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CHO cells are major hosts for the industrial production of therapeutic proteins and their production stability is of considerable economic significance. It is widely known that CHO cells can rapidly acquire genetic alterations, which affects their genetic homogeneity over time. However, the role of non-genetic mechanisms, including epigenetic mechanisms such as DNA methylation, remains poorly understood. We have now used whole-genome bisulfite sequencing to establish single-base methylation maps of eight independent CHO cell lines. Our results identify CpG islands and low-methylated regions as conserved elements with dynamic DNA methylation. Interestingly, methylation patterns were found to cluster clearly along the three main branches of CHO evolution, with no directional changes over short culture periods. Furthermore, multi-ome single-cell sequencing of 9833 nuclei from three independent cultures revealed dynamic subpopulation structures characterized by robust expression differences in pathways related to protein production. Our findings thus provide novel insights into the epigenetic landscape and heterogeneity of CHO cells and support the development of epigenetic biomarkers that trace the emergence of subpopulations in CHO cultures., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Frank Lyko reports financial support was provided by Evonik Operations GmbH. Frank Lyko reports a relationship with Evonik Operations GmbH that includes: consulting or advisory. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Epigenetics encompasses mechanisms that can alter the expression of genes without changing the underlying genetic sequence. The epigenetic regulation of gene expression is initiated and sustained by several mechanisms such as DNA methylation, histone modifications, chromatin conformation, and non-coding RNA. The changes in gene regulation and expression can manifest in the form of various diseases and disorders such as cancer and congenital deformities. Over the last few decades, high-throughput experimental approaches have been used to identify and understand epigenetic changes, but these laboratory experimental approaches and biochemical processes are time-consuming and expensive. To overcome these challenges, machine learning and artificial intelligence (AI) approaches have been extensively used for mapping epigenetic modifications to their phenotypic manifestations. In this paper we provide a narrative review of published research on AI models trained on epigenomic data to address a variety of problems such as prediction of disease markers, gene expression, enhancer-promoter interaction, and chromatin states. The purpose of this review is twofold as it is addressed to both AI experts and epigeneticists. For AI researchers, we provided a taxonomy of epigenetics research problems that can benefit from an AI-based approach. For epigeneticists, given each of the above problems we provide a list of candidate AI solutions in the literature. We have also identified several gaps in the literature, research challenges, and recommendations to address these challenges., Competing Interests: Declaration of competing interest No competing interest is declared., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Epigenetic modifications, including DNA methylation (DNAm), can play a role in the biological embedding of early-life adversity (ELA) through serotonergic mechanisms. The current study examines methylation of the CpG island in the promoter region of the stress-responsive serotonin transporter gene (SLC6A4) and is the first to jointly assess how it is influenced by ELA severity, timing, and type-specifically, deprivation and threat., Methods: We use data from 627 Youth Emotion Project study participants, recruited from two US high schools. Using adjusted linear regressions, we analyze DNA collected in early adulthood from 410 participants and ELA based on interviewer-rated responses from concurrent Childhood Trauma Interviews, adjusting for survey-measured covariates., Results: ELA robustly predicted mean CpG island SLC6A4 DNAm percent across 71 CpG sites. Each additional major-severity ELA event was associated with a 0.121-percentage-point increase (p<0.001), equating to a 0.177 standard deviation (sd) higher DNAm level (95 % CI: 0.080, 0.274) with each 1-sd higher adversity score. When modeled separately, both childhood and adolescent ELA predicted SLC6A4 DNAm. When modeled jointly, adolescent ELA was most strongly predictive, and child adversity remained significantly associated with DNAm through indirect associations via adolescent adversity. Additionally, the ELA-SLC6A4 DNAm association may vary by adversity type. Across separate models for childhood and adolescent exposures, deprivation coefficients are positive and statistically significant. Meanwhile, threat coefficients are positive and not significantly significant but do not statistically differ from deprivation coefficients. In models including all ELA dimensions, one major adolescent deprivation event is associated with a 0.222-percentage-point increased SLC6A4 DNAm (p<0.05), or a 1-sd higher deprivation score with a 0.157-sd increased DNAm., Conclusion: Results further implicate epigenetic modification on serotonergic neurotransmission via DNAm in the downstream sequelae of ELA-particularly adolescent deprivation-and support preventive interventions in adolescence to mitigate biological embedding., Competing Interests: Declaration of Competing Interest None., (Copyright © 2024. Published by Elsevier Ltd.)

Self-rated health (SRH) is a subjective indicator of overall health based on a single questionnaire item. Previous evidence found that it is a strong predictor of mortality, although the underlying mechanism is poorly understood. Epigenetic age is an objective, emerging biomarker of health, estimated using DNA methylation data at hundreds of sites across the genome. This study aimed to assess the overlap and interaction between SRH and epigenetic ageing in predicting mortality risk. We used DNA methylation data from 1059 participants in the Melbourne Collaborative Cohort Study (mean age: 69 years) to calculate three age-adjusted measures of epigenetic ageing: GrimAge, PhenoAge, and DunedinPACE. SRH was assessed using a five-category questionnaire item ("excellent, very good, good, fair, poor"). Cox models were used to assess the associations of SRH, epigenetic ageing, and their interaction, with all-cause mortality over up to 17 years of follow-up (N deaths  = 345). The association of SRH with mortality per category increase was HR = 1.29; 95%CI: 1.14-1.46. The association was slightly attenuated after adjusting for all three epigenetic ageing measures (HR = 1.25, 95%CI: 1.10-1.41). A strong gradient was observed in the association of GrimAge (P interaction  = 0.006) and DunedinPACE (P interaction  = 0.002) with mortality across worsening SRH strata. For example, the association between DunedinPACE and mortality in participants with "excellent" SRH was HR = 1.02, 95%CI: 0.73-1.43 and for "fair/poor" HR = 1.72, 95%CI: 1.35-2.20. SRH and epigenetic ageing were synergistic risk factors of mortality in our study. These findings suggest that consideration of subjective and objective factors may improve general health assessment, which has implications for the ongoing development of molecular markers of ageing., (© 2024. The Author(s).)

Stochastic epigenetic mutations (SEMs) have been proposed as novel aging biomarkers to capture heterogeneity in age-related DNA methylation changes. SEMs are defined as outlier methylation patterns at cytosine-guanine dinucleotide sites, categorized as hypermethylated (hyperSEM) or hypomethylated (hypoSEM) relative to a reference. Because SEMs are defined by their outlier status, it is critical to differentiate extreme values due to technical noise or data artifacts from those due to real biology. Using technical replicate data, we found SEM detection is not reliable: across 3 datasets, 24 to 39% of hypoSEM and 46 to 67% of hyperSEM are not shared between replicates. We identified factors influencing SEM reliability-including blood cell type composition, probe beta-value statistics, genomic location, and presence of SNPs. We used these factors in a training dataset to build a machine learning-based filter that removes unreliable SEMs, and found this filter enhances reliability in two independent validation datasets. We assessed associations between SEM loads and aging phenotypes in the Framingham Heart Study and discovered that associations with aging outcomes were in large part driven by hypoSEMs at baseline methylated probes and hyperSEMs at baseline unmethylated probes, which are the same subsets that demonstrate highest technical reliability. These aging associations were preserved after filtering out unreliable SEMs and were enhanced after adjusting for blood cell composition. Finally, we utilized these insights to formulate best practices for SEM detection and introduce a novel R package, SEMdetectR, which uses parallel programming for efficient SEM detection with comprehensive options for detection, filtering, and analysis., (© 2024. The Author(s), under exclusive licence to American Aging Association.)

Hepatic xenobiotic metabolism and transport decline with age, while intact xenobiotic metabolism is associated with longevity. However, few studies have examined the genome-wide impact of epigenetic aging on these processes. We used reduced representation bisulfite sequencing (RRBS) to map DNA methylation changes in liver DNA from mice ages 4 and 24 months. We identified several thousand age-associated differentially methylated sites (a-DMS), many of which overlapped genes encoding Phase I and Phase II drug metabolizing enzymes, in addition to ABC and SLC classes of transporters. Notable genes harboring a-DMS were Cyp1a2, Cyp2d9, and Abcc2 that encode orthologs of the human drug metabolizing enzymes CYP1A2 and CYP2D6, and the multidrug resistance protein 2 (MRP2) transporter. Cyp2d9 hypermethylation with age was significantly associated with reduced gene expression, while Abcc2 expression was unchanged with age. Cyp1a2 lost methylation with age while, counterintuitively, its expression also reduced with age. We hypothesized that age-related dysregulation of the hepatic transcriptional machinery caused down-regulation of genes despite age-related hypomethylation. Bioinformatic analysis of hypomethylated a-DMS in our sample found them to be highly enriched for hepatic nuclear factor 4 alpha (HNF4α) binding sites. HNF4α promotes Cyp1a2 expression and is downregulated with age, which could explain the reduction in Cyp1a2 expression. Overall, our study supports the broad impact of epigenetic aging on xenobiotic metabolism and transport. Future work should evaluate the interplay between hepatic nuclear receptor function and epigenetic aging. These results may have implications for studies of longevity and healthy aging., (© 2024. The Author(s).)

