

Hepatocellular carcinomas (HCC) with multinodular morphology were typically polyclonal and exhibited extensive heterogeneity. We explored the multiple-layer heterogeneity of HCC utilizing single-cell multiomics sequencing together with multiregional sampling. We found that Confluent Multi-Nodular samples exhibit more heterogeneous immune landscapes characterized by increased transcriptome heterogeneity and more complex immune-related interactions compared to Single Nodular (SN) samples. We identified differential DNA methylation patterns among distinct tumor foci in a subset of liver cancer patients. Global DNA hypomethylation in cancer cells predominantly occurs in partially methylated domains. We predicted and validated two genes, GADD45A and SNHG6, as potential drivers of DNA hypomethylation. Lastly, we demonstrated that DNA methylation distance serves as a more robust metric than gene expression distance for reconstructing tumor evolutionary trajectories.

Exposure to maternal type 1 diabetes (T1D) during pregnancy provides relative protection against T1D in the offspring. This protective effect may be driven by epigenetic mechanisms. Here we conducted an epigenome-wide blood analysis on 790 young children with and 962 children without a T1D-affected mother, and identified differential DNA methylation (q < 0.05) at multiple loci and regions. These included the Homeobox A gene cluster and 15 T1D susceptibility genes. The differential methylation was found in transcriptionally relevant regions associated with immune function, including sites previously linked to T1D-related methylation loci and protein biomarkers. Propensity scores for methylation at T1D susceptibility loci could predict the development of islet autoimmunity in offspring born to mothers without T1D. Together, these findings highlight pathways through which maternal T1D may confer protection against islet autoimmunity in offspring and suggest that environmental factors can influence T1D risk through epigenetic modifications of T1D susceptibility loci.

Background/Objectives: The global increase in the consumption of ultra-processed foods (UPFs) parallels the rise in obesity and non-communicable chronic diseases. Although several large-scale studies associate UPF intake with adverse health outcomes, the biological mechanisms remain unclear. Epigenetic alterations, such as changes in DNA methylation, may represent a potential pathway by which diet influences metabolic health. The aim of this study was to investigate whether higher UPF consumption is associated with genome-wide DNA methylation patterns in women. Methods: This was a cross-sectional observational study with exploratory epigenetic analysis. We selected 30 women, who were divided into tertiles based on their UPF consumption (expressed as a percentage of total energy intake) according to the NOVA food classification system. Dietary intake was assessed using a three-day food record. Anthropometric data, body composition and laboratory parameters were evaluated. The analysis of DNA methylation was performed utilizing DNA extracted from peripheral blood leukocytes of participants in the first and third tertiles of UPF consumption. Genome-wide methylation patterns were performed using next-generation sequencing. Results: Participants had a median (IQR) age of 31 years (26.0–36.5) and a BMI of 24.7 (23.6–35.8) kg/m2. For the epigenetic analyses, 15 women were included. Of the 30 women initially evaluated, 20 were included as they belonged to the first and third tertile of UPF consumption. Of these, five were excluded due to a low number of reads obtained by NGS. A total of 80 differentially methylated regions were identified between groups, most of which were hypomethylated in the high-UPF-intake group. Conclusions: High UPF consumption was associated with altered DNA methylation patterns, suggesting a potential epigenetic mechanism underlying the negative health effects of UPFs. This pilot study provides a model for future research with larger samples.

Peyronie's disease (PD) is a fibrosing disorder with a hereditary predisposition that can have significant impacts on quality of life. Our pilot study's objective was to determine if disease penetrance is correlated with epigenetic variations in non-plaque tissue, as compared with tissue from men with erectile dysfunction (ED), as indicated by DNA methylation. Tunica albuginea samples were collected from non-plaque tissue during penile prosthesis placement for men with ED with and without PD, with ED-only samples serving as controls. DNA methylation analyses were performed on homogenized penile tissue samples via an Illumina Human MethylationEPIC BeadChip v2 array. Using the minfi package in R, beta values were produced for all 936 990 CpG sites for each sample and subset-quantile within array normalization (SWAN) normalization was applied. Differentially methylated regions (DMRs) were found via USeq with a threshold Wilcoxon FDR score of 40. The primary outcome of the study was the prevalence and significance of DMRs between samples and controls. A total of 10 ED + PD and 12 ED-only samples were successfully processed for subsequent epigenetic analyses. Thirty-six DMRs with 60 total region-gene associations and five implicated biological processes were identified, including anterior/posterior pattern specification, chordate embryonic development, embryo development (ending in birth or egg hatching), somitogenesis, and pattern specification process. Each of the implicated biological processes are essential components of the human body's developmental biology and the specific region-gene associations suggest pathogenesis may be an early embryonic development. The identification of these epigenetic changes in non-plaque tissue suggests a systemic process related to PD that is not isolated to plaque tissue and provides further insight into PD pathogenesis. The strength of this pilot study is that it evaluated methylation changes between ED + PD and ED-only individuals using new and updated array analyses. This pilot study is limited due to a cross-sectional design that only examines differential methylation in non-plaque tissues. Identification of epigenetic differences in non-plaque tissue in men with and without PD suggests systemic changes and provides further insight into the pathogenesis of PD.

