Epigenetics

Sirtuin 6 (SIRT6) is an important regulator of DNA repair, metabolism, chromatin maintenance and longevity. SIRT6 Serine 10 phosphorylation controls SIRT6 recruitment to the sites of DNA damage. To explore the effect of SIRT6 Serine 10 phosphorylation on lifespan, we generated two SIRT6 mutant mouse strains: phospho-null S10A and phosphomimetic S10E. The S10E mutant mice demonstrated enhanced DNA repair capacity, elevated LINE1 expression and reduced lifespan in male mice compared to the wild-type and S10A mice. This result suggests that SIRT6 S10E mutation enhances DNA repair capacity at the expense of reduced LINE1 silencing leading to shorter lifespan. While both SIRT6 functions in DNA repair and chromatin maintenance are important for longevity, our results suggest that when the balance between these functions is shifted, diminished of LINE1 control has a stronger impact on lifespan than enhanced DNA repair.

Very preterm infants (<30 weeks gestation) are at elevated risk for neurodevelopmental and social-behavioral challenges. DNA methylation (DNAm) may provide a biological link between preterm birth and later behavioral outcomes. We examined associations between DNAm profiles at neonatal intensive care unit (NICU) discharge and at age 5 with Social Responsiveness Scale (SRS) scores which measure social communication, social interaction, and repetitive behaviors at age 5, including sex-specific effects, in the Neonatal Neurobehavior and Outcomes in Very Preterm Infants (NOVI) Study. Epigenome-wide buccal DNAm was profiled at NICU discharge (n=218) and at 5 years (n=188). We identified 38 neonatal and 6 age-5 CpG sites associated with SRS scores (all q<0.05) using epigenome-wide association studies (EWAS) at each time point. Several CpGs mapped to genes involved in neurodevelopment including TCF4, KLC4, CAP2, PTDSS1, ADAM12, SENP1, CHN2, SH3D19, and ITGA1, with sex-specific effects observed for CpGs in CAMTA1 and GABBR1. Enriched pathways included neurodevelopment, cytoskeletal regulation, stress-response, and metabolic processes. DNAm patterns during early life, particularly the neonatal period, were associated with social-behavioral development in very preterm children. Findings in key genes such as TCF4 and CAMTA1 highlight potential epigenetic mechanisms linking early-life biology to later behavioral challenges.

Epigenetics may play a crucial role in livestock adaptation to environmental challenges like heat stress. In recent years, a growing number of studies have investigated the epigenetic mechanisms underlying dairy cow adaptation to heat stress. However, there is still limited knowledge about the effects of heat stress on immune cells and immune-related phenotypes. Herein we aim to identify heat-stress induced DNA methylation variations on blood methylome potentially affecting regulatory regions and associated phenotypes. Blood samples were collected and peripheral blood mononuclear cell (PBMC) isolated from four cows before (D0) and after (D14) a 14-d heat stress challenge (cyclical THI 72-82) and, from four cows kept in thermoneutral conditions (THI 61-64). Heat-stressed cows had ad libitum access to diets supplemented with adequate levels of vitamin D and Ca (12,000 IU/kg of vitamin D and 0.73% Ca, respectively). To eliminate confounding effects due to differences in nutrient intake, cows maintained under thermoneutral conditions were pair-fed (PF) to their heat-stressed counterparts and received adequate concentrations of vitamin D and Ca as well. Reduced representation bisulphite sequencing (RRBS) was used to profile PBMCs methylome. Differential methylation analysis was performed using methylKit and DSS softwares ({Delta}meth [&ge;] 25%, adjusted p-value < 0.01), retaining only commonly detected differentially methylated cytosines (DMCs). A total of 2,908 DMCs were identified when comparing pre- and post-heat stress samples. After excluding 649 DMCs that were also detected under thermoneutral conditions, as these changes were likely associated with feed restriction inherent to the pair-feeding design rather than with heat stress per se, 2,259 heat stress-specific DMCs remained, predominantly hypomethylated. About half of the DMCs are annotated in intronic and intergenic regions; known to harbor regulatory elements. By intersecting the DMRs with publicly available functional annotation data, we observed hypomethylation on regulatory regions putatively affecting cows immune system. As an example, we identified a loss of methylation within an enhancer region of the MSN gene, which is involved in lymphocyte homeostasis, and a loss of methylation in the promoter region of MECP2, a well-established epigenetic regulator with a central role in chromatin organization and gene expression. These findings highlight the impact of heat stress on dairy cow immunity and provide new insights into its epigenetic regulation under environmental stress. Interpretative summaryThis study examined DNA methylation changes induced by heat stress in dairy cows to elucidate epigenetic mechanisms of thermal adaptation. Using RRBS on PBMCs, 2,259 heat stress-specific differentially methylated cytosines were identified, predominantly hypomethylated and enriched in regulatory regions. Functional annotation highlighted immune-related pathways, including hypomethylated regulatory regions near genes (e.g., MSN, ZBTB33, SLC25A5, GNAS, FAM3A, and MECP2) associated with immune function. These findings indicate that heat stress induces targeted epigenetic modifications potentially affecting immune regulation in dairy cows.