Background: As the most common sleep disorder, chronic insomnia disorder (CID) has become a global health burden to the public. However, it remains unclear about the pathogenesis of this disease. Epigenetic changes may provide important insights into the gene-environment interaction in CID. Therefore, this study was conducted to investigate the DNA methylation pattern in CID and reveal the epigenetic mechanism of this disease., Methods: In this study, whole blood DNA was extracted from 8 CID patients (the CID group) and 8 healthy controls (the control group), respectively. Besides, genome-wide DNA methylation was detected by Illumina Human Methylation 850 K Beadchip. Moreover, the sleep quality and insomnia severity were evaluated by the Pittsburgh Sleep Quality Index (PSQI) and Insomnia Severity Index (ISI), respectively., Results: A total of 369 differentially methylated positions (DMPs) and 23 differentially methylated regions (DMRs) were identified between the CID and control groups. LHX6 was identified as the most important differentially methylated gene (DMG). The Gene Ontology (GO) analysis results corroborated that DMPs were significantly enriched in 105 GO terms, including cell signaling, homogenous cell adhesion of plasma membrane adhesion molecules, nervous system development, cell adhesion, and calcium ion binding. In addition, it was demonstrated that DMPs were significantly enriched in the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, including the hippo signaling pathway, Ras signaling pathway, and vitamin B6 metabolism. The DMR-related GO analysis results revealed the positive regulation of protein kinase activities., Conclusions: DNA methylation plays a critical role in the development of CID, and LHX6 is validated to be an important DMG., Competing Interests: Declarations Ethical approval This study was approved by the Ethics Committee of the Second Affiliated Hospital of Xinxiang Medical University and performed in accordance with the Declaration of Helsinki 1964 and its later amendments or comparable ethical standards. Formal consent is not required for this study type. Conflict of interest All authors certify that they have no affiliation with or involvement in any organization or entity with any financial interest., (© 2024. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

The mostly aggressive and extremely malignant type of central nervous system is Glioblastoma (GBM), which is characterized by an extremely short average survival time of lesser than 16 months. The primary cause of this phenomenon can be attributed to the extensively altered genome of GBM, which is characterized by the dysregulation of numerous critical signaling pathways and epigenetics regulations associated with proliferation, cellular growth, survival, and apoptosis. In light of this, different genetic alterations in critical signaling pathways and various epigenetics regulation mechanisms are associated with GBM and identified as distinguishing markers. Such GBM prognostic alterations are identified in PI3K/AKT, p53, RTK, RAS, RB, STAT3 and ZIP4 signaling pathways, metabolic pathway (IDH1/2), as well as alterations in epigenetic regulation genes (MGMT, CDKN2A-p16 INK4a CDKN2B-p15 INK4b ). The exploration of innovative diagnostic and therapeutic approaches that specifically target these pathways is utmost importance to enhance the future medication for GBM. This study provides a comprehensive overview of dysregulated epigenetic mechanisms and signaling pathways due to mutations, methylation, and copy number alterations of in critical genes in GBM with prevalence and emphasizing their significance., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Background: Pulmonary arterial hypertension (PAH) is a relatively rare but severe disease with a poor prognosis. Pulmonary embolism (PE) is a serious condition that can cause sudden death. Epigenetic age acceleration (EAA) is a robust indicator derived from the DNA methylation-based epigenetic clock, which can predict the extent of aging. It has been proved that the epigenetic clock and EAA are associated with many cardiovascular diseases, while their associations with PAH and PE remain inconclusive. Our study aims to investigate the associations among these factors., Method: By harnessing summary-level data from large-scale genome-wide association studies (GWAS), we designed a two-sample bidirectional Mendelian randomization (MR) analysis to assess the causal associations between measures of three epigenetic clocks, including GrimAge acceleration (n = 34,467), Hannum Age acceleration (n = 34,449) and PhenoAge acceleration (n = 34,463) and PAH (including 125 cases and 162,837 controls), as well as PE (including 3940 cases and 480,658 controls). The inverse variance-weighted (IVW) method was used as the primary method for MR analysis. Other methods, such as MR egger and weighted mode, served as complements to the IVW approach, were also applied in the analyses. Then, the MR pleiotropy test and MR-PRESSO test, which are effective tools for quality control of MR analysis, were subsequently used to ensure the accuracy of the study., Results: The forward MR analysis indicated that all three epigenetic clocks had no significant effects on PAH or PE. The reverse analysis indicated that the onset and progression of PAH and PE had insignificant effects on three epigenetic clocks. The results of the quality control assessment confirmed that our findings were reliable., Conclusion: Our two-sample bidirectional MR analysis suggested that there is no significant association between epigenetic clocks and these two pulmonary vascular diseases., Competing Interests: Declarations. Ethics approval and consent to participate: Because it is an analysis using previously collected and published data, no additional ethics approval is needed. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Background: H 2 O 2 treatment can accelerate grape ripening and mediate changes in histone methylation levels. Histone methylation, as an epigenetic modification, is involved in regulating the expression of genes related to fruit ripening, including H3K27ac, H3K4me1, H3K27me3 and H3K4me3. Among them, H3K27me3 methylation is generally negatively regulated in development, and H3K27 methyltransferase can participate in the development process of fruit by regulate the level of H3K27me3. The H3K27 methyltransferase members in grapes are not yet clear, and a better understanding of their functions contributes to regulating fruit development., Methods and Results: By analyzing the conserved domains of the grape genome, three H3K27 methyltransferases were identified and named as VvH3K27-1, VvH3K27-2 and VvH3K27-3, respectively. Further analysis included their conserved domains, gene structure, phylogenetic relationship, protein physicochemical properties, chromosome localization, subcellular localization, and cis-acting elements in the promoter region. It is worth noting that all H3K27 methyltransferase genes have a highly conserved SET domain. VvH3K27-2 was localized in the nucleus and H 2 O 2 treatment resulted in a decrease in the expression of these genes., Conclusion: Three H3K27 methyltransferase genes were identified in grape, which are down-regulated during berry development, and their expression is inhibited by H 2 O 2 treatment. Thus, H3K27 methyltransferase genes are involved in the regulation of fruit development., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Background: In high-grade ovarian cancer (HGOC), determination of homologous recombination deficiency (HRD) status is commonly used in routine practice to predict response to platinum-based therapy or poly (ADP-ribose) polymerase inhibitors (PARPi). Here we tested the hypothesis that BRCA loss of function (LOF) due to epigenetic or genetic aberrations is a better predictor for the clinical outcome than HRD. One hundred thirty-one HGOC tissues were tested for BRCA DNA-methylation, BRCA mutations, HRD and BRCA1 mRNA expression, followed by a comprehensive survival analysis., Results: BRCA1-methylation was detected in 11% of the tumors, exclusively in BRCA1-wild-type (wt) HGOCs. BRCA1-methylated tumors (BRCA1-meth) had HRD-scores similar to those of BRCA-mutated (mut) tumors, and higher compared to unmethylated-BRCA-wt tumors (BRCA-wt-unmeth; P < 0.001). Platinum-refractory or -resistant HGOCs at first recurrence were all BRCA-unmeth cancers. Only one of the BRCA-mut cancers had a platinum-resistant recurrence. Thus, 99% of relapses in cancers with epigenetic or genetic BRCA-alterations were platinum-sensitive. Multivariate analysis confirmed BRCA-LOF as an independent predictor of progression-free survival (PFS) and overall survival (OS), whereas HRD-status had no predictive value for PFS and OS. Patients with BRCA-wt-unmeth cancers had the worst outcome compared to patients with cancers harboring epigenetic or genetic BRCA-alterations (PFS: P = 0.007; OS: P = 0.022). Most importantly, the BRCA-wt-unmeth subfraction of HRD-positive HGOCs exhibited the same poor survival as the entire HRD-negative cohort., Conclusion: In HGOC BRCA mutational status together with BRCA1-methylation exhibit the best predictive power for favorable clinical outcome and thus high sensitivity to platinum-based therapy, whereas BRCA-unrelated HRD positivity was not associated with improved platinum sensitivity., Competing Interests: Declarations. Ethics approval and consent to participate: The study was approved by the Ethics Committee of the Innsbruck Medical University (reference numbers: AN2015-0038 346/4.17; 1054/2019) and conducted in accordance with the Declaration of Helsinki Principles Consent for publication: Not applicable. Competing interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. However, the following authors receive financial support for the specified activities: KL reports travel expenses from GSK, Roche, Eisai and MSD. IT reports honoraria from Roche and Astra Zeneca; travel expenses from Eisai, GSK, PharmaMar, Roche, Astra Zeneca, Pfizer; participation on advisory boards from Astra Zeneca. VW reports honoraria from Roche, Novartis; travel expenses from Roche; participation on advisory boards from Novartis. CM reports consulting fees from Roche, Novartis, Amgen, MSD, PharmaMar, Astra Zeneca, GSK, Seagen; honoraria from Roche, Novartis, Amgen, MSD, PharmaMar, Astra Zeneca, GSK, Seagen; travel expenses from Roche, Astra Zeneca; participation on advisory boards from Roche, Novartis, Amgen, MSD, Astra Zeneca, Pfizer, PharmaMar, GSK, Seagen. AGZ reports consulting fees from Amgen, Astra Zeneca, GSK, MSD, Novartis, PharmaMar, Roche-Diagnostics, Seagen; honoraria from Amgen, Astra Zeneca, GSK, MSD, Novartis, PharmaMar, Roche, Seagen; travel expenses from Astra Zeneca, Gilead, Roche; participation on advisory boards from Amgen, Astra Zeneca, GSK, MSD, Novartis, Pfizer, PharmaMar, Roche, Seagen., (© 2024. The Author(s).)