Developmental programming: Differing impact of prenatal testosterone and prenatal bisphenol-A -treatment on hepatic methylome in female sheep.

Agricultural copper pesticide exposure and DNA methylation in Central Valley of California residents with and without Parkinson's disease.

Mapping DNA methylation signatures to identify epigenetic variation across subcortical regions of the human PTSD brain.

DNA methylation landscapes of HIV controllers: an epigenome-wide association study.

This study represents the first investigation of 5-methyl cytosine (5mC) DNA methylation patterns in sulfate-reducing bacterial (SRB) biofilms under copper (Cu) stress, utilizing Oxford Nanopore Technologies (ONT) sequencing. DNA methylation is a crucial epigenetic modification that is dynamic and regulates the signals to modulate molecular mechanisms across biological systems. The regulatory roles of DNA methylation in prokaryotic systems remain comparatively understudied than in eukaryotes. Bacteria are highly sensitive to environmental changes and therefore may utilize additional mechanisms like DNA methylation to combat the stresses. Our previous studies, utilizing microscopy and growth analyses, revealed that Oleidesulfovibrio alaskensis G20 (OA G20) biofilms responded to Cu stress. However, the DNA methylation patterns associated with this response remain unexplored, leaving a critical gap in our understanding of the epigenetic mechanisms regulating OA G20 biofilms under Cu stress. This study aims to address this knowledge gap by identifying 5mC DNA methylation in biofilms of OA G20 under Cu stress. To achieve our goal OA G20 biofilms cultivated under 30 µM-Cu ion stress along with control and sequenced through ONT sequencing. DNA methylation analysis was performed using the MicrobeMod pipeline identifying three methylated motifs: TCCG, CCCGCCCG, and CGGGAT in control (0 µM-Cu). TCCG was identified as the predominant methylated motif, with analysis revealing 78,022 genomic positions in the control condition. Of these, 61.7% exhibited 5mC modifications, 33.9% remained unmodified, and 4.4% showed uncharacterized modifications. In contrast, the 30 µM-Cu biofilm showed methylation in only two motifs, TCCG and GCANCTGCGS. Analysis of TCCG revealed 63,315 genomic positions, with 62.7% (39,706 sites) showing methylation and 33.2% (20,990 sites) remaining unmethylated. A total of 1418 common methylated positions were identified for both conditions and there were 341 and 424 genomic positions identified for motif TCCG above 75% methylation in the 0 µM and 30 µM-Cu biofilm samples, respectively. Differential methylation analysis revealed significant variations in methylation patterns across several key genes of crucial molecular pathways, important for biofilm formation, including ATP-Binding Cassettes (ABC) transporters, phosphohydrolase, flagellar biosynthesis, chemotaxis, cobalamin synthase, histidine kinase, and uncharacterized proteins.

Pediatric obesity is rising globally, and emerging evidence suggests that sleep timing may influence metabolic health through epigenetic mechanisms. This study investigated epigenome-wide DNA methylation patterns associated with bedtime in children and explored their biological relevance. Children aged 6–10 years were classified as early (≤8:30 PM) or late (&gt;8:30 PM) bedtime groups. Saliva-derived DNA was analyzed using the Illumina Infinium MethylationEPIC BeadChip Array, and the Sparse Wrapper Algorithm (SWAG) was applied to identify differentially methylated loci. A total of 1006 CpG sites, representing 571 unique genes, were significantly associated with bedtime (p &lt; 0.001). Significant methylation differences were observed between early and late bedtime groups, with ABCG2, ABHD4, MOBKL1A, AK3, SDE2, PRAMEF4, CREM, CDH4, BRAT1, and SDK1 showing the most consistent variation. Functional enrichment analyses (Gene Ontology, KEGG, and DisGeNET) conducted on the SWAG-identified gene set revealed enrichment in biological processes including peptidyl-lysin demethylation, regulation of sodium ion transport, DNA repair, and lipo-protein particle assembly. Key KEGG pathways included circadian entrainment, neurotransmission (GABAergic, dopaminergic, and glutamatergic), growth hormone synthesis, and insulin secretion. DisGeNET analysis identified associations with neurodevelopmental disorders and cognitive impairment. Cross-comparison with established sleep and obesity gene sets identified ten overlapping genes(CDH4, NR3C2, ACTG1, COG5, CAT, HDAC4, FTO, DOK7, OCLN, and ATXN1). These findings suggest that variations in bedtime during childhood may epigenetically modify genes regulating circadian rhythm, metabolism, neuronal connectivity, and stress response, potentially predisposing to later-life developmental, and metabolic challenges.

