Open research questions in Epigenetics and DNA Methylation

What the literature leaves open

• THOR methylation represents an emerging and increasingly recognized mechanism of TERT activation across cancers. Its consistent association with telomerase expression, particularly in TERT promoter mutation–negative tumors, positions it as a poten- tial unifying epigenetic biomarker. Beyond its biological relevance, THOR methylation may help refine molecular classification of cancers and provide new opportuni- ties for non-invasive diagnostics through liquid biopsy approaches. However, its clinical implementation will require further analytical validation, assay standardization, and prospective clinical studies. Future research should focus on clarifying the interplay between THOR methylation and other mechanisms of TERT activation, including TERTp mutations, copy-number alterations, structural variants, and alternative lengthening of telomeres (ALT), while evaluating its prognostic and predictive value in large, well-charac- terized clinical cohorts. Integrating THOR methylation with geno- mic, transcriptomic, and other emerging biomarkers may further define its role in precision oncology. Conflict of interest The author(s) declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

  THOR methylation as a pan-cancer mechanism of TERT activation: toward a clinically relevant epigenetic biomarker · 2026 · DOI

• Recent advances have clarified the role of epigenetic mechanisms in inflammatory airway diseases, showing that they mediate gene–environment interactions and influence Page 15 of 21 51 inflammation, tissue remodeling, and cellular transformation. This conceptual framework describes how epigenetic mechanisms, including DNA methylation, histone modifications, and ncRNAs regulate key molecular and cellular processes involved in chronic respiratory diseases such as asthma and COPD. Dysregulation of these processes contributes to chronic inflammation, immune imbalance, epithelial dysfunction, and disease progression. The dynamic and reversible nature of epigenetic mechanisms also provides opportunities for clinical applications, particularly through the development of non-invasive biomarkers detectable in airway samples, blood, or exhaled breath, useful for diagnosis, prognosis, and treatment monitoring. Today epigenetic therapies are increasingly being explored for lung diseases. Additionally, the use of miRNA mimics and antagomirs, which modulate post-transcriptional gene expression, holds potential for targeted treatments in conditions such as inflammation and fibrosis. More recently, have emerged as promising approaches to precisely reprogram disease-associated epigenetic marks, potentially offering improved specificity and reduced off-target effects compared with earlier techniques. Looking ahead, key research directions aim to translate epigenetic knowledge into clinical practice. A major focus is the integration of multi-omics approaches combining epigenomics, transcriptomics, proteomics, and metabolomics to better characterize gene–environment interaction networks driving disease, enabling the identification of novel biomarkers and therapeutic targets. Longitudinal cohort studies are also essential to clarify causal links between epigenetic changes and disease onset or progression, supporting the development of early diagnostic markers and effective interventions. Overall, personalized epigenetic medicine may improve the management of chronic respiratory diseases by tailoring therapies to each patient’s genetic and environmental profile.

  Impact of Epigenetic Modifications in Airway Diseases. Role of Inflammation, Environmental Factors and Aging · 2026 · DOI

• Circulating cell-free DNA (cfDNA) provides a minimally invasive substrate for monitoring systemic molecular changes; however, age-related LINE-1 methylation dynamics in cfDNA remain poorly characterized in non-human species.

  LINE-1 CpG methylation in canine blood and cortical DNA tracks epigenetic changes with age · 2026 · DOI

• S-adenosylmethionine (SAM), the universal methyl donor, is essential for these processes, yet how tumors sustain elevated SAM availability to support oncogenic transmethylation reactions remains poorly defined.

  NNMT Loss Drives Cancer Progression by enhancing SAM availability for mTORC1 Signaling and Chromatin Methylation · 2026 · DOI

• The incidence of early-onset colorectal cancer (CRC) has risen sharply in recent decades1, yet the biological basis underlying the distinct behavior of tumors arising in young versus aged tissues remains poorly understood.