Epigenetics governs a chromatin state regulatory system through five key mechanisms: DNA modification, histone modification, RNA modification, chromatin remodeling, and non-coding RNA regulation. These mechanisms and their associated enzymes convey genetic information independently of DNA base sequences, playing essential roles in organismal development and homeostasis. Conversely, disruptions in epigenetic landscapes critically influence the pathogenesis of various human diseases. This understanding has laid a robust theoretical groundwork for developing drugs that target epigenetics-modifying enzymes in pathological conditions. Over the past two decades, a growing array of small molecule drugs targeting epigenetic enzymes such as DNA methyltransferase, histone deacetylase, isocitrate dehydrogenase, and enhancer of zeste homolog 2, have been thoroughly investigated and implemented as therapeutic options, particularly in oncology. Additionally, numerous epigenetics-targeted drugs are undergoing clinical trials, offering promising prospects for clinical benefits. This review delineates the roles of epigenetics in physiological and pathological contexts and underscores pioneering studies on the discovery and clinical implementation of epigenetics-targeted drugs. These include inhibitors, agonists, degraders, and multitarget agents, aiming to identify practical challenges and promising avenues for future research. Ultimately, this review aims to deepen the understanding of epigenetics-oriented therapeutic strategies and their further application in clinical settings., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Wilms tumor, the most common pediatric kidney cancer, accounts for 5% of childhood cancers and is classified by stage and histological subtype. Despite high survival rates (80-85%), approximately 15% of patients experience relapse, reducing survival to around 50%. Epigenetic changes, particularly DNA methylation, play a critical role in Wilms tumor pathogenesis. This study investigates the prognostic potential of DNA methylation in stage I and II patients with favorable histology, aiming to identify early relapse biomarkers. Genome-wide methylation was assessed using methylation microarrays in tumor tissues from relapsed patients (n = 9) and those with complete responses (n = 9), alongside normal tissues (n = 3 each). Differentially methylated probes and regions were analyzed, with additional ROC and survival analyses. Real-time PCR was used to measure IGF2 and INS-IGF2 gene expression. The analysis revealed hypomethylation in intergenic regions in remission patients, identifying 14 differentially methylated positions as potential biomarkers. Increased INS-IGF2 expression was associated with relapse, suggesting its role in disease progression. While the study concentrated on stages I and II patients, where relapse rates are lower, this focus inherently led to a smaller sample size. Despite this, the findings provide valuable insights into the potential role of DNA methylation markers for monitoring disease progression and guiding personalized treatment in Wilms tumor patients., Competing Interests: Declarations. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Cytotoxic T lymphocyte antigen 4 (CTLA-4), in conjunction with PD-1 and CD28, plays a pivotal role in the modulation of T-cell activation. Specifically, CTLA-4 exerts its influence by impeding the generation of IL-2 and the proliferation of T cells. CTLA-4, being a receptor with a high affinity, engages in competitive binding with CD28 for the interaction with primary T-cell activator molecules, specifically CD80 and CD86. The appropriate functioning of T-cell activation is contingent upon maintaining a precise equilibrium between CTLA-4 and CD28. Consequently, any disruption in the expression of CTLA-4 significantly enhances the risk for a range of severe ailments, such as cancer, infectious diseases, allergies, and notably autoimmune diseases. The significance of epigenetic regulation of CTLA-4, particularly through non-coding RNAs (ncRNAs) such as microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs), has considerable weight within this particular framework. To date, there have been associations shown between various abnormalities in the expression of ncRNAs that regulate CTLA-4 and clinicopathological characteristics. Nevertheless, it is evident that there is a lack of a comprehensive investigation. Hence, the present work was undertaken to summarize the existing research on the epigenetic control of CTLA-4, with a primary emphasis on elucidating the regulatory procedures, biological processes, and clinical applications in human diseases. The objective of this review is to acquire a thorough comprehension of the relationship between RNA/lncRNA/miRNA/mRNA (CTLA-4) and its role in the progression of diverse human disorders., Competing Interests: Declarations. Conflict of interests: The authors declare no competing interests. Ethical approval: Not applicable., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

DNA methylation (DNAm) is the most commonly measured epigenetic mechanism in human populations, with most studies using Illumina arrays to assess DNAm levels. In 2023, Illumina updated their DNAm arrays to the EPIC version 2 (EPICv2), building on prior iterations, namely the EPIC version 1 (EPICv1) and 450K arrays. Whether DNAm measurements are stable across these three generations of arrays has yet not been investigated, limiting the ability of researchers-especially those with longitudinal data-to compare and replicate results across arrays. Here, we present results from a study of 30 child participants (15 male; 15 female) from the Drakenstein Child Health Study, who had DNAm measured on all three of the latest arrays: 450K, EPICv1, and EPICv2. Using these data, we created an annotation of probe quality across arrays, which includes the intraclass correlations, interquartile ranges, correlations, and array bias (i.e., the extent to which DNAm levels were explained by array type) of all CpGs. We also present results from an analysis of sex differences, where we found that CpGs with lower replicability across arrays had higher array-based variance, suggesting this variance metric help guide replication efforts. We also showed that epigenetic age estimates across arrays were more stable when using the principal component versions of epigenetic clocks. Ultimately, this collection of results provides a framework for investigating the replicability and longitudinal stability of epigenetic changes across multiple versions of Illumina DNAm arrays., Competing Interests: Declarations. Ethics approval and consent to participate: Written informed consent was obtained from mothers at enrolment and thereafter annually. The study was approved by the Human Research Ethics Committee of the Faculty of Health Sciences, University of Cape Town, by Stellenbosch University and the Western Cape Provincial Research committee (HREC UCT REF 401/2009; HREC UCT REF 525/2012) [17]. Consent for publication: Not applicable. Competing interests: The authors declare that they have no competing interests., (© 2024. The Author(s).)

Background: Sudden unexplained death (SUD) is a devastating event in the young. Despite efforts to identify causal genetic variants, many cases remain unexplained after genetic screening. This study aimed to investigate an alternative potential contributor to SUD by studying the human methylome using the MethylationEPIC v2.0 BeadChip kit in heart tissue from SUD cases. The genome-wide methylation results of the SUD cases were compared to the results of a control cohort. The SUD cases were divided into three main groups based on their autopsy reports, heart morphology and histopathology (primaryN: macroscopically and histologically normal heart; primaryCM: macroscopically or histologically abnormal heart, suspected cardiomyopathies; and secondary: myocardial damage due to other underlying conditions). The main focus of this study was to identify differentially methylated regions (DMRs) between the case groups and the control cohort., Results: We identified DMRs for both the primaryN and primaryCM groups, whereas the secondary group yielded no such results. In the primaryN cases, the corresponding genes for each DMR led to the identification of genes with common biological pathways. Some were associated with heart morphology (e.g. heart outflow tract morphogenesis or trabecular morphogenesis), but the majority belonged to more general cellular regulatory pathways (e.g. transcription coactivator activity, long non-coding RNAs, etc.). Although no common pathways were found for the primaryCM group, some common regulatory molecular functions were identified, such as p53 binding and transcription coactivator activity., Conclusions: Our study is the first to investigate the whole human methylome in heart tissue of SUD cases. We propose that there are observable differences in the methylation patterns of the case groups that may have contributed to SUD. Still, further studies are required to improve our understanding of the impact of methylation levels on SUD risk and to pinpoint methylation-based screening opportunities for SUD relatives., Competing Interests: Declarations. Ethics approval and consent to participate: Ethical approval for this study was provided by the local ethics committee in Zurich (KEK-No. 2013-0086), and the study was conducted in full conformance with Swiss laws and regulations. The requirements of the local ethics committee included written informed consent of family members. If no family members were available, the cases were anonymized. Consent for publication: All authors of the manuscript have read and agreed to its content and consent to publish it. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Background: Cancer-associated fibroblasts (CAFs) are essential components of the tumor microenvironment and play a critical role in cancer progression. Numerous studies have identified significant molecular differences between CAFs and normal tissue-associated fibroblasts (NAFs). In this study, we isolated CAFs and NAFs from liver tumors and conducted a comprehensive analysis of their DNA methylation profiles, integrating our finding with data from studies on other cancer types., Results: Our analysis revealed that several CAF samples exhibited aberrant DNA methylation patterns, which corresponded with altered gene expression levels. Notably, DNA methylation at liver CAF-specific CpG sites was linked to survival outcomes in liver cancer datasets. An integrative analysis using publicly available datasets from various cancer types, including lung, prostate, esophageal, and gastric cancers, uncovered common epigenetic abnormalities across these cancers. Among the consistently altered CpGs were cg09809672 (EDARADD), cg07134930 (HDAC4), and cg05935904 (intergenic). These methylation changes were associated with prognosis across multiple cancer types., Conclusion: The activation of CAFs by the tumor microenvironment seems to be associated with distinct epigenetic modifications. Remarkably, similar genomic regions tend to undergo hypomethylation in CAFs across different studies and cancer types. Our findings suggest that CAF-associated DNA methylation changes hold potential as prognostic biomarkers. However, further research and validation are necessary to develop and apply such signatures in a clinical setting., Competing Interests: Declarations. Ethics approval and consent to participate: Liver biopsies were received from the Clinic for General, Visceral, Children and Transplantation Surgery at the University Hospital of RWTH Aachen after informed and written consent and following the guidelines of the Ethic Committee for the Use of Human Subjects at the University of Aachen (Permit number: EK 206/09). Consent for publication: Not applicable. Availability of data and materials: DNA methylation profiles generated in this study can be accessed from the Gene expression omnibus (GEO) under accession number GSE255123; the RNA-sequencing data are accessible under GSE255122. The following tokens have been created to allow review of the records GSE255123 (qxahsaionterlqr) and GSE255122 (khmjmegabnmjnqn). Competing interests: W.W. is involved in the company Cygenia GmbH ( www.cygenia.com ) that can provide service for epigenetic analysis to other scientists. Apart from this, the authors have no competing interests to declare., (© 2024. The Author(s).)

