Epigenetics

Family environments may contribute to childrens long-term health through biological processes, including epigenetic regulation such as DNA methylation (DNAm). However, most studies in this area focus on Euro-American populations while also rarely including fathering data. The current study investigated childrens blood DNAm associations with positive (father caregiving) and negative (parental conflict) family dynamics in a smaller-scale subsistence society living in the Congo Basin rainforest. We measured DNAm from dried blood spots of 54 children (mean age=8.48 years) and conducted three epigenome-wide association studies aimed at discovering differential co-methylated regions (CMRs) associated with family dynamics. Via path models, we investigated the health implications and shared contribution of family factors of the identified CMRs. Differential DNAm associated with family dynamics was localized to genes related to stress, immunology, development, and aging, thus possibly linking to childrens physical health and were simultaneously connected to other family factors such as number of siblings. Our findings suggested similarities in biological embedding of family factors across socio-ecologically diverse contexts.

The existence and functional relevance of mitochondrial DNA methylation remain controversial. Here, we systematically profiled cytosine methylation and hydroxymethylation across human brain and blood tissues spanning healthy and malignant states using orthogonal sequencing approaches that avoid chemical conversion during library preparation. While nuclear DNA exhibited canonical methylation patterns, mitochondrial DNA consistently showed negligible signal, indistinguishable from background technical noise. By mapping cytosine-guanine sites between mitochondrial DNA and nuclear-embedded mitochondrial sequences, we demonstrate the potential of these nuclear counterparts to confound not only cytosine methylation but also hydroxymethylation measurements, corroborating and extending prior findings implicating nuclear contamination as a potential source of apparent mitochondrial epigenetic signals. Additional technical factors that inflate apparent mtDNA methylation signals were identified, including sequence context biases, flow cell chemistries, and coverage-dependent discrepancies between the heavy and light strands. Collectively, these results provide convergent evidence against the presence of biologically meaningful cytosine methylation or hydroxymethylation in mitochondrial DNA. These findings caution against interpreting apparent mtDNA methylation signals in human adult tissues as meaningful without rigorous orthogonal validation and comprehensive consideration of technical and analytical confounding factors.

BackgroundDNA methylation can be profiled using multiple technologies that vary in resolution, coverage and cost. Yet systematic benchmarks across these methods remain scarce. MethodsWe compared six widely used technologies -- Illumina EPIC array, TWIST, Whole-Genome Enzymatic Conversion (WGEC), Reduced Representation Bisulfite Sequencing (RRBS), long-read genome sequencing (LR-GS) with Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT) -- using Genome in a Bottle (GIAB) reference samples and ten samples derived of blood and fibroblast cultures of 5 individuals. We assessed CpG coverage, consistency of differentially methylated cytosine (DMC) detection and genomic annotation, with particular attention to overlapping signals across assays. ResultsDespite major differences in assay design, all technologies consistently identified DMCs enriched in promoter and intronic regions, highlighting these loci as robust hotspots of epigenetic variability. Annotation redundancy strongly influenced initial interpretations, with CpG island-related categories largely disappearing once annotations were collapsed to unique features. Sequencing-based methods (WGEC, TWIST, ONT) achieved the most comprehensive coverage, whereas EPIC arrays reproducibly captured promoter-associated differences despite limited scope. ONT sequencing enabled direct, long-read-based methylation profiling with phasing capability and showed strong concordance with short-read sequencing methods after coverage filtering, but required higher and more uniform coverage to achieve reproducible CpG-level agreement. PacBio methylation profiles showed a coverage-dependent discrepancy, with cross-platform concordance plateauing in GIAB samples despite high mean coverage, indicating residual technology-specific biases beyond simple coverage effects. ConclusionsCross-platform benchmarking yields coherent biological insights when coverage and annotation redundancies are carefully addressed. Practically, EPIC arrays remain valuable for promoter-focused cohort studies, WGEC and TWIST enable genome-wide discovery and ONT provides unique phasing and multimodal potential. This comparative framework can guide method selection and support more robust interpretation of DNA methylation data across diverse platforms.