  Aging restricts colorectal tumor growth by epigenetically silencing developmental gene programs · 2026 · DOI

• 1024, 1025 There remains a lack of evidence on how health care utilization changed during the pandemic for individuals living with Alzheimer’s and other dementias. Although the new drug treatments have been shown to slow cognitive decline, the aggregate impact on health care use and costs is not yet known. There is high interest in brain health programs despite limited research awareness, with cost a primary driver of program participation Despite low awareness of current clinical research on lifestyle programs and brain health, there is robust enthusiasm for such programs among Americans age 40 and older. Building a Coordinated Framework to Advance Brain Health Across the Lifespan This year’s Special Report reveals that Americans value brain health, feel motivated to protect it and want guidance they can trust but face gaps in knowledge, access and system-level frameworks to do so. 104 Americans age 40 and older worry about Alzheimer’s disease and dementia, but are slightly less convinced of the role of healthy lifestyle behaviors in lowering their risk Worry about Alzheimer’s disease and other dementia is widespread, yet many adults remain uncertain about whether adopting healthy lifestyle behaviors can reduce their risk of dementia.

  Structural Disadvantage in Adolescence and Biological Aging in Early Midlife · 2026 · DOI

• This finding has direct implications for the design and evaluation of DMR 640 detection frameworks, as it indicates that sensitivity to effect size is insufficient 641 for the reliable identification of biologically meaningful methylation changes 642 between groups.

  glmmDMR reveals replicate-level methylation variance as a major determinant of false-positive DMR detection · 2026 · DOI

• Sepsis survivors exhibit divergent clinical trajectories, including rapid recovery (RAP) or progression to chronic critical illness (CCI), yet how these outcomes are linked to epigenetic repression remains poorly defined.

  Divergent chromatin remodeling in post-sepsis MDSCs underlies MHC class II repression in CCI · 2026 · DOI

• Therefore, the extent to which GEN and VitC contribute to potentiating ROS-induced epigenome remodeling through their actions on DNMTs and TETs remains to be quantitatively determined.

  Mild oxidative stress and dietary epigenetic modulators direct DNA methylation remodeling toward stress-resilience pathways · 2026 · DOI

• Nonetheless, fur- ther studies are warranted to inspect whether KIT over- expression is also evident in other types of cancer (other than AMLs) which are responsive to LSD1i.

  KIT-dependent acute myeloid leukemias are responsive to LSD1 inhibition · 2026 · DOI

• Despite this urgent challenge, a systematic, model-agnostic framework for adapting existing epigenetic clocks to HTS-based cfDNA data remains lacking.

  A Robust and Integrated Framework for Cross-platform Adaptation of Epigenetic Clocks in Cell-free DNA Sequencing · 2026 · DOI

• Polycomb Repressive Complex 2 (PRC2) propagates H3K27me3 through EED-dependent allosteric activation, yet how cells modulate the magnitude of this positive-feedback response remains poorly understood.

  EZH2 Serine 21 Phosphorylation Restrains Compact-State PRC2 Activation and H3K27me3 Propagation · 2026 · DOI

• Since breast cancer MLL3 mutations are often truncating mutations that lead to protein degradation, whether the MLL3 tumor suppressor activity depends on its catalytic activity or non-catalytic chromatin adaptor function remains unclear.

  MLL3 adaptor function, not methyltransferase catalytic activity, is essential for breast tumor suppression · 2026 · DOI

• Future research should examine epigenetic changes associated with persisting symptoms in long COVID, investigate downstream effects of DNAm changes on other -omics, and consider longer follow-up periods to further elucidate the molecular mechanisms underlying SARS-CoV-2 induced epigenetic changes.

  DNAm landscape up to 4 months post SARS-CoV-2 infection: insights from four population-based cohorts · 2026 · DOI

• It is well known that EZH2-directed H3K27me3 modification is one of the main contributors to tumorigenesis; however, how it affects the DNA mismatch repair (MMR) system and the genesis of extrachromosomal circular DNA (eccDNA) remains unknown.

  EZH2 deficiency suppresses colorectal cancer progression by inhibiting the mismatch repair pathway and consequently reducing extrachromosomal circular DNA formation · 2026 · DOI

• The zinc finger transcription factor Kaiso recognizes methylated CpG dinucleotides at silenced promoters and imprinted loci, but how it engages methylated DNA within the nucleosome remains unclear.