Chromatin states play important roles in the maintenance of cell identities, yet their spatial patterns remain poorly characterized at the organism scale. We developed a systematic approach to analyzing spatial epigenomic data and then applied it to a recently published spatial-CUT&Tag dataset that was obtained from a mouse embryo. We identified a set of spatial genes whose H3K4me3 patterns delineate tissue boundaries. These genes are enriched with tissue-specific transcription factors, and their corresponding genomic loci are marked by broad H3K4me3 domains. Integrative analysis with H3K27me3 profiles showed coordinated spatial transitions across tissue boundaries, which is marked by the continuous shortening of H3K4me3 domains and expansion of H3K27me3 domains. Motif-based analysis identified transcription factors whose activities change significantly during such transitions. Taken together, our systematic analyses reveal a strong connection between the genomic and spatial variations of chromatin states. A record of this paper's transparent peer review process is included in the supplemental information., Competing Interests: Declaration of interests R.F. is a scientific founder and advisor of IsoPlexis, Singleron Biotechnologies, and AtlasXomics., (Copyright © 2024 Elsevier Inc. All rights reserved.)

In recent years, epigenetic modifications have been strongly linked to tumor development, with histone modifications representing a key epigenetic mechanism. In addition, non-coding RNAs (ncRNAs) play a critical role in regulating cancer-related pathways. The abnormal interaction between histone modifications and ncRNAs, both pivotal epigenetic regulators, has been widely observed across various cancer types. Here, we systematically explore the molecular mechanisms through which histone modifications and ncRNAs contribute in the pathogenesis of digestive system cancers, and aberrant ncRNA-mediated histone modifications manipulate various biological behaviors of tumor cells including proliferation, migration, angiogenesis, etc. In addition, we provide new insights into diagnostic, prognostic markers, therapeutic targets and chemoradiation resistance for digestive system cancers from the epigenetic perspective., Competing Interests: Declarations Ethics approval and consent to participate Not applicable. Consent for publication By submitting my article I agree to pay the APC in full if my article is accepted for publication (unless it is covered by an institutional agreement or journal partner, or a full waiver has been granted). Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Background: DNA methylation is a critical regulatory mechanism of gene expression, influencing various human diseases and traits. While traditional expression quantitative trait loci (eQTL) studies have helped elucidate the genetic regulation of gene expression, there is a growing need to explore environmental influences on gene expression. Existing methods such as PrediXcan and FUSION focus on genotype-based associations but overlook the impact of environmental factors. To address this gap, we present MOSES (methylation-based gene association), a novel approach that utilizes DNA methylation to identify environmentally regulated genes associated with traits or diseases without relying on measured gene expression., Results: MOSES involves training, imputation, and association testing. It employs elastic-net penalized regression models to estimate the influence of CpGs and SNPs (if available) on gene expression. We developed and compared four MOSES versions incorporating different methylation and genetic data: (1) cis-DNA methylation within 1 Mb of promoter regions, (2) both cis-SNPs and cis-CpGs, 3) both cis- and a part of trans- CpGs (±5Mb away) from promoter regions), and 4) long-range DNA methylation (±10 Mb away) from promoter regions. Our analysis using nasal epithelium and white blood cell data from the Epigenetic Variation and Childhood Asthma in Puerto Ricans (EVA-PR) study demonstrated that MOSES, particularly the version incorporating long-range CpGs (MOSES-DNAm 10 M), significantly outperformed existing methods like PrediXcan, MethylXcan, and Biomethyl in predicting gene expression. MOSES-DNAm 10 M identified more differentially expressed genes (DEGs) associated with atopic asthma, particularly those involved in immune pathways, highlighting its superior performance in uncovering environmentally regulated genes. Further application of MOSES to lung tissue data from idiopathic pulmonary fibrosis (IPF) patients confirmed its robustness and versatility across different diseases and tissues., Conclusion: MOSES represents an innovative advancement in gene association studies, leveraging DNA methylation to capture the influence of environmental factors on gene expression. By incorporating long-range CpGs, MOSES-DNAm 10 M provides superior predictive accuracy and gene association capabilities compared to traditional genotype-based methods. This novel approach offers valuable insights into the complex interplay between genetics and the environment, enhancing our understanding of disease mechanisms and potentially guiding therapeutic strategies. The user-friendly MOSES R package is publicly available to advance studies in various diseases, including immune-related conditions like asthma., Competing Interests: Declarations Ethical approval and consent to participate The Epigenetic Variation and Childhood Asthma in Puerto Ricans (EVA-PR) study was approved by the institutional review boards of the University of Puerto Rico (San Juan, PR) and the University of Pittsburgh (Pittsburgh, PA). Written parental consent and participant assent were obtained for individuals younger than 18, while consent was obtained from participants 18 years and older. Consent for publication Not applicable. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Epilepsy, affecting approximately 1% of the global population, manifests as recurring seizures and is heavily influenced by genetic factors. Recent advancements in genetic technologies have revolutionized our understanding of epilepsy's genetic landscape. Key studies, such as the discovery of mutations in ion channels (e.g., SCN1A and SCN2A), neurotransmitter receptors (e.g., GABRA1), and synaptic proteins (e.g., SYNGAP1, KCNQ2), have illuminated critical pathways underlying epilepsy susceptibility and pathogenesis. Genome-wide association studies (GWAS) have identified specific genetic variations linked to epilepsy risk, such as variants near SCN1A and PCDH7, enhancing diagnostic accuracy and enabling personalized treatment strategies. Moreover, epigenetic mechanisms, including DNA methylation (e.g., MBD5), histone modifications (e.g., HDACs), and non-coding RNAs (e.g., miR-134), play pivotal roles in altering gene expression and synaptic plasticity, contributing to epileptogenesis. These discoveries offer promising avenues for therapeutic interventions aimed at improving outcomes for epilepsy patients. Genetic testing has become essential in clinical practice, facilitating precise diagnosis and tailored management approaches based on individual genetic profiles. Furthermore, insights into epigenetic regulation suggest novel therapeutic targets for developing more effective epilepsy treatments. In summary, this review highlights significant progress in understanding the genetic and epigenetic foundations of epilepsy. By integrating findings from key studies and specifying genes involved in epigenetic modifications, we underscore the potential for advanced therapeutic strategies in this complex neurological disorder, emphasizing the importance of personalized medicine approaches in epilepsy management., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Systemic lupus erythematosus (SLE) is an autoimmune disease with protean manifestations. There is little understanding of why some organs are specifically impacted in patients and the mechanisms of disease persistence remain unclear. While much work has been done characterizing the DNA methylation status in SLE, there is less information on histone modifications, a more dynamic epigenetic feature. This study identifies two histone marks of activation and the binding of p300 genome-wide in three cell types and three clinical subsets to better understand cell-specific effects and differences across clinical subsets., Results: We examined 20 patients with SLE and 8 controls and found that individual chromatin marks varied considerably across T cells, B cells, and monocytes. When pathways were examined, there was far more concordance with conservation of TNF, IL-2/STAT5, and KRAS pathways across multiple cell types and ChIP data sets. Patients with cutaneous lupus and lupus nephritis generally had less dramatically altered chromatin than the general SLE group. Signals also demonstrated significant overlap with GWAS signals in a manner that did not implicate one cell type more than the others., Conclusions: The pathways identified by altered histone modifications and p300 binding are pathways known to be important from RNA expression studies and recognized pathogenic mechanisms of disease. NFκB and classical inflammatory pathways were strongly associated with increased peak heights across all cell types but were the highest-ranking pathway for all three antibodies in monocytes according to fgsea analysis. IL-6 Jak/STAT3 signaling was the most significant pathway association in T cells marked by H3K27ac change. Therefore, each cell type experiences the disease process distinctly although in all cases there was a strong theme of classical inflammatory pathways. Importantly, this NFκB pathway, so strongly implicated in the patients with generalized SLE, was much less impacted in monocytes when cutaneous lupus was compared to the general SLE cohort and also less impacted in lupus nephritis compared to general SLE. These studies define important cell type differences and emphasize the breadth of the inflammatory effects in SLE., Competing Interests: Declarations Ethical approval and consent to participate The Johns Hopkins University School of Medicine Institutional Review Board approved this study. IRB00216558. UPENN Institutional Review Board for healthy controls was 705906. Patient consent All patients consented for this study. Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

TET2 is a critical gene that regulates DNA methylation, encoding a dioxygenase protein that plays a vital role in the regulation of genomic methylation and other epigenetic modifications, as well as in hematopoiesis. Mutations in TET2 are present in 7%-28% of adult acute myeloid leukemia (AML) patients. Despite this, the precise mechanisms by which TET2 mutations contribute to malignant transformation and how these insights can be leveraged to enhance treatment strategies for AML patients with TET2 mutations remain unclear. In this review, we provide an overview of the functions of TET2, the effects of its mutations, its role in clonal hematopoiesis, and the possible mechanisms of leukemogenesis. Additionally, we explore the mutational landscape across different AML subtypes and present recent promising preclinical research findings., (© 2024. The Author(s).)