BACKGROUNDDNA methylation is the most prevalent epigenetic modification in human cells and undergoes dynamic changes during cell differentiation, disease progression, and aging. Here, we introduce Locus-Enriched Mapping Of Nucleotide methylation (LEMONmethyl-seq): an optimized, cost-effective pipeline for single-nucleotide detection of DNA methylation using locus-specific amplification and long-read DNA sequencing. RESULTSWe apply LEMONmethyl-seq to profile DNA methylation of endogenous gene promoters across different cell types along with DNA methylation establishment and long-range propagation induced by CRISPR epigenome editing technologies. We profile dynamic changes in DNA methylation patterns on transposable element genomic loci during global epigenetic resetting in stem cells, and we identify site-specific enrichment of non-canonical CpH methylation on genomic sites in stem cells and cultured neurons. Lastly, we apply LEMONmethyl-seq to profile DNA methylation across the MGMT promoter, a clinical biomarker for glioblastoma. We identify additional differentially methylated sites correlated with chemotherapeutic sensitivity, which may be clinically relevant. CONCLUSIONSTogether, LEMONmethyl-seq serves as a cost-effective, long-read DNA methylation sequencing pipeline that advances methods for detecting DNA methylation patterns and dynamics in mammalian cells. We envision its broad use for studying chromatin pathways, diagnostics, and therapeutic applications.

Prenatal alcohol exposure (PAE) can lead to a range of deficits falling under the umbrella of Fetal Alcohol Spectrum Disorder (FASD), which included higher risk for adverse neurodevelopmental and mental health outcomes. Although the biological mechanisms underlying the link between PAE and mental health remain unclear, DNA methylation (DNAm), an epigenetic modification responsive to environmental exposures, may explain these relationships. Here, we applied a two-sample Mendelian randomization (MR) framework to assess whether DNAm loci previously associated with PAE or FASD are linked to 11 psychiatric outcomes. Using summary statistics from the Genetics of DNA Methylation Consortium (GoDMC) mQTL database and large-scale GWAS, we analyzed DNAm loci from two epigenome-wide association studies: one examining FASD by Lussier et al. (2018) and one examining PAE patterns by Sharp et al. (2018). A total of 106 associations (Lussier) and 28 associations (Sharp) reached nominal significance (p<0.05) and passed sensitivity tests, with several surviving multiple testing correction. Notably, schizophrenia and bipolar disorder had the highest number of associated loci across both studies. Functional analysis showed that DNAm loci were enriched in signaling pathways, embryonic development, and neuron differentiation. Regional enrichment analysis revealed that FASD-related loci were more likely to occur in enhancer and south shore, implicating distal regulatory elements. PAE patterns conferred heterogeneous effects on DNAm and mental health risk, underscoring the complexity of timing-specific epigenetic vulnerability. These findings offer novel insights into the potential mechanism of DNAm linking PAE to mental health, and demonstrate the utility of MR in epigenetic epidemiology.