  Kaiso reads methylated CpGs at nucleosome entry/exit and displaces the H3 tail · 2026 · DOI

• Research on DNMTs in the context of diabetes is still in its early stages. Most studies to date have primarily focused on documenting aberrant DNMT expression and subsequent changes in DNA methylation associated with various dia- betic complications. The mechanisms underlying dysregu- lated DNMT expression in diabetes are still not thoroughly investigated. Key questions remain unanswered: How does 1 3Molecular Biology Reports (2026) 53:802 802 Page 10 of 14 diabetes induce abnormal DNMTs expression? And how do DNMTs, in turn, further influence the initiation and progres- sion of diabetes and its complications? The precise mecha- nisms have not been fully elucidated. Moreover, given that alterations in DNMTs activity can affect genome-wide DNA methylation status, their impact on gene expression and diabetic complications is complex and highly context-dependent. The mechanisms likely vary across different tissues and types of diabetic complica- tions. Therefore, further investigation into the regulatory mechanisms of DNMTs in diabetes and its complications, along with elucidation of their associated signalling path- ways, may reveal novel molecular targets for therapeutic intervention. In addition to these mechanistic considerations, a grow- ing body of literature has begun to explore therapeutic strategies directly targeting DNMTs in diabetes and its complications. Pharmacological inhibitors of DNMTs, such as 5-azacytidine and decitabine, have been widely used in cancer therapy to reactivate silenced genes by reducing global DNA methylation [59]. Recent evidence suggests that these agents may also hold promise for diabetic com- plications. For example, DNMT inhibitors have been shown to ameliorate diabetic nephropathy by reducing extracellu- lar matrix accumulation and inflammatory gene expression, and to improve cardiac function in diabetic cardiomyopa- thy models by reversing hyperglycemia-induced aberrant methylation of protective genes [90, 91]. However, several challenges remain, including the lack of tissue specificity, potential off-target effects, and the risk of global hypo- methylation leading to genomic instability. Future efforts should focus on developing next-generation DNMT inhibi- tors with isoform selectivity or targeted delivery systems, as well as combining epigenetic therapy with conventional glucose-lowering treatments to achieve synergistic effects. Moreover, natural compounds and dietary factors that modulate DNMT activity (e.g., polyphenols, curcumin) rep- resent another promising avenue for safer, long-term epi- genetic interventions in diabetes. A particularly intriguing link concerns the role of vita- min B12 and one-carbon metabolism in regulating DNMT expression and activity. Vitamin B12 is an essential cofac- tor for methionine synthase, which converts homocysteine to methionine, the precursor of SAMe, the primary methyl donor for DNMT-catalyzed DNA methylation [13]. There- fore, vitamin B12 deficiency can limit SAMe availability, potentially impairing normal DNA methylation patterns. Paradoxically, in certain diabetic conditions, vitamin B12 deficiency has been associated with DNMT upregulation and regional DNA hypermethylation, suggesting a complex, tissue-specific adaptive response rather than a simple lin- ear relationship. Emerging evidence indicates that maternal vitamin B12 status influences offspring‘s risk of insulin resistance and adiposity, partly through epigenetic modifi- cations involving DNMTs [92]. In diabetic patients, metfor- min use is well known to lower vitamin B12 levels, raising the possibility that metformin-induced B12 deficiency may inadvertently contribute to aberrant DNMT activity and epigenetic dysregulation [93]. These observations open up critical new questions: Can vitamin B12 supplementation reverse DNMT-mediated hypermethylation in diabetic tis- sues? Does the combination of DNMT inhibitors with B12 optimization offer synergistic benefits? And what is the role of other one-carbon nutrients (folate, choline, betaine) in modulating DNMTs in diabetes?

  Role of the DNA methyltransferases in diabetes mellitus and Its complications · 2026 · DOI

• With the encouraging findings reported in this review, several limitations are acknowledged. The studies show considerable var- iation in the study designs, patient populations, sample types, and analytical methods. This heterogeneity precluded direct compari- sons and prevented formal meta–analysis. Many studies used retrospective or case–control designs, with small sample sizes, which increases the risk of selection bias and limits the robustness of the conclusions. Reproducibility was further limited by method- inconsistencies. Differences in sample handling, ological normalisation strategies, assay calibration, and the definition of diagnostic cut–off values decreased comparability across studies. Additionally, most studies evaluated biomarkers at a single time point, with few comparing changes over time or across disease stages. Importantly, the lack of large, prospective, blinded validation studies remains a major obstacle to clinical adoption.