Proliferation of cytotrophoblasts (CTBs) and their differentiation into invasive extravillous trophoblasts (EVTs) are critical processes in early placental development. Defects in these processes are associated with adverse pregnancy outcomes, including recurrent pregnancy loss (RPL). There is evidence that reduced expression of the maintenance DNA methyltransferase DNMT1 in the placenta occurs in pregnancy loss and that RPL is associated with aberrant DNA methylation patterns. Therefore, we investigated the role of DNMT1 in human trophoblast growth and differentiation. Using human trophoblast stem cells (hTSCs), an in vitro analog to CTBs, we found that shRNA-mediated knockdown of DNMT1 led to decreased hTSC proliferation, genome-wide reductions in methylation, broad changes in gene expression, and impaired EVT differentiation. Transcriptome profiling of DNMT1-deficient hTSCs and hTSC-derived EVTs highlighted aberrant cytokine expression, drawing a connection to prior reports of immunological dysfunction in RPL. Finally, using a catalytic DNMT1 chemical inhibitor, we demonstrate the canonical methyltransferase activity of DNMT1 is essential for EVT differentiation and invasion. This study identifies new roles for DNMT1 in trophoblasts and addresses the molecular basis of the associations between DNMT1 expression, altered DNA methylation profiles, and RPL.

Tobacco smoking induces DNA methylation (DNAm) changes in blood and other tissues, which may influence chronic health outcomes. However, the breadth of smoking-related DNAm changes remains unmapped, offering a space for employing novel technologies. To expand our understanding of smoking impacts on DNAm, we conducted an epigenome-wide association study (EWAS) comparing ever smokers to never smokers, using blood from a multiethnic U.S. study population (n=887). We employed the newly developed Illumina Methylation Screening Array (MSA) covering 269,094 unique sites, including 123,776 CpGs not assayed in previous EWAS. Trans-ethnic meta-analysis identified 152 differentially methylated positions (DMPs) associated with ever-smoking status (n=764); European-specific analysis yielded 129 DMPs (n=674), including 106 overlapping with trans-ethnic analysis. A separate, large-scale replication EWAS (n=2,190) confirmed 91 trans-ethnic and 77 European-specific DMPs. Among our findings, we identified 61 DMPs at CpGs novel to the MSA platform, including near both new and known smoking-associated genes. Most notably, we uncovered a dense cluster of 12 DMPs within a 1117 bp region of ECEL1P1, forming the most long-lasting, persistent smoking-associated DMR ever detected, even among former smokers who quit decades prior. We also detected new signals at AHRR, a well-known locus for smoking-related DNAm changes. eFORGE analysis revealed that detected smoking-associated DNAm changes are predominantly located in hematopoietic stem and progenitor cell (HSPC) DNase I hotspots, aligning with gene set enrichment analyses that highlighted pathways related to hematopoietic stem cell differentiation. Our findings suggest that HSPCs serve as a reservoir for an epigenetic memory of smoking. Additionally, we observed short-term cell-specific smoking-associated DNAm changes in myeloid cells. Our results demonstrate the utility of the MSA in expanding our knowledge of both transient and persistent environmental exposure-associated DNAm changes.

IntroductionChronic inflammation has been implicated in lung carcinogenesis. Prospective studies have linked higher circulating C-reactive protein (CRP), an acute-phase inflammation marker, to higher lung cancer risk in predominantly smoking populations but lower risk in never smokers. We evaluated DNA methylation-based inflammation risk scores (DNAm-IRSs), which may capture longer-term immune-inflammatory and exposure-related biology, with lung cancer risk among never smokers. MethodsWe evaluated six DNAm-IRSs, including four CRP-based scores (IRSLigthart, IRSWielscher, IRSLinear_Hillary, IRSElnet_Hillary), in 683 risk-set-sampled case-control pairs nested in the Shanghai Womens Health Study (n=74,941). We estimated hazard ratios (HRs) and 95% confidence intervals (CIs) using conditional logistic regression. We examined DNAm-derived leukocyte composition and circulating immune-inflammatory proteins to characterize DNAm-IRS biology. ResultsCirculating CRP correlated positively with IRSLigthart (r=0.19), IRSWielscher (r=0.13), and IRSElnet_Hillary (r=0.30), but inversely with IRSLinear_Hillary (r=-0.02). Per standard deviation increase, IRSLigthart was associated with lower lung cancer risk (HR=0.85, 95% CI: 0.76-0.95), and IRSWielscher with lower risks of lung cancer (HR=0.87, 95% CI: 0.77-0.97) and adenocarcinoma (HR=0.83, 95% CI: 0.71-0.97). Associations persisted after adjustment for leukocyte composition and strengthened after adjustment for DNAm pack-years, an epigenetic smoking index that may capture combustion-related exposures beyond active smoking. Inverse associations were more evident among women with lower DNAm pack-years, although formal interaction tests were not statistically significant. Both scores were positively associated with acute-phase inflammation, IFN-{gamma}/effector trafficking, and higher CD8+ T-cell proportions. ConclusionsAmong never smokers, selected CRP-related DNAm-IRSs were associated with lower lung cancer risk and were linked to immune features consistent with antitumor activity.