BackgroundEpigenetic clocks based on DNA methylation (DNAm) are widely used indicators of biological aging; however, most established models have been developed using EPIC arrays and non-Japanese populations. The Methylation Screening Array (MSA), a cost-efficient platform with reduced CpG content, has not been evaluated for its capacity to support biological age estimation and biomarker prediction in Japanese cohorts. MethodsDNAm profiles and clinical laboratory measurements were obtained from 166 Japanese participants for model development; an independent cohort of 48 individuals processed at a separate institute was used for validation. A linear regression model was trained using the Elastic Net method to predict phenotypic age from MSA-derived methylation data, and a two-stage modeling (residual learning) framework integrating EPIC-based clock predictions with MSA-specific residual predictions was evaluated. Additional models were constructed to examine the predictability of 59 clinical biomarkers and their log-transformed variants, including sex-stratified analyses. ResultsThe MSA-based model accurately predicted phenotypic age in the validation dataset; prediction performance improved when the EPIC-based estimates were incorporated through the residual learning framework. Several clinical biomarkers, particularly those related to leukocyte composition and sex hormone regulation, were also predicted from the MSA data, although some markers were strongly affected by sex. Some of the nine constituent phenotypic age biomarkers were not individually predicted. ConclusionsMSA methylation profiles contain sufficient biological information for reliable prediction of epigenetic aging markers in Japanese individuals. These findings demonstrate the feasibility of applying cost-efficient MSA-based DNAm profiling for biological age prediction and provide a methodological foundation for expanding epigenetic biomarker applications in Japan.

Epigenetic mechanisms regulate gene-expression by altering the structure of the chromatin without modifying the underlying DNA sequence. Histone post-translational modifications (PTMs) are critical epigenetic signals that influence transcriptional activity, promoting or repressing gene-expression.Understanding the impact of individual PTMs and the combinatorial effects is essential to deciphering gene regulatory mechanisms.In this study,we analyzed the ChIP-seq data for 26 PTMs in yeast, examining the PTM intensities gene-wise from positions-3 to 8 in each gene.Using XGBoost classifiers, we predicted gene transcription rates and identified key histone modifications and nucleosomal positions that are critical in gene-expression using explainability measures (such as SHAP). Our study provides a comprehensive insight into the histone modifications, their positions and their combinations that are most critical in gene regulation in yeast.The proposed explainable Machine Learning models can be easily extended to other model organisms to provide meaningful insights into gene regulation by epigenetic mechanisms.

DNA methylation is a key epigenetic mechanism influencing gene regulation and cellular identity. In skeletal muscle, methylation contributes to fiber-type specification, metabolic programming, and satellite cell function, with evidence of sex-specific differences. Here, we investigated whether spatial regionalization of gene expression along the proximal-distal axis of the tibialis anterior (TA) is mirrored by corresponding patterns of DNA methylation. Using MeDseq on TA sections from muscles previously analyzed by spatial transcriptomics, we profiled methylation across transcriptional start sites (TSS), gene bodies, and regulatory elements. Despite robust spatial differences in transcriptomes, methylation patterns were largely uniform along the proximal-distal axis, indicating that DNA methylation does not underlie regional gene expression in adult TA muscle. In contrast, sex emerged as the primary determinant of methylation variation. Male muscles exhibited widespread hypermethylation at TSS, gene-bodies and regulatory regions, corresponding with sex-specific transcriptional programs, including glycolytic fiber enrichment in males and oxidative fiber markers in females. Notably, chromatin- and methylation-associated regulators such as Setd7, Gsk3a, and Bmyc were upregulated in males, suggesting mechanisms linking transcriptional control to epigenetic state. These findings highlight that while spatial gene expression is transcriptionally driven, sex-specific epigenetic programs dominate adult skeletal muscle, underscoring the need to consider sex in multi-omic studies of muscle biology.