  Liquid biopsy–based epigenetic signatures for early detection of prostate cancer: a systematic review · 2026 · DOI

• Although we identified DMGs as a result of variations in the timing of hypoxic stress, our results may be con- founded by the use of whole-body tissue samples. Many methylation patterns are specific to the tissue type and therefore are potentially obscured in our analyses. Addi- tionally, the use of methylRAD-sequencing in our study resulted in lower coverage than other standard methyla- tion sequencing methods, such as bisulfite sequencing. While this method allowed us to identify differences across treatments, we likely are not capturing the entirety of the methylome. However, the primary goal of this study was to characterize temporal patterns of DNA methylation in response to hypoxia, rather than detecting all possible methylation occurrences. Because Methyl- RAD targets the same restriction sites across all samples, we are confident that the differences we observe reflect biological changes due to treatments and timepoints, however the constraints of this method means there are likely other epigenetic effects not captured in this study. Lastly, the functional role of methylation in most inver- tebrates, especially oysters, remains unclear. While we observed differences in methylation between treatments, we can only speculate how methylation may influence gene regulation. In the future, studies would benefit from analyzing methylation and gene expression concur- rently in tissue-specific contexts to better understand this relationship.

  Prior exposure to hypoxia alters DNA methylation patterns in the eastern oyster · 2026 · DOI

• This review highlighted the intimate and bidirectional relationship between mitochondrial function and the epigenetic landscape, with a particular focus on oogenesis and early embryogenesis. The reciprocal coupling of mitochondrial metabolism and chromatin-modifying processes emerges as a central determinant of developmental competence, influencing transcriptional reprogramming, chromatin architecture, and metabolic adaptation during critical windows such as oocyte maturation, ZGA, and lineage specification [5, 93, 102]. Accumulating evidence that mitochondrial metabolites and mtDNA methylation can shape nuclear epigenetic states, and vice versa, suggests that mitochondrial integrity is fundamental for the successful execution of early developmental programs. At the same time, the susceptibility of mtDNA to oxidative damage, its distinctive replication dynamics, and the apparent bimodal distribution of mtDNA methylation raise the possibility that functionally distinct mitochondrial subpopulations are selectively preserved for transmission to the next generation [86, 103–105]. Perturbations in mtDNA copy number or mutation burden can trigger metabolic rewiring and epigenetic derangements with lasting effects on gene expression and organismal metabolism, as illustrated by links between embryonic mtDNA depletion, PPARα hypermethylation, and altered lipid homeostasis in adulthood [8, 57, 106]. Collectively, these findings point to a coordinated mitochondrial–nuclear epigenetic axis that is particularly critical during the maternal-to-embryo transition and has direct implications for reproductive aging and maternally inherited mitochondrial disease. Conceptually, the emerging view that mitochondrial genomes may sense environmental and metabolic cues and relay this information intergenerationally via epigenetic mechanisms reshapes our understanding of reproductive biology. Such a framework provides a plausible basis for how perturbations during gametogenesis or early embryogenesis could be stably encoded in mitochondrial and nuclear epigenomes and later manifest as altered disease susceptibility. These insights also underscore the need to maintain mitochondrial integrity during gamete and embryo handling, including assisted reproductive technologies, as an avenue to improve reproductive outcomes and limit the transmission of mitochondrial defects. Taken together, current evidence supports several broadly shared features of mito–epigenome crosstalk across mammalian systems.

  Mitochondria and the epigenome: maternal co-inheritance and crosstalk from oocyte to embryo · 2026 · DOI

• Epigenetic alterations play central roles in chemoresistance by regulating gene expression, signaling pathways, and CSC properties, thereby significantly affecting tumor drug sensitivity. Epigenetic drugs targeting these mechanisms, including DNMTi, HDACi, and emerging BET protein or RNA-modifying enzyme inhibitors, have shown potential to reverse resistance and enhance chemotherapy efficacy in preclinical studies and early clinical trials (Suraweera et al., 2025; Sun et al., 2023). Notably, combination strategies with conventional chemotherapeutics can intervene at multiple resistance levels, providing new avenues to overcome tumor chemoresistance. Frontiers in Cell and Developmental Biology 06 frontiersin.org Jiang et al.

  Epigenetic regulation in cancer chemoresistance and combined therapeutic strategies · 2026 · DOI

• The comparative functional redundancy and specificity among JmjC family members in regulating DNA damage responses and mutagenesis has not been thoroughly characterized. Selective inhibition or genetic ablation studies comparing multiple JmjC demethylases in parallel under defined DNA damage conditions would clarify their individual and compensatory roles in genomic stability.