Inflammation underlies many conditions causing excess morbidity and mortality among people with HIV (PWH). A handful of single-trait epigenome-wide association studies (EWAS) have suggested that inflammation is associated with DNA methylation (DNAm) among PWH. Multi-trait EWAS may further improve statistical power and reveal pathways in common between different inflammatory markers. We conducted single-trait EWAS of three inflammatory markers (soluble CD14, D-dimers and interleukin-6) in the Veterans Aging Cohort Study (n = 920). The study population was all male PWH with an average age of 51 years, and 82.3% self-reported as Black. We then applied two multi-trait EWAS methods-CPASSOC and OmniTest-to combine single-trait EWAS results. CPASSOC and OmniTest identified 189 and 157 inflammation-associated DNAm sites, respectively, of which 112 overlapped. Among the identified sites, 56% were not significant in any single-trait EWAS. Top sites were mapped to inflammation-related genes including IFITM1, PARP9 and STAT1. These genes were significantly enriched in pathways such as "type I interferon signaling" and "immune response to virus." We demonstrate that multi-trait EWAS can improve the discovery of inflammation-associated DNAm sites, genes and pathways. These DNAm sites might hold the key to addressing persistent inflammation in PWH., (© 2024. The Author(s).)

Background: Asthma, a complex respiratory disease, presents with inflammatory symptoms in the lungs, blood, and other tissues. We investigated the relationship between DNA methylation and 35 clinical markers of asthma., Methods: The Illumina Infinium EPIC v1 methylation array was used to evaluate 742,442 CpGs in whole blood from 319 participants from 94 families. They were part of the Netherlands Twin Register from families with at least one member suffering from severe asthma. Repeat blood samples were taken after 10 years from 182 individuals. Principal component analysis on the clinical asthma markers yielded ten principal components (PCs) that explained 92.8% of the total variance. We performed epigenome-wide association studies (EWAS) for each of the ten PCs correcting for familial structure and other covariates., Results: 221 unique CpGs reached genome-wide significance at timepoint 1 after Bonferroni correction. PC7, which correlated with loadings of eosinophil counts and immunoglobulin levels, accounted for the majority of associations (204). Enrichment analysis via the EWAS Atlas identified 190 of these CpGs to be previously identified in EWASs of asthma and asthma-related traits. Proximity assessment to previously identified SNPs associated with asthma identified 17 unique SNPs within 1 MB of two of the 221 CpGs. EWAS in 182 individuals with epigenetic data at a second timepoint identified 49 significant CpGs. EWAS Atlas enrichment analysis indicated that 4 of the 49 were previously associated with asthma or asthma-related traits. Comparing the estimates of all the significant associations identified across the two time points yielded a correlation of 0.81., Conclusion: We identified 270 unique CpGs that were associated with PC scores generated from 35 clinical markers of asthma, either cross-sectionally or 10 years later. A strong correlation was present between effect sizes at the 2 timepoints. Most associations were identified for PC7, which captured blood eosinophil counts and immunoglobulin levels and many of these CpGs have previous associations in earlier studies of asthma and asthma-related traits. The results point to a robust DNA methylation profile as a new, stable biomarker for asthma., (© 2024. The Author(s).)

The chromosomal theory of inheritance dictates that genes on the same chromosome segregate together while genes on different chromosomes assort independently 1 . Extrachromosomal DNAs (ecDNAs) are common in cancer and drive oncogene amplification, dysregulated gene expression and intratumoural heterogeneity through random segregation during cell division 2,3 . Distinct ecDNA sequences, termed ecDNA species, can co-exist to facilitate intermolecular cooperation in cancer cells 4 . How multiple ecDNA species within a tumour cell are assorted and maintained across somatic cell generations is unclear. Here we show that cooperative ecDNA species are coordinately inherited through mitotic co-segregation. Imaging and single-cell analyses show that multiple ecDNAs encoding distinct oncogenes co-occur and are correlated in copy number in human cancer cells. ecDNA species are coordinately segregated asymmetrically during mitosis, resulting in daughter cells with simultaneous copy-number gains in multiple ecDNA species before any selection. Intermolecular proximity and active transcription at the start of mitosis facilitate the coordinated segregation of ecDNA species, and transcription inhibition reduces co-segregation. Computational modelling reveals the quantitative principles of ecDNA co-segregation and co-selection, predicting their observed distributions in cancer cells. Coordinated inheritance of ecDNAs enables co-amplification of specialized ecDNAs containing only enhancer elements and guides therapeutic strategies to jointly deplete cooperating ecDNA oncogenes. Coordinated inheritance of ecDNAs confers stability to oncogene cooperation and novel gene regulatory circuits, allowing winning combinations of epigenetic states to be transmitted across cell generations., (© 2024. The Author(s).)

The last decade has seen remarkable progress in the characterization of methylation clocks that can serve as indicators of biological age in humans and many other mammalian species. While the biological processes of aging that underlie these clocks have remained unclear, several clues have pointed to a link to developmental mechanisms. These include the presence in the vicinity of clock CpG sites of genes that specify development, including those of the Hox (homeobox) and polycomb classes. Here we discuss how recent advances in programmatic theories of aging provide a framework within which methylation clocks can be understood as part of a developmental process of aging. This includes how such clocks evolve, how developmental mechanisms cause aging, and how they give rise to late-life disease. The combination of ideas from evolutionary biology, biogerontology and developmental biology open a path to a new discipline, that of developmental gerontology (devo-gero). Drawing on the properties of methylation clocks, we offer several new hypotheses that exemplify devo-gero thinking. We suggest that polycomb controls a trade-off between earlier developmental fidelity and later developmental plasticity. We also propose the existence of an evolutionarily-conserved developmental sequence spanning ontogenesis, adult development and aging, that both constrains and determines the evolution of aging., Competing Interests: Declaration of Competing Interest J.P.M. is CSO of YouthBio Therapeutics, an advisor/consultant for the Longevity Vision Fund, 199 Biotechnologies, and NOVOS, and the founder of Magellan Science Ltd, a company providing consulting services in longevity science., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Direct lineage reprogramming into dopaminergic (DA) neurons holds great promise for the more effective production of DA neurons, offering potential therapeutic benefits for conditions such as Parkinson's disease. However, the reprogramming pathway for fully reprogrammed DA neurons remains largely unclear, resulting in immature and dead-end states with low efficiency. In this study, using single-cell RNA sequencing, the trajectory of reprogramming DA neurons at multiple time points, identifying a continuous pathway for their reprogramming is analyzed. It is identified that intermediate cell populations are crucial for resetting host cell fate during early DA neuronal reprogramming. Further, longitudinal dissection uncovered two distinct trajectories: one leading to successful reprogramming and the other to a dead end. Notably, Arid4b, a histone modifier, as a crucial regulator at this branch point, essential for the successful trajectory and acquisition of mature dopaminergic neuronal identity is identified. Consistently, overexpressing Arid4b in the DA neuronal reprogramming process increases the yield of iDA neurons and effectively reverses the disease phenotypes observed in the PD mouse brain. Thus, gaining insights into the cellular trajectory holds significant importance for devising regenerative medicine strategies, particularly in the context of addressing neurodegenerative disorders like Parkinson's disease., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Post-translational modifications on the histone H3 tail regulate chromatin structure, impact epigenetics, and hence the gene expressions. Current chemical modulation tools, such as unnatural amino acid incorporation, protein splicing, and sortase-based editing, have allowed for the modification of histones with various PTMs in cellular contexts, but are not applicable for editing native chromatin. The use of small organic molecules to manipulate histone-modifying enzymes alters endogenous histone PTMs but lacks precise temporal and spatial control. To date, there has been no achievement in modulating histone methylation in living cells with spatiotemporal resolution. In this study, a new method is presented for temporally manipulating histone dimethylation H3K9me2 using a photo-responsive inhibitor that specifically targets the methyltransferase G9a on demand. The photo-caged molecule is stable under physiological conditions and cellular environments, but rapidly activated upon exposure to light, releasing the bioactive component that can immediately inhibit the catalytic ability of the G9a in vitro. Besides, this masked compound could also efficiently reactivate the inhibition of methyltransferase activity in living cells, subsequently suppress H3K9me2, a mark that regulates various chromatin functions. Therefore, the chemical system will be a valuable tool for manipulating the epigenome for therapeutic purposes and furthering the understanding of epigenetic mechanisms., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Longevity, the length of an organism's lifespan, is impacted by environmental factors, metabolic processes, and genetic determinants. The base excision repair (BER) pathway is crucial for maintaining genomic integrity by repairing oxidatively modified base lesions. Nei-like DNA Glycosylase 1 (NEIL1), part of the BER pathway, is vital in repairing oxidative bases in G-rich DNA regions, such as telomeres and promoters. Hence, in this comprehensive review, it have undertaken a meticulous investigation of the intricate association between NEIL1 and longevity. The analysis delves into the multifaceted aspects of the NEIL1 gene, its various RNA transcripts, and the diverse protein isoforms. In addition, a combination of bioinformatic analysis is conducted to identify NEIL1 mutations, transcription factors, and epigenetic modifications, as well as its lncRNA/pseudogene/circRNA-miRNA-mRNA regulatory network. The findings suggest that the normal function of NEIL1 is a significant factor in human health and longevity, with defects in NEIL1 potentially leading to various cancers and related syndromes, Alzheimer's disease, obesity, and diabetes., (© 2024 Wiley‐VCH GmbH.)