IntroductionEarly-life adversity (ELA) encompasses a range of environmental stressors, including physical, emotional, and social challenges that can affect health during the critical early developmental period. Extensive research has linked ELA to negative long-term health outcomes, yet the underlying biological mechanisms remain poorly understood. The current study investigates how early institutional care changes the epigenetic landscape in young adults. The study also provides insights into role of DNA methylation as a potential mediator for disease susceptibility and altered health trajectories. Materials and MethodsDNA was extracted from blood samples obtained from 111 individuals (71 Controls; 40 ELA) who were part of the EpiPath cohort. DNA methylation was measured using the Infinium Methylation EPIC v2.0 BeadChip. Results3,785 differentially methylated CpG loci were identified in the ELA group in comparison with the control group (FDR <0.05). Pathway enrichment analysis highlighted biological processes involved in metabolic regulation, stress response, and neurodevelopment, with novel pathways such as GTPase-mediated signalling, efferocytosis and glucuronosyltransferase emerging as potential drivers of the ELA phenotype. A subset of 28 CpG loci was used to develop an epigenetic signature, which showed a significant association with the development of chronic diseases in ELA-exposed individuals. ConclusionThis study reinforces Barkers concept of sensitive periods and it underscores the enduring impact of ELA in shaping long-term health outcomes. The persistence of DNA methylation patterns decades after exposure to ELA, and their clear association with the resultant phenotype confirms that stable epigenetic imprints play a potential role in long-term disease risk and resilience.

BackgroundStressful life events (SLEs) across the life course have been associated with cognitive decline, but evidence on their cumulative impact and potential modifiers remains limited. We aimed to examine the associations between SLE exposure in childhood, adulthood, or both life stages and cognitive trajectories, and to investigate whether these associations vary by sex and level of education. MethodsWe used data from the China Health and Retirement Longitudinal Study (CHARLS), a nationally representative cohort of adults aged 45 years and older. Participants with complete data on SLEs, cognitive function, and covariates were included (n=5,922). SLEs were assessed retrospectively for childhood and adulthood periods. Cognitive function was measured using a composite score (range 0-21) across three waves (2011-2015). Linear mixed-effects models were used to examine longitudinal associations, adjusting for sociodemographic factors, health behaviors, and chronic health conditions. Interaction analyses were performed to explore effect modification by sex and education. ResultsOf 5,922 participants, 2,428 (41.0%) reported no SLEs, 1,374 (23.2%) experienced childhood-only SLEs, 1,294 (21.8%) experienced adulthood-only SLEs, and 826 (14.0%) had cumulative SLE exposure (both childhood and adulthood). In fully adjusted longitudinal models, cumulative SLE exposure showed the strongest association with cognitive decline ({beta} = -0.52, 95% CI -0.69 to -0.35, p<0.001), followed by childhood-only ({beta} = -0.34, -0.48 to -0.20, p<0.001) and adulthood-only exposure ({beta} = -0.22, -0.37 to -0.07, p<0.01). Sex significantly moderated the associations for childhood-only and cumulative exposure (interaction {beta} = -0.21, 95% CI -0.36 to -0.05, p<0.01; interaction {beta} = -0.25, 95% CI -0.43 to -0.07, p<0.01), with women showing greater cognitive vulnerability than men. Higher educational attainment buffered against single-period stress effects but provided only partial protection against cumulative life-course adversity. ConclusionLife-course stress exposure, particularly when cumulative, is associated with accelerated cognitive decline in Chinese middle-aged and older adults. Women show greater vulnerability to stress-related cognitive effects, whereas higher education appears to buffer against such impacts across both sexes. These findings underscore the importance of sex-sensitive, education-informed approaches to stress reduction and cognitive health promotion across the life course.