Background Complex traits are influenced by numerous variants, most of which have regulatory effects on gene expression that can be mediated by DNA methylation. Molecular QTL mapping is an approach that aims to dissect these effects. However, obtaining molecular phenotypes on a large scale is challenging, particularly in livestock species. In cattle, an epigenotyping array called EpiChip has recently been developed in the European RUMIGEN project. The EpiChip, which contains 43,317 CpG sites distributed all over the bovine genome, enables large-scale measurement of DNA methylation. This study aims to characterize the genetic determinism of blood DNA methylation in cows by estimating heritability and mapping cis- and trans-methylation QTLs (meQTLs). Results Whole blood samples from 4,457 genotyped Holstein cows were epigenotyped. Across all CpG sites, the heritability estimates averaged 24.6%. The local meQTL mapping at sequence-level for variable CpG sites (SD > 2.5%; n = 28,806) detected cis-meQTLs for 80.1% of the CpG sites, with sentinel SNPs located close to their associated CpGs. A two-step analysis was also conducted to identify long-range associations, with a particular focus on trans-meQTL hotspots. First, we identified CpG-SNP trans-associations using medium-density genotypes (50k SNPs) that revealed 31,846 SNPs with significant effects on 1 to 530 trans-CpG sites. Then, regions associated with at least 34 independent trans-CpGs were retained defining 31 hotpots. For each hotspot, a local sequence-level GWAS was conducted using the first principal component derived from the associated trans-CpGs. Out of the 31 detected hotspots, three were located close to transcription factor genes (RUNX1, NFIC and FOXA3) for which the associated trans-CpGs were enriched for the corresponding binding motif. Two other hotspots were located within KDM5A and KDM5B, and their corresponding trans-CpGs were strongly overrepresented in H3K4me3 narrow peaks in blood as well as in other tissues. Conclusions By identifying functional candidate genes associated with blood DNA methylation in cattle, these findings provide new insights into the regulatory architecture of DNA methylation in mammals, highlighting the value of large-scale molecular data from livestock populations.

BackgroundLeukocyte telomere length (LTL) from cord blood is a marker of biological aging and long-term systemic health. Exposure to essential and toxic metals has been shown to influence LTL in a sexually dimorphic manner. However, little is known about the interplay between early-life longitudinal biodynamic patterns of these elements and cord blood LTL, as well as potential sex differences. MethodsFrom an ongoing longitudinal birth cohort study in Mexico City, we used available tooth samples from 231 children (129 males and 102 females) to generate 16 elemental weekly time series of direct fetal intensities from the second trimester through four to five months after birth. We analyzed the dentine growth rings using Inductively Coupled Plasma Mass Spectrometry to generate time-resolved elemental intensities. The elements included were Li, Mg, Ca, Mn, Co, Ni, Cu, Zn, As, Sr, Mo, Cd, Sn, Ba, Pb, and Bi. LTL was measured in cord blood using qPCR. We used cross-recurrence quantification analysis and entropy-complexity-based measures to generate time-resolved features that quantify the synchronization of elemental biodynamics. A stability-selection approach using five-fold cross-validation of regularized ridge regression was used for feature selection, and covariate-adjusted linear models were used to estimate associations with LTL. FindingsThe biodynamic interaction of Mg-Co and Mn-Sn was identified as the most stable feature among male and female children, respectively. In males, higher vertical entropy (i.e., a measure of higher variability) of Mg-Co temporal biodynamics was associated with shorter LTL ({beta}[95%CI]: -0.9[-0.14,-0.03]; p-value<0.01), but not in females ({beta}[95%CI]:-0.02[-0.10,0.06]; p-value=0.60); whereas higher recurrence rate (i.e., a measure of higher synchronicity) of Mn-Sn temporal biodynamics was associated with longer LTL ({beta}[95%CI]: 0.09[0.02,0.16]; p-value=0.01), in females but not in males ({beta}[95%CI], 0.03[-0.04, 0.09]; p-value=0.39). InterpretationWe demonstrate that time-varying multi-elemental synchronization of early-life elemental biodynamics, a potential marker of homeostatic balance, may be associated with cord blood-based telomere length in a sexual dimorphic manner.