Abstract: Epigenetics has gained considerable interest as potential mediators of molecular alterations that could underlie the prolonged sensitization of nociceptors, neurons, and glia in response to various environmental stimuli. Histone acetylation and deacetylation, key processes in modulating chromatin, influence gene expression; elevated histone acetylation enhances transcriptional activity, whereas decreased acetylation leads to DNA condensation and gene repression. Altered levels of histone deacetylase (HDAC) have been detected in various animal pain models, and HDAC inhibitors have demonstrated analgesic effects in these models, indicating HDACs' involvement in chronic pain pathways. However, animal studies have predominantly examined epigenetic modulation within the spinal cord after pain induction, which may not fully reflect the complexity of chronic pain in humans. Moreover, methodological limitations have previously impeded an in-depth study of epigenetic changes in the human brain. In this study, we employed [ 11 C]Martinostat, an HDAC-selective radiotracer, positron emission tomography to assess HDAC availability in the brains of 23 patients with chronic low back pain (cLBP) and 11 age-matched and sex-matched controls. Our data revealed a significant reduction of [ 11 C]Martinostat binding in several brain regions associated with pain processing in patients with cLBP relative to controls, highlighting the promising potential of targeting HDAC modulation as a therapeutic strategy for cLBP., (Copyright © 2024 International Association for the Study of Pain.)

Mutations in the isocitrate dehydrogenase (IDH) gene are recognized as the key drivers in the oncogenesis of astrocytoma and oligodendroglioma. However, the significance of IDH mutation in tumor maintenance and malignant transformation has not been elucidated. We encountered a unique case of IDH-mutant astrocytoma that, upon malignant transformation, presented two distinct intratumoral components: one IDH-wildtype and one IDH-mutant. The IDH-wild-type component exhibited histological findings similar to those of small cell-type glioblastoma with a higher Ki-67 index than the IDH-mutant component. Despite their genetic divergence, both components exhibited similar comprehensive methylation profiles within the CpG island and were classified into methylation class of "Astrocytoma, IDH-mutant; High Grade" by the German Cancer Center (DKFZ) classifier v11.4. Phylogenetic analysis demonstrated that the IDH-wildtype component emerged as a subclonal component of the primary tumor. Detailed molecular analyses revealed that the loss of the IDH mutation was induced by the hemizygous loss of the entire arm of chromosome 2, on which IDH1 gene is located. Notably, the IDH-wild-type subclones uniquely acquired CDKN2A/B homozygous deletion and PDGFRA amplification, which is a marker of the aggressive phenotype of astrocytoma, IDH-mutant. Because these genetic abnormalities can drive oncogenic pathways, such as the PI3K/AKT/mTOR and RB signaling pathway, IDH-mutant gliomas that acquired these mutations were no longer dependent on the initial driver mutation, the IDH mutation. Molecular analysis of this unique case provides insight that in a subset of astrocytoma, IDH-mutant that acquired these genetic abnormalities, IDH mutation may not play a pivotal role in tumor growth and acquisition of these genetic abnormalities may contribute to the acquisition of resistance to IDH inhibitors., (© 2024. The Author(s).)

Background: Facioscapulohumeral dystrophy (FSHD) is a myopathy characterized by the loss of repressive epigenetic features affecting the D4Z4 locus (4q35). The assessment of DNA methylation at two regions (DUX4-PAS and DR1) of D4Z4 locus proved to be an effective method to detect epigenetic signatures compatible with FSHD. The present study aims at validating the employment of this method into clinical practice and improving the protocol by refining the classification thresholds of 4qA/4qA patients. To this purpose, 218 subjects with clinical suspicion of FSHD collected in 2022-2023 were analyzed. Each participant underwent in parallel the traditional FSHD molecular testing (D4Z4 sizing) and the proposed methylation assay. The results provided by both analyses were compared to evaluate the concordance and calculate the performance metrics of the methylation test., Results: Among the 218 subjects, the 4q variant type distribution was 54% 4qA/4qA, 43% 4qA/4qB and 3% 4qB/4qB. The methylation analysis was performed only on carriers of at least one 4qA allele. After refining the classification threshold, the test reached the following performance metrics: sensitivity = 0.90, specificity = 1.00 and accuracy = 0.93. These results confirmed the effectiveness of the methylation assay in identifying patients with genetic signature compatible with FSHD1 and FSHD2 based on their DUX4-PAS and DR1 profile, respectively. The methylation data were also evaluated with respect to the clinical information., Conclusions: The study confirmed the ability of the method to accurately identify methylation profiles compatible with FSHD genetic signatures considering the 4q genotype. Moreover, the test allows the detection of hypomethylated profiles in asymptomatic patients, suggesting its potential application in identifying preclinical conditions in patients with positive family history and FSHD genetic signatures. Furthermore, the present work emphasizes the importance of interpreting methylation profiles considering the patients' clinical data., (© 2024. The Author(s).)

Background: Colorectal cancer is a public health issue and was the third leading cause of cancer-related death worldwide in 2022. Early diagnosis can improve prognosis, making screening a central part of colorectal cancer management. Blood-based screening, diagnosis and follow-up of colorectal cancer patients are possible with the study of cell-free circulating tumor DNA. This study aimed to identify novel DNA methylation biomarkers of colorectal cancer that can be used for the follow-up of patients with colorectal cancer., Methods: A DNA methylation profile was established in the Gene Expression Omnibus (GEO) database (n = 507) using bioinformatics analysis and subsequently confirmed using The Cancer Genome Atlas (TCGA) database (n = 348). The in silico profile was then validated on local tissue and cell-free DNA samples using methylation-specific digital PCR in colorectal cancer patients (n = 35) and healthy donors (n = 35)., Results: The DNA methylation of COL25A1 and METAP1D was predicted to be a colorectal cancer biomarker by bioinformatics analysis (ROC AUC = 1, 95% CI [0.999-1]). The two biomarkers were confirmed with tissue samples, and the combination of COL25A1 and METAP1D yielded 49% sensitivity and 100% specificity for cell-free DNA., Conclusion: Bioinformatics analysis of public databases revealed COL25A1 and METAP1D DNA methylation as clinically applicable liquid biopsies DNA methylation biomarkers. The specificity implies an excellent positive predictive value for follow-up, and the high sensitivity and relative noninvasiveness of a blood-based test make these biomarkers compatible with colorectal cancer screening. However, the clinical impact of these biomarkers in colorectal cancer screening and follow-up needs to be established in further prospective studies., (© 2024. The Author(s).)

Background: DNA methylation profiling may provide a more accurate measure of the smoking status than self-report and may be useful in guiding clinical interventions and forensic investigations. In the current study, blood DNA methylation profiles of nearly 800 Polish individuals were assayed using Illuminia EPIC and the inference of smoking from epigenetic data was explored. In addition, we focused on the role of the AHRR gene as a top marker for smoking and investigated its responsiveness to other lifestyle behaviors., Results: We found > 450 significant CpGs associated with cigarette consumption, and overrepresented in various biological functions including cell communication, response to stress, blood vessel development, cell death, and atherosclerosis. The model consisting of cg05575921 in AHRR (p = 4.5 × 10 -32 ) and three additional CpGs (cg09594361, cg21322436 in CNTNAP2 and cg09842685) was able to predict smoking status with a high accuracy of AUC = 0.8 in the test set. Importantly, a gradual increase in the probability of smoking was observed, starting from occasional smokers to regular heavy smokers. Furthermore, former smokers displayed the intermediate DNA methylation profiles compared to current and never smokers, and thus our results indicate the potential reversibility of DNA methylation after smoking cessation. The AHRR played a key role in a predictive analysis, explaining 21.5% of the variation in smoking. In addition, the AHRR methylation was analyzed for association with other modifiable lifestyle factors, and showed significance for sleep and physical activity. We also showed that the epigenetic score for smoking was significantly correlated with most of the epigenetic clocks tested, except for two first-generation clocks., Conclusions: Our study suggests that a more rapid return to never-smoker methylation levels after smoking cessation may be achievable in people who change their lifestyle in terms of physical activity and sleep duration. As cigarette smoking has been implicated in the literature as a leading cause of epigenetic aging and AHRR appears to be modifiable by multiple exogenous factors, it emerges as a promising target for intervention and investment., (© 2024. The Author(s).)

DNA lies at the heart of the central dogma of life. Altering DNA can modify the flow of information in fundamental cellular processes such as transcription and translation. The ability to precisely manipulate DNA has led to remarkable advances in treating incurable human genetic ailments and has changed the landscape of biological research. Genome editors such as CRISPR-Cas nucleases and TALENs have become ubiquitous tools in basic and applied biological research and have been translated to the clinic to treat patients. The specificity and modularity of these genome editors have made it possible to efficiently engineer genomic DNA; however, underlying principles governing editing outcomes in eukaryotes are still being uncovered. Editing efficiency can vary from cell type to cell type for the same DNA target sequence, necessitating de novo design and validation efforts. Chromatin structure and epigenetic modifications have been shown to affect the activity of genome editors because of the role they play in hierarchical organization of the underlying DNA. Understanding the nuclear search mechanism of genome editors and their molecular interactions with higher order chromatin will lead to improved models for predicting precise genome editing outcomes. Insights from such studies will unlock the entire genome to be engineered for the creation of novel therapies to treat critical illnesses.