The causal relationship between smoking and colorectal cancer (CRC) remains unclear. In this study, a cigarette smoke-exposed mouse model demonstrated that smoking significantly increased CRC incidence by inducing gut microbiota dysbiosis and altering related metabolites. Smoke exposure reduced beneficial bacteria (e.g., Lactobacillus), increased harmful bacteria (e.g., Firmicutes and Clostridium), elevated metabolites such as histamine, and suppressed the tumor suppressor genes PARG, CPT2, and ALDH1A1, thereby promoting tumor development. Functional assays in CRC cell lines further confirmed that CPT2 knockdown enhanced malignant phenotypes, including proliferation, migration, and invasion. Clinical analysis showed that these genes were markedly downregulated in smoking-related CRC patients, with strong diagnostic value (AUC > 0.8). ConclusionSmoking promotes CRC by inducing gut microbiota dysbiosis, metabolic reprogramming, and suppression of tumor suppressor genes, particularly CPT2. These findings highlight the importance of smoking cessation in CRC prevention and provide potential biomarkers for early diagnosis and therapeutic intervention.

BackgroundSmall-cell lung cancer (SCLC) represents 15% of lung cancers and with a 5-year survival rate under 7% remains one of the deadliest malignancies. Although initially responsive to chemotherapy, rapid recurrence and resistance are common. Epigenetic modifications, particularly DNA methylation, contribute to tumor progression and therapy resistance. Guadecitabine, a hypomethylating agent (HMA), has shown promising clinical activity when combined with carboplatin in preclinical models. We evaluated the combination of guadecitabine with carboplatin as a second-line treatment for extensive-stage SCLC (NCT03913455). Here we report methylome changes in peripheral blood mononuclear cell (PBMCs) collected at baseline and during treatment from patients on the trial. ResultsPMBC DNA was analyzed using Infinium HumanMethylationEPIC v1.0 bead chips. Data were processed and differentially methylated positions (DMPs) were identified and analyzed for pathway enrichment using bioinformatic approaches and immune deconvolution analyses were conducted to investigate the impact on immune cell composition. Direct comparison of PBMCs between cycle 2 day 5 (C2D5; post-treatment) vs cycle 1 day 1 (C1D1; pre-treatment) revealed a greater number of hypomethylated DMPs (380 DMPs in C2D5 vs C1D1 PBMCs; p < 0.05, |{beta}| > 20%). Moreover, when first compared with normal PBMCs from cancer-free controls, the number of hypomethylated DMPs was even greater in C2D5 than in C1D1 (1,771 vs 237 DMPs, respectively; p < 0.05, |{beta}| > 20%). Long interspersed nucleotide elements-1 (LINE-1) were also significantly hypomethylated in PBMCs after HMA treatment (C2D5), compared to C1D1. Pathway analysis of hypomethylated DMPs revealed significant alterations in key signaling pathways including NF-{kappa}B, Rho GTPase, pulmonary fibrosis, and p75 NTR in C1D1 vs C2D5. When normal PBMCs were compared to C1D1 PBMCs, changes in IL-3 signaling, Fc{gamma} receptor-mediated phagocytosis, and molecular mechanisms of cancer were observed. Deconvolution analysis revealed a significantly higher percentage of monocytes in C1D1 PBMCs vs normal PBMCs. However, after HMA treatment, percentages of monocytes and B cells decreased, while eosinophil percentage increased in C1D1 compared to C2D5 PBMCs. ConclusionIn the first study on the global impact of HMA treatment on PBMC methylomes in SCLC patients, DNA methylation changes associated with biological pathways related to PBMC function reveal shifts in distinct immune cell populations. SummaryMethylome changes in peripheral blood mononuclear cell (PBMCs) from small cell lung cancer (SCLC) patients treated with an epigenetic therapy revealed global hypomethylation and altered cancer signaling processes associated with tumor progression, immune response, therapy resistance and significant change in the proportion of immune cells. Integrating blood-based methylation biomarkers into clinical trials of epigenetic therapy and methylomic analysis of PBMCs provides direct monitoring of treatment effects in cancer patients, which may improve patient selection and enable real-time response assessment in patients receiving hypomethylating agents.