DNA methylation research has vastly expanded over the past decade, producing a wealth of epigenome-wide association studies, biomarker algorithms such as epigenetic clocks, technical performance analyses, and functional annotations for CpG sites. However, these resources remain fragmented across dozens of databases and supplementary files within manuscripts, forcing researchers to spend time and effort on data cleaning and integration prior to meaningful analyses. No single resource currently unifies this information into a centralized, easy-to-query framework. Here, we present CpG Atlas, a curated relational database that integrates 18 distinct annotation layers encompassing over 1.2 million CpG sites across all four generations of Illumina methylation arrays (HM450K, EPIC v1, EPIC v2, and MSA). Built on a snowflake schema with a canonical probe identifier hub implemented in SQL, CpG Atlas consolidates over 800,000 CpG-trait associations, results from Mendelian randomization analyses, CpG membership across 81 epigenetic clocks, array manifest information, and probe reliability data. It further includes specialized layers such as solo-WCGW, CoRSIVs, PRC2 binding, transposon and retroelement annotations, tissue-specific differentially methylated positions across 17 tissues, and hallmarks of aging and cancer. To maximize utility and ease of use, the database is paired with an interactive web tool and a natural language-to-SQL query interface, enabling users to quickly perform complex multi-dimensional queries. Detailed documentation about every data source and table is also provided, facilitating the identification and interpretation of relevant studies. We demonstrate the utility of CpG Atlas through two case studies: a systematic enrichment analysis revealing distinct functional signatures across 16 epigenetic clocks, and an iterative biomarker discovery workflow for IBD that leverages cross-layer integration. Because it is readily scalable simply by adding or updating tables in the database, CpG Atlas provides a continuously evolving and extensible infrastructure for the epigenetics community that supports collaborative research, interpretable biomarker development, and integrative analyses across the growing landscape of epigenetic data.

Enhancers are key epigenetic regulatory elements that orchestrate spatiotemporal gene expression and are critical in mammalian development, gene regulation, and disease. Histone modifications such as H3K4me1 (a canonical enhancer mark) and H3K27ac (which distinguishes active enhancers) remain poorly characterized during early mammalian embryogenesis. Using low-input CUT&RUN (Cleavage Under Targets and Release Using Nuclease) with input as low as 50 cells, this study profiles genome-wide H3K4me1 and H3K27ac patterns in mouse oocytes and pre-implantation embryos. Both marks are enriched in distal regions and exhibit distinct sequence preferences and reprogramming dynamics in pre-implantation embryos. H3K27ac is reprogrammed at the 2-cell stage and marks active enhancers, while H3K4me1 is remodeled at the 4-cell stage and co-localizes with H3K27ac, overlapping with accessible chromatin regions. Interestingly, the co-localization of H3K4me1 and H3K27ac is also detected in promoter regions, where they exhibit a mutually exclusive pattern with H3K4me3. Three enhancer types-active (H3K4me1/H3K27ac), primed (H3K4me1), and poised (H3K4me1/H3K27me3)-are dynamically remodeled during maternal-to-zygotic transition (MZT), with active enhancers increasing significantly after zygotic genome activation. Furthermore, genome-wide super-enhancers are identified and mainly enriched in promoters. The differences in gene expression at different stages may be related to the specific motifs enriched by super-enhancers.