Epigenetic modifications control gene expression and are essential for turning genes on and off to regulate and maintain differentiated cell types. Epigenetics are also modified by a multitude of environmental exposures, including diet and pollutants, allowing an individual's environment to influence gene expression and resultant phenotypes and clinical outcomes. These epigenetic modifications due to gene-environment interactions can also be transmitted across generations, raising the possibility that environmental influences that occurred in one generation may be transmitted beyond the second generation, exerting a long-lasting effect. In this review, we cover the known mechanisms of epigenetic modification acquisition, reprogramming and persistence, animal models and human studies used to understand multigenerational epigenetic transmission, and examples of environmentally induced epigenetic change and its transmission across generations. We highlight the importance of environmental health not only on the current population but also on future generations that will experience health outcomes transmitted through epigenetic inheritance., (© 2024. The Author(s).)

Background: The role of epigenetics in cardiovascular diseases has paved the way for innovative therapeutic approaches. Investigating epigenetic changes using cell-free DNA (cfDNA) holds substantial promise beyond mere diagnostics, especially for heart-related conditions like acute myocardial infarction (AMI), where obtaining tissue samples is a challenge. This study explores the methylation patterns of cfDNA in AMI patients and compares them with genomic DNA (gDNA) from the same individuals, aiming to evaluate the effectiveness of cfDNA as a valuable resource for studying heart-related diseases., Methodology: We generated global methylome profiles of cfDNA and gDNA from 25 AMI patients using EM-Seq. Tissue deconvolution analysis was performed to estimate tissue specificity based on the methylation patterns. Differentially methylated loci were identified and explored to understand AMI pathophysiology., Results: Comparative analysis of cfDNA and gDNA methylation patterns in AMI patients reveals cfDNA holds more significance than gDNA. Principal component analysis revealed distinct clusters for cfDNA and gDNA, indicating distinct methylome profiles. cfDNA originated from multiple sources, predominantly from neutrophils (~ 75%) and about 10% from the left atrium, highlighting cardiac-specific changes. In contrast, immune cells are the major source of gDNA, indicative of inflammatory responses. Gene set enrichment analysis (GSEA) associates cfDNA methylation patterns with pathways related to cardiac muscle contraction, inflammation, hypoxia, and lipid metabolism. The affected genes include G protein-coupled receptors (GHSR, FFAR2, HTR1A, and VIPR2) that are part of the cAMP signaling pathway., Conclusion: Epigenetic changes in cfDNA are more specific to cardiac tissue compared to those in gDNA, providing better insights into the molecular mechanisms involved in AMI. Genes that are differentially methylated in cfDNA and regulate core pathways, such as cAMP signaling, could be targeted for clinical applications, including the development of effective biomarkers and therapeutic targets., (© 2024. The Author(s).)

Background: Lack of insight into factors that determine purity and quality of human iPSC (hiPSC)-derived neo-cartilage precludes applications of this powerful technology toward regenerative solutions in the clinical setting. Here, we set out to generate methylome-wide landscapes of hiPSC-derived neo-cartilages from different tissues-of-origin and integrated transcriptome-wide data to identify dissimilarities in set points of methylation with associated transcription and the respective pathways in which these genes act., Methods: We applied in vitro chondrogenesis using hiPSCs generated from two different tissue sources: skin fibroblasts and articular cartilage. Upon differentiation toward chondrocytes, these are referred to as hFiCs and hCiC, respectively. Genome-wide DNA methylation and RNA sequencing datasets were generated of the hiPSC-derived neo-cartilages, and the epigenetically regulated transcriptome was compared to that of neo-cartilage deposited by human primary articular cartilage (hPAC)., Results: Methylome-wide landscapes of neo-cartilages of hiPSCs reprogrammed from two different somatic tissues were 85% similar to that of hPACs. By integration of transcriptome-wide data, differences in transcriptionally active CpGs between hCiC relative to hPAC were prioritized. Among the CpG-gene pairs lower expressed in hCiCs relative to hPACs, we identified genes such as MGP, GDF5, and CHAD enriched in closely related pathways and involved in cartilage development that likely mark phenotypic differences in chondrocyte states. Vice versa, among the CpG-gene pairs higher expressed, we identified genes such as KIF1A or NKX2-2 enriched in neurogenic pathways and likely reflecting off target differentiation., Conclusions: We did not find significant variation between the neo-cartilages derived from hiPSCs of different tissue sources, suggesting that application of a robust differentiation protocol such as we applied here is more important as compared to the epigenetic memory of the cells of origin. Results of our study could be further exploited to improve quality, purity, and maturity of hiPSC-derived neo-cartilage matrix, ultimately to realize introduction of sustainable, hiPSC-derived neo-cartilage implantation into clinical practice., (© 2024. The Author(s).)

Background: Malignant cells exhibit reduced period circadian regulator 3 (PER3) expression. However, the underlying mechanisms of variations in PER3 expression in cancers and the specific function of PER3 in tumor progression remain poorly understood., Results: We explored multiple public databases, conducted bioinformatics analyses, and performed in vitro and in vivo experiments for validation. We found PER3 expression was decreased in most types of cancers, and PER3 downregulation was associated with a poor prognosis in 8 types of cancer. PER3 promoter methylation levels were increased in 11 types of cancer. Promoter hypermethylation (CpG islands [CGIs] cg12258811 and cg14204433) correlated with decreased PER3 expression in six cancers (breast invasive carcinoma, colon adenocarcinoma, head and neck squamous cell carcinoma, kidney renal papillary cell carcinoma [KIRP], lung adenocarcinoma [LUAD], and uterine corpus endometrial carcinoma). CGI cg12258811 hypermethylation was associated with reduced survival time and advanced cancer stages. Moreover, the bisulfite pyrosequencing assay confirmed CGI cg12258811 hypermethylation and its negative correlation with PER3 expression. In vitro and in vivo experiments demonstrated that PER3 inhibited KIRP and LUAD progression. Decitabine enhanced PER3 expression and inhibited KIRP cell functions by reducing promoter (cg12258811) methylation level., Conclusions: Our findings advanced the mechanistic understanding of variations in PER3 expression in cancers and confirmed the tumor-associated function of PER3 hypermethylation and downregulation., (© 2024. The Author(s).)

T cells are dynamically regulated immune cells that are implicated in a variety of diseases ranging from infection, cancer and autoimmunity. Recent advancements in sequencing methods have provided valuable insights in the transcriptional and epigenetic regulation of T cells in various disease settings. In this review, we identify the key sequencing-based methods that have been applied to understand the transcriptomic and epigenomic regulation of T cells in diseases., (© 2024. The Author(s).)

Accurate grading of IDH-mutant gliomas defines patient prognosis and guides the treatment path. Histological grading is challenging, and aside from CDKN2A/B homozygous deletions in IDH-mutant astrocytomas, there are no other objective molecular markers used for grading. RNA-sequencing was conducted on primary IDH-mutant astrocytomas (n = 138) included in the prospective CATNON trial, which was performed to assess the prognostic effect of adjuvant and concurrent temozolomide. We integrated the RNA-sequencing data with matched DNA-methylation and NGS data. We also used multi-omics data from IDH-mutant astrocytomas included in the TCGA dataset and validated results on matched primary and recurrent samples from the GLASS-NL study. Since discrete classes do not adequately capture grading of these tumours, we utilised DNA-methylation profiles to generate a Continuous Grading Coefficient (CGC) based on classification scores from a CNS-tumour classifier. CGC was an independent predictor of survival outperforming current WHO-CNS5 and methylation-based classification. Our RNA-sequencing analysis revealed four distinct transcription clusters that were associated with (i) upregulation of cell cycling genes; (ii) downregulation of glial differentiation genes; (iii) upregulation of embryonic development genes (e.g. HOX, PAX, and TBX) and (iv) upregulation of extracellular matrix genes. The upregulation of embryonic development genes was associated with a specific increase of CpG island methylation near these genes. Higher grade IDH-mutant astrocytomas have DNA-methylation signatures that, on the RNA level, are associated with increased cell cycling, tumour cell de-differentiation and extracellular matrix remodelling. These combined molecular signatures can serve as an objective marker for grading of IDH-mutant astrocytomas., (© 2024. The Author(s).)