BackgroundThe histone methyltransferase EZH2, enzymatic core of the trimeric polycomb repressive complex 2 (PRC2), has been shown to promote small cell lung cancer (SCLC) survival through epigenetic silencing of multiple targets including Class I MHC molecules (HLA-A/B) and DNA repair factors (SLFN11). Treatment of SCLC cells with EZH2 inhibitors in vitro can reactivate expression of these genes and result in therapeutic response to immune checkpoint inhibition (ICI) and chemotherapy. Here, we investigate the impact of EZH1/2 dual inhibition on 3D chromatin structure and its relationship to transcriptional regulation in neuroendocrine (NE) SCLC. ResultsEmploying Micro-C, a micrococcal nuclease-based 3D genome mapping technique, we show that EZH1/2 inhibition with Valemetostat induced significant changes at multiple genome organizational levels (compartment, topological associated domain, and chromatin loop) without incurring cell death in NE SCLC. Alterations in 3D genome permissive for transcriptional activation were correlated with increased chromatin accessibility (ATAC-sequencing) and expression of target genes (transcriptome profiling). Known transcription factor motif discovery revealed enrichment of non-NE motifs (e.g., REST) in regions with gained chromatin accessibility in Valemetostat-treated cells, consistent with results from gene set enrichment analysis demonstrating NE to non-neuroendocrine lineage shift. Notably, EZH1/2 inhibition reactivated Class I MHC expression by facilitating enhancer-promoter looping. ConclusionOur results demonstrate that repression of a subset of EZH2 targets including Class I MHC genes is affected through modulation of 3D genome structure to the level of chromatin looping and further support clinical investigation of EZH2 inhibition in boosting therapeutic efficacy of ICI in SCLC patients.

BackgroundPost-stroke cognitive impairment (PSCI) affects nearly 30% of stroke survivors and significantly impairs functional recovery. Brain-derived neurotrophic factor (BDNF)-tropomyosin receptor kinase-{beta} (Trk{beta}) signalling is crucial for synaptic plasticity and cognitive function. While altered expression of truncated TRK{beta}-T1 isoforms has been linked to stroke, the contribution of the TRK{beta}-SHC isoform to PSCI in humans remains poorly understood. ObjectivesThis study aimed to (i) assess isoform-specific expression changes of NTRK2 associated with PSCI, (ii) evaluate the role of an isoform-specific genetic variant in disease susceptibility, and (iii) identify DNA methylation changes regulating NTRK2 expression (if any). MethodsGene expression levels of three major NTRK2 isoforms and MEK2 were analyzed in peripheral blood mononuclear cells from 19 PSCI patients, 21 post-stroke cognitively normal (PSCN) individuals, and 11 healthy controls. Expression data were correlated with raw memory scores and MEK2 expression. DNA methylation profiles of NTRK2 and its transcriptional regulators were assessed using whole-genome bisulfite sequencing. ResultsTRK{beta}-FL expression was significantly reduced in stroke patients compared with controls. In contrast, TRK{beta}-SHC expression was elevated in PSCN individuals relative to PSCI cases and showed a positive correlation with MEK2 expression and memory performance. No significant association was observed between rs65339833 and cognitive subdomains. Gene body hypermethylation, but not promoter methylation, was detected in NTRK2 and its regulatory genes. ConclusionsElevated TRK{beta}-SHC expression may contribute to preserved cognitive function following stroke. DNA methylation status of NTRK2 may regulate alternative splicing and thus represent a novel therapeutic avenue for preventing or mitigating PSCI.