Objective: To characterize genome-wide DNA methylation patterns associated with sepsis using the Infinium Methylation EPIC v2.0 platform and to evaluate the feasibility of pooled methylation profiling in a pilot critical care cohort. Design: Single-center pilot epigenome-wide association study using pooled whole-blood genomic DNA and pool-level bioinformatic analysis. Setting: Academic medical center. Patients: Fifty critically ill adults enrolled within 48 hours of illness onset and 20 healthy controls. Interventions: None. Measurements and Main Results: Critically ill patients required mechanical ventilation and/or vasopressor support. Sepsis was defined according to Sepsis-3 criteria. Seventy individual samples were organized into 14 intended pools of 5 individuals each: 7 sepsis pools, 3 critically ill non-septic pools, and 4 healthy-control pools. One critically ill non-septic pool was excluded because of poor DNA quality, yielding 13 analyzable pools. For the primary pooled comparison, 7 sepsis pools were compared with 6 non-sepsis comparator pools comprising 2 critically ill non-septic and 4 healthy-control pools. After quality control and preprocessing with SeSAMe, 876,094 CpG sites were retained. The initial pool-level screen identified 170,897 candidate differentially methylated regions. Application of stringent secondary filters (false discovery rate <= 1%, absolute delta-beta >= 7.5%, and >= 5 CpGs per region) yielded a high-confidence subset with marked directional skewing, including 155 hypomethylated and 32 hypermethylated regions in sepsis. Differentially methylated region-associated genes were enriched in myeloid leukocyte activation, myeloid leukocyte-mediated immunity, defense response to bacterium, neutrophil granule biology, and hematopoietic cell lineage pathways. Additional signals involved microRNA-associated targets, ribosome biogenesis, RNA processing, long noncoding RNAs, and previously uncharacterized loci. Conclusions: In this pilot pooled EPIC v2.0 study, sepsis was associated with a biologically coherent, predominantly hypomethylated methylation signature enriched in myeloid and host-defense pathways. These findings support the feasibility of pooled methylation profiling for discovery-oriented sepsis biobank studies but should be interpreted as hypothesis-generating given the pool-level design, limited effective sample size, heterogeneous comparator group, and lack of direct validation against individual-level methylation profiles.

CD4+FOXP3+ regulatory T (Treg) cells maintain self-tolerance, restrain immune responses during inflammatory stimuli, and promote tissue function and repair. Treg cell lineage identity, stability, and function depend on specific DNA methylation patterns maintained by the epigenetic regulator, UHRF1. Aging disrupts DNA methylation patterns necessary for Treg cell-mediated lung repair in a cell-autonomous manner. Nevertheless, whether maintenance DNA methylation is necessary for age-related Treg cell transcriptional and methylation programs is unknown. Here, we performed transcriptional and DNA methylation profiling on young and old Treg cells isolated from mice with chimeric Treg cell-specific loss of UHRF1. We observed cell-autonomous, age-related alterations in transcriptional and DNA methylation signatures that were dependent on UHRF1. We conclude that maintenance DNA methylation is required for age-related alterations in Treg cell transcriptional and DNA methylation signatures.

Maternal heat stress (HS) is an emerging risk factor for adverse pregnancy outcomes, yet how gestational heat exposure causes persistent placental dysfunction remains unclear. In the present study, we established a murine HS model (38.5{degrees}C, 2.5 h/day, E0-E12.5) followed by thermal recovery to E17.5. HS reduced fetal weight during early gestation and caused persistent fetal growth restriction after recovery, despite partial placental weight restoration. Histological analyses revealed early reductions in the junctional and labyrinth zones, followed by sustained labyrinthine deficiency and compensatory junctional zone expansion. Consistently, HS impaired placental vascularization, with reduced vessel length and area, decreased CD31 and -SMA abundance, and altered angiogenesis-related gene expression. HS also triggered oxidative stress, weakened antioxidant capacity, disrupted anti-inflammatory signaling, reduced tight junction protein expression, and compromised barrier integrity. Mechanistically, HS induced excessive endoplasmic reticulum stress, accompanied by increased CHOP, phosphorylated ERK, and cleaved caspase-3. In conclusion, our data unveil a heat-induced placental insufficiency program that restricts fetal growth through vascular, redox, barrier, and ERS-MAPK-apoptotic remodeling. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/730306v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@177be8forg.highwire.dtl.DTLVardef@7faa52org.highwire.dtl.DTLVardef@1870971org.highwire.dtl.DTLVardef@7d1705_HPS_FORMAT_FIGEXP M_FIG C_FIG