Low-dose aspirin (LDA) is an intervention recommended to prevent the development of hypertensive disorders of pregnancy (HDP) in high-risk pregnancies. Maternal conditions such as HDP have been associated with cord blood epigenetic changes including those related to cardiovascular processes; however, it is unclear whether maternal aspirin therapy may impact neonatal epigenetics in otherwise healthy high-risk pregnancy.This study aimed to determine if maternal LDA exposure in utero leads to altered DNA methylation in umbilical cord blood cells in term neonates compared with controls not exposed to aspirin, and to identify if these methylation changes alter key pathways in the development of chronic disease.Umbilical cord blood was collected from 10 neonates without LDA exposure and 13 neonates with LDA exposure in utero. Patients with hypertensive disorders of pregnancy, COVID-19, and chorioamnionitis were excluded. Genomic DNA was isolated from umbilical cord blood cells and genome-wide DNA methylation was performed using Illumina Methylation EPIC assay.A total of 155 differentially methylated loci (81 genes were hypermethylated and 74 were hypomethylated) were identified in LDA-exposed neonatal umbilical cord blood compared with the control group. Important canonical pathways identified by Ingenuity Pathway Analysis (IPA) were related to Th1 and Th2 signaling and classical (M1) macrophage activation. The genes affected by LDA exposure were associated with cardiac and renal systems.LDA exposure led to differential DNA methylation in umbilical cord blood. The differentially methylated genes were related to inflammatory pathways as well as cardiac and renal toxicity pathways. LDA exposure in utero may promote altered health programming in the neonate in areas impacting cardiovascular health. · Maternal aspirin exposure is associated with differential DNA methylation in cord blood.. · Cord blood epigenetic changes associated with maternal aspirin relate to anti-inflammatory pathways.. · Research on potential protective impact of maternal aspirin on neonatal epigenetics is warranted..

ViMIC 2.0 is an updated database that provides comprehensively curated data on virus mutations (VMs), viral integration sites (VISs), and multi-omics datasets related to human diseases. Leveraging expanding public data, ViMIC 2.0 significantly enhanced data scale, diversity, and analytical capabilities compared to the previous version. In terms of data volume, the number of virus types has increased from 8 to 28, VM entries have grown from 31 712 to 64 168, virus-related diseases expanded from 77 to 177, literature rose from 2539 to 6433, and omics datasets have substantially increased from 28 sets of single expression profile data to 255 sets of multi-omics data. In addition, ViMIC 2.0 has updated 9409 VISs, 173 048 sequences, newly incorporated sequencing types such as single-cell transcriptomic sequencing (scRNA-seq), and genome binding/occupancy profiling. Regarding the visualization module, ViMIC 2.0 now provides results of differential gene expression analysis for bulk RNA-seq or array, cell type annotation and gene feature plot for scRNA-seq data, and differential methylation analysis for methylation profiling, as well as peak annotation for ChIP-seq/ChIP-on-chip/ATAC-seq data. In summary, ViMIC 2.0 serves as a user-friendly, up-to-date, and well-maintained resource for the virology research community. ViMIC 2.0 is freely accessible at http://www.biomedinfo.cn/ViMIC2.0/index.php.