Yin Yang 1 (YY1) is a multifunctional transcription factor and mammalian Polycomb Group (PcG) protein critical for lymphocyte development. While YY1 is essential for early T-cell development and survival, the underlying epigenetic mechanisms by which YY1 regulates early T-cell development are not fully understood. Herein, we utilized the YY1 PcG function conditional knockout mouse model (Yy1-/{Delta}REPO) by CRISPR/Cas9 to further dissect the underlying mechanisms. Yy1-/{Delta}REPO mice show early T cell development blockage at the double-negative (DN) 3 to single positive T cell transition with expansion of the DN3 population. Yy1-/{Delta}REPO DN3 cells are highly proliferative, but more prone to apoptosis, leading to reduced single positive T cells output. The genetic network governing T cell differentiation is deregulated in Yy1-/{Delta}REPO DN3 T cells. The YY1 REPO deletion leads to downregulation of DNA demethylase enzyme Tet1 and Tet2 expressions in DN3 cells with no change of Tet3. Pharmacologic inhibition of TET catalytic activity blocked DN-to-DP progression at the DN3 stage, whereas re-expression of TET2 catalytic domain in Yy1-/{Delta}REPO DN thymocytes partially rescued T cell differentiation. Together, our study demonstrates that YY1-mediated PcG function is essential for the DN3 to SP T cell transition and YY1-TET2 axis promotes proper DN3 differentiation.

Background Prenatal exposure to pesticides and psychosocial factors often co-occurs, particularly in low- and middle-income settings, yet their joint effects on epigenetic age acceleration (EAA) in early life remain unknown. We investigated the joint associations of prenatal pesticides metabolites and psychosocial factors on EAA in the first five years of life in the South African Drakenstein Child Health Study. Methods In 643 mothers, we measured 11 urinary pesticide metabolites and seven psychosocial factors during the second trimester of pregnancy. Child DNA methylation was measured in whole blood at ages 1, 3, and 5 years. EAA was estimated using the Horvath, Skin & Blood Horvath (skinHorvath), and Wu epigenetic clocks. Longitudinal associations were estimated using generalized estimating equations, adjusted for confounders. Joint mixture associations were evaluated using weighted quantile sum regression (WQS) and quantile g-computation (QGCOMP). Results The joint prenatal exposure mixture was positively associated with Wu ({beta} per one quintile increase in the mixture [95% CI]: 0.41 years [0.15, 0.80]), skinHorvath (0.11 years [0.06, 0.16]), and Horvath EAA (0.31 years [0.20, 0.46]) over time using WQS. Psychosocial factors, particularly food insecurity, physical interpersonal violence, and stress biomarkers, contributed most to the total mixture effect for all clocks. Pyrethroid metabolites PBA and TDCCA were top pesticide contributors to Wu EAA. Pathway enrichment analyses of clock-specific CpGs revealed distinct biological architectures, with the Wu clock enriched for neurodevelopmental and immune pathways, and metabolic pathways for the Horvath clock. Discussion Joint prenatal exposure to pesticides and psychosocial factors was associated with increased EAA across early childhood, with psychosocial factors contributing the most to the total effect. These findings highlight the importance of assessing chemical and non-chemical stressors jointly and clock-specific biological interpretation in epigenetic aging research.

Child maltreatment is a leading cause of pediatric morbidity and mortality, potentially propagated by DNA methylation (DNAm) changes. We conducted an EWAS meta-analysis (n=175, 554,979 Illumina EPICv1/EPICv2 sites) in buccal swabs from three hospital-based studies of children with traumatic injuries, stratified by study group to include 1) any traumatic injury, 2) fractures, and 3) traumatic brain injuries. Empirical bayes-moderated linear models tested differential DNAm with M-values, followed by near-promoter gene set enrichment analysis. Abuse was associated with methylation at 11 sites (q<0.10), including enhancers of neuroblast differentiation-associated AHNAK, immunomodulators SCGB1A1 and CCL26, exon 5 of LAMP1, essential for lysosomal function and cytotoxicity, and RGS7, a GTPase essential for synaptic transmission. Enriched biological processes included cranial skeletal system and connective tissue development, neural structure and function, immune regulation, gene expression, and metabolism. Our findings suggest that early abuse may epigenetically affect both proximal injury responses and longer-lived systemic biological dysregulation.