Non-small cell lung cancer (NSCLC) remains one of the leading causes of cancer-related mortality, partly because it is often diagnosed at advanced stages and frequently develops resistance to platinum-based chemotherapy. We previously showed that MAFG becomes derepressed following miR-7 hypermethylation, promoting platinum resistance in NSCLC and ovarian cancer cell lines. Although MAFG is a well-established regulator of oxidative stress, recent evidence in melanoma and colorectal cancer suggests an additional role as a regulator of methylator phenotypes. However, how MAFG reshapes the lung cancer epigenome remains unknown. Here, we investigated the contribution of MAFG to DNA methylation remodeling by combining CRISPR/Cas9-mediated MAFG deletion with CpG-Methyl-Array profiling, followed by expression (qPCR) and methylation (qMSP) validation in tumor cell lines. Our translational approach integrated aptahistochemistry using MAFG-specific aptamers in 127 NSCLC patients, methylation analysis in 35 fresh-frozen tumors and 40 FFPE samples, and interrogation of TCGA methylation datasets. MAFG loss reduced promoter methylation of LIF and MAFG itself. Importantly, these effects were subtype-specific, with MAFG expression and methylation displaying distinct transcriptional programs in LUAD versus LUSC, and prognostic associations restricted to KRAS-mutated adenocarcinomas. In NSCL in silico and in house cohorts, lower MAFG methylation and higher MAFG protein levels were both associated with worse prognosis. In summary, our findings identify MAFG as a regulator of DNA methylation in NSCLC and support the use of MAFG DNA methylation, or protein levels as clinically relevant prognostic biomarkers, particularly in lung adenocarcinoma.

Human brain DNA methylation is shaped by inherited genetic variation and cumulative environmental experience, yet how these influences partition the methylome remains poorly resolved in postmortem cohorts with modest sample sizes and limited ancestral diversity. To map this architecture in an underrepresented population, we analyzed whole-genome bisulfite sequencing and genotype array data from 168 admixed Black American adults from the BrainSEQ consortium across three brain regions. We adapted and benchmarked SNP-based elastic-net modeling to classify variably methylated regions (VMRs) by local SNP-explained methylation variation, an approach that provided stable classification at the modest sample sizes of postmortem brain cohorts, where conventional methods are underpowered. Using this framework, we partitioned 31,143 VMRs into high and low SNP-explained classes and evaluated their generalizability in a multi-ancestry cohort of Black American and non-Hispanic white American donors. High SNP-explained VMRs were concentrated in distal intergenic sequences and, at the highest heritability levels, were enriched for H3K9me3, quiescent/repressive chromatin states and LINE/L1 elements, linking genetically anchored methylation to repeat-associated repressive chromatin across the human brain. A small subset overlapping Activity-by-Contact-defined enhancers was linked to candidate immune-related genes, including MHC class II loci. By contrast, low SNP-explained VMRs were more gene-proximal and enriched for active regulatory elements. Metadata-associated VMRs showed region-, exposure-, and donor-group-dependent enrichment across SNP-explained classes, including substance use and sociodemographic variables. Together, these findings show that the most genetically anchored component of the human brain methylome is concentrated in repressive, repeat-rich chromatin compartments involved in heterochromatin maintenance and repeat silencing, distinct from the gene-proximal, exposure-associated variation less explained by nearby SNPs. By resolving this architecture in an underrepresented population, this work clarifies how inherited variation structures the brain methylome and, given the established role of these compartments in neuronal aging, informs the interpretation of epigenomic mechanisms relevant to neuropsychiatric and neurodegenerative diseases.