Investigating the phenotypic contribution of gene-spliced isoforms to tumor cellular heterogeneity can facilitate the development of innovative strategies for precision medicine. Serine protease 3 (PRSS3), a trypsin-like protease with four spliced variants (PRSS3-SVs: PRSS3-V1 to V4), plays diverse biological roles in cancer progression. Herein, we systematically analyzed the expression patterns and functional implications of PRSS3-SVs in gastric cancer (GC) using an integrative approach that combined bioinformatic analyses, CpG site-specific methylation detection, splice-specific qPCR, isoform-based methodologies, and multiple functional assays. Our findings revealed that differentially expressed PRSS3 isoforms, predominantly PRSS3-V1 and PRSS3-V2, are regulated by intragenic methylation and exert pleiotropic roles in GC. Overexpression of PRSS3 transcripts suppressed GC cell proliferation via the NF-κB signaling pathway, while exerting distinct effects on matrix metalloproteinase-associated cell migration and invasion. Clinically, patients with low PRSS3-V1 or high PRSS3-V2 expression exhibited poorer survival outcomes, and the expression difference between these two transcripts was identified as an independent prognostic indicator for GC patients. Epigenetically, differential methylation patterns within PRSS3 enabled stratification of GC patients into subgroups characterized by either high-methylation with low-expressed PRSS3-SVs or low-methylation with high-expressed PRSS3-SVs. The UHRF1/DNMT1 complex was found to mediate CpG site methylation and regulate PRSS3 transcripts, particularly silencing PRSS3-V1 through intragenic CpG methylation. This methylation pattern was associated with reduced survival rates and further validated its correlation with tumor metastasis in an independent cohort (n = 243). Our study elucidates that methylation-regulated alternative splicing contributes to phenotypic heterogeneity in GC, highlighting its potential advantage over differentially expressed genes in improving stratification strategies for precision oncology.

Allergic rhinitis (AR) is a common immune-mediated chronic inflammatory disease with a complex pathogenesis involving multiple responses of the immune system and epigenetic changes. In recent years, DNA methylation, a key epigenetic mechanism, has been shown to play an important role in the onset and development of AR. GuoMinKang (GMK) have been used in the treatment of AR through their multi-component properties. However, its specific epigenetic regulatory mechanisms have not been fully investigated. The aim of this study was to explore the epigenetic regulatory mechanisms of the traditional Chinese medicine compound GMK in the treatment of AR. By analysing DNA methylation and transcriptome data from AR patients, we identified differentially methylated regions (DMRs) and differentially expressed genes (DEGs) associated with AR. Through network pharmacology analysis, we screened the active components of GMK and their potential target genes, particularly those related to DNA methyltransferases (DNMTs). An AR mouse model was established to observe the behavioural and pathological changes of the nasal mucosa of mice after drug administration; the expression of IgE cytokines was detected by ELISA, and the expression of nasal mucosa genes was verified by qPCR. Total IgE (tIgE) levels were significantly reduced in AR patients after GMK treatment, suggesting a possible role in immunomodulation. Our analysis revealed that GMK was able to restore aberrant methylation patterns in AR patients by modulating specific DNA methylation regions. Through differential methylation analysis, we identified 10 genes whose methylation levels were significantly restored to normal after GMK treatment and which already showed significant differential expression in AR patients, particularly in immune regulation and epithelial cell function. These genes include LERP, NFIA, etc., suggesting that they may play a key role in the onset and development of AR. Further through target prediction and network pharmacological analyses, we confirmed that the active ingredients of GMK (e.g., quercetin, coumarin, and geranylgeranyl) may exert their epigenetic regulatory functions by targeting the protein activity of DNMTs. In vivo experiments showed that GMK reduced the number of nose scratching and sneezing in mice, glandular hyperplasia in the nasal mucosa was alleviated, with a reduction in the number and volume of glands, and serum tIgE levels were reduced. The increase in LERP expression in the AR model was reduced after treatment with GMK, and the change in NFIA expression was not significant. It suggests that GMK may regulate LERP activity through DNMTs to alleviate allergic symptoms. This study reveals the potential therapeutic mechanism of the traditional Chinese medicine compound GMK in regulating AR through epigenetic mechanisms. These findings provide a theoretical basis and molecular foundation for the clinical application of GMK in AR and open up new research directions.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-21298-7.