In many eukaryotic species, DNA is methylated at the 5 position of cytosine to form 5mC, predominantly within CG dinucleotides. Despite being conserved since the dawn of eukaryotic life, 5mC is often lost from individual lineages, suggesting that it may have detrimental effects. One such effect is genotoxicity, through the effect of 5mC on the process of cytosine deamination and its repair. Additionally, enzymes that introduce 5mC (DNA methyltransferases, DNMTs) can also damage DNA through alkylation and oxidative stress, but how these genotoxic effects combine to influence mutagenesis is unclear. To investigate how mutagenesis changes upon methylation of CG dinucleotides we introduced high levels of CG methylation into the bacteria E. coli. 5mC induction increased mutation at CG dinucleotides consistent with increased C to T mutations. We also discovered that 5mC induction led to increased mutations at AT base pairs, specifically in the absence of the alkylation repair enzyme AlkB. This effect was specific to certain E. coli strains and was not dependent on the DNA repair enzyme RecA, so its exact mechanism remains unclear. Together, our work highlights multiple mutagenic consequences of DNMT expression, which might act as selective pressures for organisms to lose 5mC across evolution.

BackgroundGene expression changes in response to developmental and environmental cues rely on cis-regulatory sequence elements (cREs). BRG1/BRM-Associated Factors (BAF) chromatin remodeling complexes maintain chromatin accessibility at many cREs, enabling binding by transcription factors (TFs). However, cREs exhibit a broad range of sensitivity to loss of BAF function, and the basis of this variability remains unknown. ResultsTo identify the characteristics of BAF-dependent cREs, we mapped chromatin accessibility changes following acute pharmacologic BAF inhibition in GM12878 lymphoblastoid cells. We integrated these results with over 100 TF and histone modification ChIP-seq datasets and used machine learning to identify features that predict chromatin accessibility changes. We found that Activator Protein 1 (AP-1) factors and lymphoid lineage-defining TFs including RUNX3 and PU.1 predicted BAF-dependence. Strikingly, we found that cREs bearing the chromatin signature of "primed" enhancers - enriched for H3K4me1 but lacking H3K27ac - were significantly more sensitive to BAF inhibition than typical active enhancers. As primed enhancers are known to facilitate transcriptional responses to stimuli, we tested the requirement of BAF activity in these responses. Acute BAF inhibition was sufficient to prevent both chromatin and transcriptional responses to interferon gamma and dexamethasone. cREs which normally gained accessibility in response to stimulation failed to do so with BAF inhibition, and these cREs were linked to genes with suppressed transcriptional induction. ConclusionsCollectively, our results demonstrate a requirement for continuous BAF activity to enable stimulus response and suggest that defective signal responsiveness may be a pathogenic mechanism in disease states caused by loss-of-function mutations in BAF subunits.

Early life adversity (ELA) has a well-established link to mental health disorders later in life, yet the molecular mechanisms behind this relationship are incompletely understood. The Future of Families Child Wellbeing Study (FFCWS) provides an opportunity to examine experience encoding in the genome through functional changes in DNA methylation (DNAm) in a cohort enriched in subjects exposed to ELA. We investigated epigenome-wide differences in DNAm across thirteen early-life exposures in salivary samples from FFCWS participants. We calculated differential methylation associations with disease-linked genetic variants, evaluated tissue-specific gene expression, and assessed the persistence of DNAm changes from ages 9 to 15 years. Using data from the mSTARR-seq assay, we characterized methylation-dependent regulatory activity. Differential methylation in the FFCWS validated prior results and identified new genomic regions associated with child adversity. Differential methylation occurs in genomic regions likely to impact gene expression, and affected genes are expressed in disease-relevant tissues. We also identified association of genetic variants associated with downstream disorders near differential methylation, including depression, alcohol and substance use, and anxiety disorders. Overall, cumulative ELA is associated with specific DNAm changes, functional regulation, and persistence over time. Our findings indicate that ELA-associated differential methylation in the FFCWS does not simply occur at random, but in genomic regions that are functional. Our results support the conclusion that altered DNAm represents a biological link between early life experience and later health outcomes.