The X chromosome is often excluded from studies analyzing associations between traits and DNA methylation. In females, one copy of most genes on the X is inactivated (X-chromosome inactivation; XCI) through DNA methylation of the gene promoter on the inactive X. This leads to challenges in analyzing and interpreting DNA methylation data patterns. Particularly for sex-biased diseases and traits, there may be many loci of interest on the X chromosome, which contains about 5% of the genome. To address the need for appropriate analysis of DNA methylation data on the X chromosome, we develop a statistical approach to infer locus-specific escape from XCI sensitive to phenotype or covariate values. Performance of this method is illustrated by analysis of data from two sex-biased traits: rheumatoid arthritis which is 3-fold more common in females, and recurrent venous thromboembolism which occurs 2.5 times more often in males. Analyses of these two datasets identify new trait-associated loci on the X chromosome, demonstrate the capabilities of the new method for both bisulfite sequencing data and Illumina EPIC data, suggest at least one locus where variable escape may explain a sex-specific disease association, and rule out variable escape as a potential explanation at other loci. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=176 HEIGHT=200 SRC="FIGDIR/small/732395v1_ufig1.gif" ALT="Figure 1"> View larger version (52K): org.highwire.dtl.DTLVardef@1fd6c70org.highwire.dtl.DTLVardef@da4ee8org.highwire.dtl.DTLVardef@729512org.highwire.dtl.DTLVardef@98edb1_HPS_FORMAT_FIGEXP M_FIG C_FIG Created with BioRender (bioRender.com)

The information content of the genome has been extensively analyzed. However, a comparable quantitative framework for DNA methylation is still lacking. Without such quantification, the magnitude of this regulatory and dynamic epigenetic structure remains conceptually imprecise, even though methylation dysregulation is strongly linked to disease-related phenotypes and altered cellular identity. Here we address this gap by applying Shannon information theory to DNA methylation. We first consider methylation marks as binary or probabilistic regulatory states and estimate the theoretical upper-bound information capacity of the human methylome under simplifying assumptions. We then progressively refine this estimate by incorporating biologically relevant constraints, including methylation bias, bimodal methylation distributions, local CpG correlation, genomic regulatory class, and cell-type-discriminative methylation patterns. This approach allows us to distinguish between theoretical methylation capacity, statistical methylation entropy, and biologically interpretable regulatory information. Finally, we consider methylation information from a discriminative perspective, analyzing its contribution to distinguishing cell types and regulatory cellular states. Within this framework, mutual information between methylation patterns and cell identity provides a biologically constrained estimate of methylations role as an epigenetic identity code. Our layered analysis reconciles megabit-scale methylome capacity with compact, biologically interpretable identity signatures. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=113 SRC="FIGDIR/small/735086v1_ufig1.gif" ALT="Figure 1"> View larger version (73K): org.highwire.dtl.DTLVardef@f0f0fdorg.highwire.dtl.DTLVardef@5d8a1eorg.highwire.dtl.DTLVardef@116debdorg.highwire.dtl.DTLVardef@79530e_HPS_FORMAT_FIGEXP M_FIG C_FIG

During embryonic development bipotential gonads differentiate into either testes or ovaries under the direction of mutually exclusive gene networks. DNA methylation has been shown to regulate gene expression and to play a role in many developmental processes. This includes sex differentiation in which DNA methylation is linked to control of key sex genes. Whether DNA methylation regulates sexual differentiation in species where environmental influences such as temperature are involved is less clear. We conducted a genome-wide study in embryonic gonads of the central bearded dragon (Pogona vitticeps), a lizard with temperature induced sex reversal, and compared DNA methylation patterns to gene expression profiles at a stage of early sex differentiation. Overall, sex reversed ZZf females were found to have lower global methylation than both canonical sexes, ZZm males and ZWf females. We found that the expression of a key gene in sex differentiation, Amh, is regulated via DNA methylation. Amh is a driver of testis differentiation and is repressed in genetically determined females, as well as in temperature sex-reversed females, by hypermethylation around its transcription start site. Although the trigger of ovary determination is different in both groups of females, one by genetic complement and one by a temperature signal, downstream regulation of gene expression converges and seems to follow similar mechanisms.