Endosperm is a key evolutionary innovation associated with the origin of angiosperms (flowering plants). This altruistic seed tissue supports the growth and development of the embryo by mediating the relationship of the mother plant as a nutrient source to the compatriot embryo as a nutrient sink. The endosperm is the primary site of gene imprinting in plants (where expression of an allele in offspring depends on which parent it was inherited from) and of parent-specific epigenetic modifications like DNA methylation, which are differentially patterned during male and female gamete development. Experimental results from a phylogenetically-wide array of monocot and eudicot plants suggest these parent-of-origin effects are a common feature across angiosperms. However, information about genetic imprinting and epigenetic modifications in seeds of angiosperm lineages whose origins predate the monocot-eudicot divergence (such as Nymphaeales, water lilies) is extremely limited. Additionally, Nymphaeales are an intriguing lineage in which to investigate seed genetic and epigenetic phenomena as they are characterized by diploid endosperm and a maternal storage tissue (perisperm), both of which are unusual across angiosperm diversity. Here, we examined DNA methylation and genetic imprinting using two reproductively compatible water lily sister-species, Nymphaea thermarum and N. dimorpha. Our results suggest that maternally-expressed imprinted genes and differential DNA methylation of maternally and paternally inherited endosperm genomes are an ancestral condition for endosperm, whereas other seed characters like seed provisioning strategies, endosperm ploidy, and paternally-expressed imprinted genes might have evolved as coinciding, opposing strategies in the evolutionary dialogue over parental control of offspring development.

Idiopathic dilated cardiomyopathy (iDCM) is a multifactorial disease with a complex pathogenesis involving diverse molecular mechanisms. Among these, epigenetic mechanisms, including both DNA methylation and microRNAs (miRNAs)-mediated regulation, play an important role in determining the disease phenotype. However, the interplay between the DNA methylome and the miRNA transcriptome in iDCM remains largely unexplored. We conducted a cross-cohort multiomic integrative analysis of left ventricular (LV) tissue samples from iDCM patients and control (CNT) donors. DNA methylation profiling was performed using the Infinium MethylationEPIC BeadChip, whereas ncRNA-seq was used to assess transcriptomic changes. We identified a subset of three miRNAs exhibiting both differential methylation in their promoter regions and differential expression in their primary and mature forms. Notably, the miRNA hsa-miR-433-3p (r = 0.671, p < 0.01), which is involved in fibrotic pathways, appear to be significantly correlated with the left ventricular ejection fraction (LVEF), an established echocardiographic marker of cardiac function. This study enhances our understanding of the epigenetic mechanisms shaping the miRNA transcriptomic landscape in iDCM, suggesting potential roles for these miRNAs in cardiac dysfunction and myocardial fibrosis.

Disclosure: L. Zoff: None. F.M. Garcia: None. G. Veneruzzo: None. G. Aschettino: None. M.C. Mattone: None. E.B. Berensztein: None. C. Terada: None. N. Perez Garrido: None. R.M. Marino: None. C. Alonso: None. G. Guercio: None. A. Belgorosky: None. M.S. Baquedano: None.Being born SGA in 46, XY DSD patients without molecular diagnosis and with no specific disorders of undermasculinization have a higher prevalence than expected for the general population. Numerous links have been made between infant growth restriction and specific epigenetic alterations, including changes in DNA methylation, thereby implicating such regulation in appropriate growth and development. Moreover, several studies have suggested that SGA children present with permanent alterations in the epigenome due to the programming of fetal tissues. The Growth hormone (GH)/ insulin-like growth factor type 1 (IGF1) and Insulin axis, crucial in human growth and development, is involved in adrenogonadal differentiation in mice. We hypothesized that epigenetic changes in the GH/IGFs and Insulin axis, could mediate the association between being born SGA and with DSD in humans. Our aim was to evaluate both the levels and patterns of methylation across the regulatory regions of GHR, IGF1R, and INSR genes in foreskin (FT) from 46,XY DSD children born either AGA (AGADSD-FT, n=7; 0.8-7.37y) or SGA (SGADSD-FT, n=7; 1.3-7.3y) vs age-matched control children (C-FT; n=7, 0.9-7y) using targeted deep-amplicon bisulfite sequencing on a MiSeq system. Sequencing libraries of 4 promoter CpG-rich regions for GHR, 3 for IGF1R and 5 for INSR (104, 103 and 141 CpG sites, respectively) were analyzed for mean methylation values of all CpG sites using amplikyzer2 software. The average CpG methylation levels of the SP1-responsive IGF1R proximal promoter [GRCh38:15:98648387: 98648592:1 (-152 to +53)] and the 5´UTR regulatory region [GRCh38:15:98649214: 98649496:1 (+676 to +958)] were significantly higher in SGADSD-FT than in C-FT and AGADSD-FT (Kruskal Wallis test, p<0.05). In locus-by-locus comparative analyses, higher methylation levels of CpG-23, CpG-17, CpG-15, CpG-13, and CpG-11 within the unique Sp1-dependent initiator motif of the IGF1R gene were detected in SGADSD-FT compared with C-FT and AGADSD-FT, p<0.05. The mean methylation levels of INSR promoter regions [GRCh38:19:7294796:7295022:-1 (-608 to -381) and GRCh38:19:7294410:7294702:1 (-288 to +5)], covering the binding site of activating transcription factors, were higher in DSD 46, XY patients, both in SGADSD-FT and in AGADSD-FT than in C-FT (p<0.05). There were no significant differences in GHR promoter methylation among the three FT groups. Promoter DNA methylation is a fundamental epigenetic mark associated with transcriptional repression during development. According to our findings, it could be speculated that epigenetic repression, mainly of IGF1R, might contribute to mediate the association between being born SGA and 46XYDSD in humans. Nevertheless, whether or not the epigenetic changes observed in postnatal foreskin are present at the time of sexual differentiation during intrauterine life must be determined.Presentation: Saturday, July 12, 2025