  JmjC Histone Demethylases: Beyond Histone Lysine Demethylation · 2026 · DOI

• The role of 2-oxoglutarate-dependent oxygenase activity in JmjC demethylase-mediated regulation of non-epigenetic pathways has not been systematically investigated. Structure-activity relationships for small-molecule inhibitors targeting the 2-oxoglutarate binding pocket across different JmjC family members, and their effects on both histone and non-histone substrate demethylation, require comprehensive kinetic and cellular validation studies.

  JmjC Histone Demethylases: Beyond Histone Lysine Demethylation · 2026 · DOI

• The structural basis for substrate recognition and catalytic selectivity among different JmjC demethylase family members remains underexplored. Crystal structures of JmjC enzymes in complex with their diverse non-histone substrates, particularly those containing modifications beyond lysine methylation, are sparse and would clarify the mechanistic determinants of isoform-specific activity.

  JmjC Histone Demethylases: Beyond Histone Lysine Demethylation · 2026 · DOI

• The substrate specificity of JmjC histone demethylases for non-histone substrates remains incompletely characterized. While evidence suggests these enzymes can demethylate proteins beyond histones, systematic substrate profiling of JmjC demethylases against the full cellular proteome and identification of specific lysine and arginine methylation sites targeted by individual family members is needed.

  JmjC Histone Demethylases: Beyond Histone Lysine Demethylation · 2026 · DOI

Most-cited papers in Epigenetics and DNA Methylation

• Role of Histone H3 Lysine 27 Methylation in Polycomb-Group Silencing · Science · 2002 · 3,260 citations
• Epigenetic Reprogramming in Mammalian Development · Science · 2001 · 2,488 citations
• The Nuclear DNA Base 5-Hydroxymethylcytosine Is Present in Purkinje Neurons and the Brain · Science · 2009 · 2,406 citations
• Environmental Influences on the Epigenome: Exposure- Associated DNA Methylation in Human Populations · Annual Review of Public Health · 2018 · 556 citations
• Small-Magnitude Effect Sizes in Epigenetic End Points are Important in Children’s Environmental Health Studies: The Children’s Environmental Health and Disease Prevention Research Center’s Epigenetics Working Group · Environmental Health Perspectives · 2017 · 256 citations
• Cancer epigenetics: from laboratory studies and clinical trials to precision medicine · Cell Death Discovery · 2024 · 239 citations
• H3K18 Lactylation Potentiates Immune Escape of Non–Small Cell Lung Cancer · Cancer Research · 2024 · 225 citations
• HBO1 catalyzes lysine lactylation and mediates histone H3K9la to regulate gene transcription · Nature Communications · 2024 · 207 citations
• Causality-enriched epigenetic age uncouples damage and adaptation · Nature Aging · 2024 · 176 citations
• Histone lactylation drives CD8+ T cell metabolism and function · Nature Immunology · 2024 · 158 citations

Most recent work

• A tumor-suppressive role of the PRC1 Polycomb epigenetic complex in the maintenance of adult Drosophila intestinal stem cell identity · bioRxiv · 2026
• JmjC Histone Demethylases: Beyond Histone Lysine Demethylation · CHIMIA International Journal for Chemistry · 2026
• Structural Disadvantage in Adolescence and Biological Aging in Early Midlife · JAMA Network Open · 2026
• The mitochondrial unfolded protein response in human microglia disrupts neuronal-glial communication and promotes senescence · bioRxiv · 2026
• Blood DNA Methylation Patterns Across Carotid, Coronary, and Peripheral Atherosclerosis · Journal of the American College of Cardiology · 2026
• DNA Methyltransferase Inhibitors in Triple-Negative Breast Cancer: Mechanisms, Limitations, and Therapeutic Potential · Journal of Research in Pharmacy · 2026
• DNA hypermethylation of nonspecific cytotoxic cell receptor protein 1 and poor prognosis of pancreatic cancer · Oncology Letters · 2026
• Sex-specific accelerated epigenetic aging in the Alzheimer's disease spectrum · Journal of Alzheimer’s Disease · 2026
• Mitochondria and the epigenome: maternal co-inheritance and crosstalk from oocyte to embryo · Cell Communication and Signaling · 2026
• Abstract 3213: Utility of TAPS+: a positive-readout methylation sequencing approach for high-fidelity epigenetic profiling · Cancer Research · 2026

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