Background: Epigenetic clocks are markers of biological aging that may vary in their sensitivity to environmental stressors and lifestyle modifiers. To evaluate the utility of these biomarkers as sensors of the human exposome, we investigated how they respond to two powerful and opposing exposures: smoking, a source of oxidative stress, and the antioxidant-rich Mediterranean diet. Objectives: We assessed the sensitivity of eleven epigenetic clocks to diet and smoking and evaluated whether Mediterranean diet adherence modifies associations between smoking and epigenetic aging. Methods: We analysed 928 participants (mean age 41 years, 59% male) from the Airwave Health Monitoring Study. Linear regression models assessed associations between Mediterranean Diet Score (MDS) and epigenetic age acceleration (EAA), alongside smoking status and blood cotinine. Interaction terms between smoking status and MDS were included to detect dietary attenuation of smoking-related EAA. Models were adjusted for demographic, socioeconomic, lifestyle, and psychological covariates. Results: Higher MDS was associated with lower EAA for GrimAge ({beta} = -0.07 SD; 95% CI: -0.13, -0.01) and Bernabeu ({beta} = -0.08 SD; 95% CI: -0.14, -0.02) after false discovery rate correction. Smoking was strongly associated with increased EAA, particularly for GrimAge, Bernabeu, and DunedinPACE. Among current smokers, effect sizes were greater in those with lower dietary adherence (e.g. GrimAge: 1.79 SD, 95% CI: 1.54, 2.04) compared with those with higher adherence (1.35 SD, 95% CI: 1.01, 1.68; P_interaction < 0.001). Similar attenuation patterns were observed for Bernabeu. Higher intake of fruits, vegetables, and whole grains contributed most to the attenuation of smoking-related EAA. Conclusions: Our findings indicate that certain epigenetic clocks effectively capture the tension between harmful and protective exposures within the exposome. Rather than suggesting that diet neutralises the risks of tobacco, these results demonstrate that specific clocks are sensitive enough to monitor how lifestyle factors modify molecular responses to environmental toxins. This highlights the value of second-generation clocks in quantifying biological resilience.

Chronically stressing male rodents can induce stress-specific epigenetic changes in sperm that contribute to altered offspring phenotypes. Whether similar phenomena occur in men is unclear. This study addresses this knowledge gap by analyzing sperm microRNAs (miRNAs) from 51 men exposed to various levels of adult trauma including crime, disaster, and physical or sexual violence, quantified by the Trauma History Questionnaire (THQ), a measure of risk for Post-Traumatic Stress Disorder (PTSD). Four sperm miRNAs, miR-532-3p, 491-5p, 375-3p and 361-3p correlated positively with mens THQ scores, showing 4X to 130X over expression in sperm from the most highly traumatized men. These changes were independent of mens adverse childhood experiences (ACEs), which we previously linked to decreased miR-34/449 in their sperm; and sperm miR-34/449 levels were not associated with THQ scores. Injecting these 4 miRNAs into fertilized mouse oocytes at levels comparable to those found in men reporting high THQ scores yielded offspring with elevated anxiety-and depression-like phenotypes. This finding differs from the stress related phenotypes we observed in offspring of mice fertilized by sperm with reduced levels of miR-34/449. Consistent with only a small subset of men with high THQ scores developing PTSD, we observed no statistically significant increase in overall anxiety or depression among this highly traumatized group, however there were indications of increased sleeplessness, appetite and concentration difficulties and negative self-concept among this group. Nevertheless, almost all men reporting high THQ scores had elevated levels of all 4 of these miRNAs in their sperm, suggesting these trauma-induced epigenetic changes may raise mental health risks in the offspring of men with only subtle mental health problems. Since [~]20 % of men report either THQ or ACE scores in the ranges linked here and in our earlier study to changes in sperm miRNAs that in mice lead to elevated levels of stress-related behaviors, a large human population with an elevated risk of transmitting stress-related traits to their offspring likely exists.