ABSTRACT Recent studies have shown that newborns conceived using assisted reproductive technology (ART) exhibited significantly different DNA methylation (DNAm) profiles at birth compared to those conceived naturally. Of note was the observation of increased DNAm at the promoter region of BRCA1/NBR2 in ART-conceived newborns. However, it remains unclear if these DNAm differences persist after birth. Using the Norwegian Mother, Father, and Child Cohort Study (MoBa), a large-scale population-based pregnancy cohort with extensive longitudinal data collected through biological samples and questionnaires, we generated longitudinal DNAm data for 105 ART-conceived and 250 naturally conceived children at birth and at ages 3–22 years. DNAm differences in the BRCA1/NBR2 promoter between ART- and naturally conceived children, at birth and postnatally, were tested using linear mixed model with adjustment for maternal and newborn covariates. While ART-conceived children showed subtle hypermethylation at birth and postnatally, the differences diminished over time and did not remain statistically significant after multiple testing correction. Our findings suggest that subtle hypermethylation at the BRCA1/NBR2 promoter in ART-conceived children may represent an ART-associated epigenetic signature, although further studies in larger populations are needed to clarify its persistence and significance.

BackgroundEpigenetic variations, particularly in response to environmental factors, play a crucial role in shaping immune identity and function in hematopoietic cells. This study investigates interindividual differences in DNA methylation among dairy cows, with the aim of enhancing our understanding of the adaptive capacity essential for sustainable animal production. We conducted whole-genome sequencing and DNA methylation analysis using enzymatic methyl-seq on whole blood from 60 Holstein cows. The study included five phenotype groups: mastitis, lameness, infertility, metabolic disorders and healthy controls.ResultsAmong the 50 million CpG sites, 5.1% were identified as variable methylated cytosines (VMCs) and 94.9% as conserved methylated cytosines (CMCs). VMCs displayed variability in distal promoter regions, suggesting potential plasticity in the associated genes, while CMCs exhibited a bimodal methylation pattern near the transcription start site, indicative of tissue-specific functions. Notably, we identified motif enrichments related to genes potentially expressed in blood. An age-related analysis revealed a 1.4% faster decline in CMCs methylation compared to VMCs. Additionally, disease risk assessment may be achievable using as few as 586 methylation biomarkers, which could be used to select which cows to keep in the herd for additional lactation.ConclusionOur results suggest a dual role for VMCs and CMCs: while the stability of conserved sites is potentially associated with essential functions in cell development and homeostasis, variable sites may be involved in dynamically regulating gene transcription in response to internal or external stimuli. These insights underscore the epigenome’s role in immune regulation and adaptive resilience in cattle.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12864-025-12112-9.

In epigenetic analysis, the identification of differentially methylated regions (DMRs) typically involves the detection of consecutive CpGs groups that show significant changes in their average methylation levels. However, the methylation state of a genomic region can also be characterized by a mixture of patterns (epialleles) with variable frequencies, and the relative proportions of such patterns can provide insights into its mechanisms of formation. Traditional methods based on bisulfite conversion and high-throughput sequencing, such as Illumina, owing to the read size (150 bp) allow epiallele frequency analysis only in high CpG density regions, limiting differential methylation studies to just 50% of the human methylome. Nanopore sequencing, with its long reads, enables the analysis of epiallele frequency across both high and low CpG density regions. Here, we introduce a novel computational approach, PoreMeth2, an R library that integrates epiallelic diversity and methylation frequency changes from nanopore data to identify DMRs, providing insights into their possible mechanisms of formation, and annotate them to genic and regulatory elements. We apply PoreMeth2 to cancer and glial cell data sets, providing evidence of its advance over other state-of-the-art methods and demonstrating its ability to distinguish epigenomic alterations with a strong impact on gene expression from those with weaker effects on transcriptional activity.