Background: Multi-locus imprinting disturbance (MLID) with methylation defects in various differentially methylated regions (DMRs) has recently been identified in approximately 150 cases with imprinting disorders (IDs), and deleterious variants have been found in genes related to methylation maintenance of DMRs, such as those encoding proteins constructing the subcortical maternal complex (SCMC), in a small fraction of patients and/or their mothers. However, integrated methylation analysis for DMRs and sequence analysis for MLID-causative genes in MLID cases and their mothers have been performed only in a single study focusing on Beckwith-Wiedemann syndrome (BWS) and Silver-Russell syndrome (SRS) phenotypes., Results: Of 783 patients with various IDs we have identified to date, we examined a total of 386 patients with confirmed epimutation and 71 patients with epimutation or uniparental disomy. Consequently, we identified MLID in 29 patients with epimutation confirmed by methylation analysis for multiple ID-associated DMRs using pyrosequencing and/or methylation-specific multiple ligation-dependent probe amplification. MLID was detected in approximately 12% of patients with BWS phenotype and approximately 5% of patients with SRS phenotype, but not in patients with Kagami-Ogata syndrome, Prader-Willi syndrome, or Angelman syndrome phenotypes. We next conducted array-based methylation analysis for 78 DMRs and whole-exome sequencing in the 29 patients, revealing hypomethylation-dominant aberrant methylation patterns in various DMRs of all the patients, eight probably deleterious variants in genes for SCMC in the mothers of patients, and one homozygous deleterious variant in ZNF445 in one patient. These variants did not show gene-specific methylation disturbance patterns. Clinically, neurodevelopmental delay and/or intellectual developmental disorder (ND/IDD) was observed in about half of the MLID patients, with no association with the identified methylation disturbance patterns and genetic variants. Notably, seven patients with BWS phenotype were conceived by assisted reproductive technology (ART)., Conclusions: The frequency of MLID was 7.5% (29/386) in IDs caused by confirmed epimutation. Furthermore, we revealed diverse patterns of hypomethylation-dominant methylation defects, nine deleterious variants, ND/IDD complications in about half of the MLID patients, and a high frequency of MLID in ART-conceived patients., (© 2024. The Author(s).)

Osteosarcoma (OS) is the most frequent primary malignant bone tumour, whose heterogeneity represents a major challenge for common antitumour therapies. Inflammatory cytokines are known to be necessary for OS progression. Therefore, to optimise therapy, it is important to discover reliable biomarkers by identifying the mechanism generating OS and investigating the inflammatory pathways that support the undifferentiated state. In this work, we highlight the differences of epigenetic activities of IL-1β and TNFα, and the susceptibility of TET-1 enzymatic inhibition, in tumour progression of three different OS cell lines. Investigating DNA methylation of IL-6 promoter and determining its expression, we found that TET enzymatic inhibition influences proliferation induced by inflammatory cytokines in OS cell lines. Moreover, Bobcat 339 treatment blocks IL-1β epigenetic action on IL-6 promoter, while only partially those of TNFα as well as inhibits IL-1β-dependent epithelial-mesenchymal transition (EMT) process, but only partially those of TNFα. In conclusion, this work highlights that IL-1β and TNFα have different effects on DNA demethylation in OS cell lines, making DNA methylation a potential biomarker of disease. Specifically, in IL-1β treatment, TET-1 inhibition completely blocks tumour progression, while in TNFα actions, it is only partially effective. Given that these two inflammatory pathways can be therapeutic targets for treating these tumours, knowledge of their distinct epigenetic behaviours can be useful for developing precise and specific therapeutic strategies for this disease., (© 2024. The Author(s).)

Targeted integration (TI) Chinese hamster ovary (CHO) platforms are commonly used for protein expression. However, the impact of epigenetic modifications on protein expression in TI cell lines remains elusive since almost all the epigenetic studies focus on random integration (RI) of the gene of interest and only within the promoter region. To address the impact of epigenetic modifications on TI CHO cells, we utilized a standard mAb-1 to identify and characterize TI clones with the same transgene copy numbers but different levels of transgene transcription and titer. Surprisingly, while CMV promoters were not methylated and histone acetylation/methylation was present, these epigenetic markers did not trend with mRNA transcription and protein expression in our TI model. Instead, ATAC-seq data analysis revealed that differences in chromatin accessibility within the TI site could be a major factor impacting these observed differences. However, neither chromatin accessibility nor histone acetylation/methylation profiles in early cultures were predictive of high-expressing clones early during the CLD process. Finally, modulation of the histone profiles (H3K27ac and H3K4me3) at the CMV promoters within the TI integration site using dCas9 fusion proteins was not effective in further increasing mAb titers which could have been likely due to interference of the dCas9 fusion proteins with transcription from the CMV promoters. Overall, our data suggests increasing chromatin accessibility at the TI site is the most effective way to increase mRNA transcription and hence, productivity in TI cell lines., (© 2024 Wiley‐VCH GmbH.)

Fokienia hodginsii (F. hodginsii), belonging to the genus Fokienia of the Cupressaceae. F. hodginsii has significant application value due to its wood properties and great research value in evolutionary studies as a gymnosperm. However, the genome of F. hodginsii remains unknown due to the large size of gymnosperms genome. Pacific Bioscience sequencing, Hi-C mapping, whole-genome Bisulfite Sequencing (BS-Seq), long-read isoform sequencing (Iso-Seq), direct RNA sequencing (DRS), quantitative proteomics, and metabonomics analysis are employed to facilitate genome assembly, gene annotation, and investigation into epigenetic mechanisms. In this study, the 10G F. hodginsii genome is assembled into 11 chromosomes. Furthermore, 50 521 protein-coding genes are annotated and determined that 65% of F. hodginsii genome comprises repetitive sequences. It is discovered that transposable element (TE)-including introns is associated with higher expression. The DNA methylome of F. hodginsii reveals that xylem has a higher DNA methylation level compared to callus. Moreover, DRS reveals the significant alterations in RNA full-length ratio, which potentially associated with poly(A) length (PAL) and alternative polyadenylation (APA). Finally, the morphology measurement and metabonomics analysis revealed the difference of 14 cultivars. In summary, the genomes and epigenetics datasets provide a molecular basis for callus formation in the gymnosperm family., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

The lymph node is the most common site of distant metastasis of cervical squamous cell carcinoma (CSCC), which elicits dismal prognosis and limited efficiency for treatment. Elucidation of the mechanisms underlying CSCC lymphatic metastasis would provide potential therapeutic strategies for nodal metastatic of CSCC. Here, based on in vivo lymphatic metastasis screening model, a circular RNA is identified that is termed as lymph node metastasis associated circRNA (LNMAC), is markedly upregulated in lymphatic metastatic CSCC and correlated with lymph node metastasis. Overexpression of LNMAC dramatically augments the metastatic capability of CSCC cells to the lymph node via inducing lymphangiogenesis. Mechanistically, LNMAC epigenetically upregulates fibroblast growth factor 2 (FGF2) expression by directly associating with histone acacetylase 1 (HDAC1), preventing Importin α6/8-mediated nuclear translocation of HDAC1 and eliciting histone H3K27ac-induced FGF2 transcriptional activation. Treatment with 3F12E7, an anti-FGF2 monoclonal antibody, effectively inhibits LNMAC-induced CSCC lymphatic metastasis. Taken together, these findings indicate that LNMAC plays a crucial role in FGF2-mediated lymphangiogenesis and lymphatic metastasis, highlighting that LNMAC might be a therapeutic target for lymph node metastasis in CSCC patients., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

In forensic investigations, semen samples are a common form of biological evidence, especially in cases involving sexual assault. Therefore, accurately estimating the age of an individual is crucial in criminal cases. This study presents a novel age estimation model based on semen-specific CpG methylation patterns. A multiplex panel was developed, consisting of 12 CpG sites (PARP14, C5orf25, cg23488376, MXRA5, PFKFB3, DLL1, NOX4, cg12837463, TTC7B, KCNA7, NKX2-1, and SYNE4), which exhibit strong correlations with age. Additionally, this study investigates the resilience of these methylation markers under simulated environmental challenges. We collected ejaculate samples from a diverse cohort of 115 male individuals, aged 20-71 years, who underwent deoxyribonucleic acid extraction and bisulfite conversion. Methylation levels of the selected CpG sites were assessed using a SNaPshot assay, which revealed significant correlations with chronological age. We developed and validated two robust age estimation models through stepwise and enter regression analyses, achieving reliable accuracy with mean absolute errors ranging from 3.81 to 4.1 years. Additionally, the study also investigated the robustness of semen stains under diverse environmental conditions, including fabric type, washing, hematin exposure, and UV-C light. The selected methylation markers demonstrated remarkable resilience despite the challenges posed by washing procedures and environmental exposure, confirming their potential for age estimation in forensic genetics. This research presents successful age estimation models, emphasizing the strong correlations between methylation levels and chronological age. The proposed methodology's accuracy is affirmed through model validation on an independent test set, while also highlighting the resilience of semen stains on fabrics under varying storage and washing conditions., (© 2024 The Author(s). ELECTROPHORESIS published by Wiley‐VCH GmbH.)

Epigenetic regulation of metabolism profoundly influences cell fate commitment. During osteoclast differentiation, the activation of RANK signaling is accompanied by metabolic reprogramming, but the epigenetic mechanisms by which RANK signaling induces this reprogramming remain elusive. By transcriptional sequence and ATAC analysis, this study identifies that activation of RANK signaling upregulates PRMT6 by epigenetic modification, triggering a metabolic switching from fatty acids oxidation toward glycolysis. Conversely, Prmt6 deficiency reverses this shift, markedly reducing HIF-1α-mediated glycolysis and enhancing fatty acid oxidation. Consequently, PRMT6 deficiency or inhibitor impedes osteoclast differentiation and alleviates bone loss in ovariectomized (OVX) mice. At the molecular level, Prmt6 deficiency reduces asymmetric dimethylation of H3R2 at the promoters of genes including Ppard, Acox3, and Cpt1a, enhancing genomic accessibility for fatty acid oxidation. PRMT6 thus emerges as a metabolic checkpoint, mediating metabolic switch from fatty acid oxidation to glycolysis, thereby supporting osteoclastogenesis. Unveiling PRMT6's critical role in epigenetically orchestrating metabolic shifts in osteoclastogenesis offers a promising target for anti-resorptive therapy., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)