Epigenetics

Biological sex regulates fundamental neurobiology, as well as the etiology and prevalence of neuropsychiatric disorders. Cyclin-dependent kinase 5 (Cdk5) is a neuronally enriched kinase that regulates synaptic plasticity, neuronal homeostasis, and hippocampal-dependent memory. While Cdk5 protein activity is necessary and sufficient to promote memory in male rodents, its role in females and its gene regulation in either sex remain poorly understood. In males, Cdk5 protein inhibition impairs fear memory. We previously showed that fear conditioning activates Cdk5 gene expression and increases permissive chromatin acetylation in male, but not female hippocampus. We hypothesize that Cdk5 gene repression would impair fear memory in males. We developed an excitatory neuron-specific, CRISPR/dCas9-HDAC3 epigenetic editing tool to target histone acetylation at the endogenous Cdk5 promoter. This strategy reduced histone acetylation and decreased Cdk5 mRNA, protein, and kinase activity in both sexes. Interestingly, Cdk5 repression in hippocampal neurons impaired fear and spatial memory in both male and female mice. Targeted deacetylation also evicted the transcription factor CREB1 from the Cdk5 promoter, revealing a link between histone acetylation and Cdk5 transcriptional activation. These findings demonstrate that Cdk5 acetylation in neurons is necessary for hippocampal memory in both sexes, providing new insight into sex-specific epigenetic regulation of memory.

Mediation analysis quantifies how an exposure affects an outcome through an intermediate variable. We extend mediation analysis to capture the cumulative effects of longitudinal predictors on longitudinal outcomes. Our proposed model examines how mediators transmit the effects of the current and previous exposure on the current outcome. We construct a least-squared estimator for cumulative indirect effect (CIE) and used three approaches (exact form, delta method, and bootstrap procedure) to estimate its standard error (SE). The estimator of CIE is unbiased with no unmeasured confounding and independent model errors between mediator model and outcome model at all time points, as shown in statistical inference and in simulations. While three SE estimates are numerically similar, bootstrap procedure is recommended due to its simplicity in implementation. We apply this method to Framingham Heart Study offspring cohort to assess if DNA methylation mediates the association of alcohol consumption with systolic blood pressure over two time points. We identify two CpGs (cg05130679 and cg05465916) as mediators and construct a composite DNA methylation score from 11 CpGs, which mediates for 39% of the cumulative effect. In conclusion, we propose an unbiased estimator for CIE. Future studies will investigate the missingness in mediators and outcomes.

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.

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.

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.

BackgroundFathers adolescent smoking and overweight affect respiratory health in offspring, suggesting that paternal puberty exposures may influence offspring biological ageing through preconception epigenetic mechanisms. MethodsWe analyzed epigenetic age acceleration using four validated epigenetic clocks derived from blood DNA methylation in 892 RHINESSA offspring (mean age 27 years), linked to parental data on smoking and body shapes from RHINE/ECRHS. Linear regression examined parental smoking initiation ([&le;]15 or >15 years) and overweight body shape (childhood/puberty or age 30) in relation to offspring epigenetic age acceleration, adjusting for offspring sex, age and parental socioeconomic status. Sensitivity analyses accounted for offspring smoking and BMI. ResultsPCHorvath ({beta} 1.53; 95% CI 0.02, 2.9), PCGrimAge (1.21; 0.03, 2.1), DunedinPACE (0.04; -0.001, 0.1) and PCPhenoAge (1.92; -0.3, 4.2) were accelerated in daughters of fathers who started smoking [&le;]15 years. Likewise, PCHorvath (2.25; 1.2, 3.3), PCGrimAge (1.36; -0.2, 2.9), DunedinPACE (0.07; 0.01, 0.1) and PCPhenoAge (3.11; 1.8, 4.4) were accelerated in daughters and sons of fathers who had been overweight in childhood and puberty. These results remained largely unchanged after additional adjustments or stratification in sensitivity analyses. No associations were found for maternal smoking or overweight in puberty. ConclusionsEpigenetic ageing is accelerated in offspring of fathers who smoked or were overweight in puberty, independent of offspring lifestyle. These findings suggest that adolescent boys environment and lifestyle may be critical for next-generation health. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=104 SRC="FIGDIR/small/26352444v1_fig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@1eea189org.highwire.dtl.DTLVardef@1af41f4org.highwire.dtl.DTLVardef@1132932org.highwire.dtl.DTLVardef@f5ba2c_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1.C_FLOATNO Graphical abstract Legend to graphical abstract Figure Fathers smoking or overweight during puberty was associated with accelerated epigenetic aging in offspring (n=892), independent of the offsprings own lifestyle. No such pattern was observed for maternal puberty exposures, or when paternal exposures occurred after puberty. Male puberty may be a critical window for next-generation health. C_FIG

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.

BackgroundDNA methylation-based biomarkers have been widely used to predict biological age; however, most blood-derived data have been used in most existing models, and whether cheek mucosa can serve as an alternative indicator for methylation-based estimation of aging-related and clinical phenotypes is unclear. MethodsDNA methylation profiles from cheek mucosa and whole blood of 186 Japanese adults were analyzed using Illumina Infinium Methylation Screening Array (MSA). Models were constructed to predict chronological age, phenotypic age, and clinical laboratory biomarkers from cheek mucosa- and blood-derived methylation data. In addition to applying the ordinary elastic net method, a two-stage residual learning method incorporating existing blood-based epigenetic clocks was applied for more accurate prediction of biological age. Sex-stratified analyses and comparisons of selected CpG features across sexes and tissues were performed. ResultsCheek mucosa-derived MSA methylation data enabled accurate prediction of chronological age (R = 0.965) and phenotypic age (R = 0.964) using the two-stage method. The performance gain achieved by the two-stage approach was greater for phenotypic age than for chronological age. Multiple clinical laboratory biomarkers could be predicted using cheek mucosa-derived methylation data, particularly after sex stratification, including inflammatory, metabolic, thyroid-related, and sex hormone-related markers. Most biomarkers that could be predicted using blood-derived methylation data were also predicted using cheek mucosa-derived methylation data. However, the CpG sites selected for prediction showed minimal overlap across sexes and tissues despite overlap in the corresponding predictable phenotypes. ConclusionsCheek mucosa-derived DNA methylation profiles measured using the MSA can predict chronological age, phenotypic age, and multiple clinically relevant laboratory biomarkers, supporting the utility of cheek mucosa as a less invasive alternative for methylation-based assessment of biological aging and systemic physiological state.

Vitamin D signaling has recognized roles in female reproductive physiology, but its effects at the chromatin level in endometrial stromal cells are still unclear. Here, we investigated how the active form of vitamin D, 1,25-dihydroxyvitamin D3, or calcitriol, influences the accessible chromatin landscape of human endometrial stromal cells. Assay for transposase-accessible chromatin using sequencing (ATAC-seq) was performed on T-HESCs treated with either a vehicle or 1,25(OH)2D3. Ligand treatment increased overall chromatin accessibility, shown by higher ATAC-seq signal intensity, while causing only minor changes in the total number of called peaks. Peak annotation revealed that accessible regions were spread across both promoter-proximal and distal genomic areas. Integrating this data with CUT&RUN and RNA sequencing showed that most vitamin D-responsive cistromic modifications and transcripts were linked to nearby open chromatin, though fewer were associated with regions that were significantly differentially accessible. These results suggest that 1,25(OH)2D3-dependent transcription mainly occurs within a permissive, pre-accessible chromatin environment. This study offers new evidence that active vitamin D influences the epigenomic landscape of human endometrial stromal cells, establishing the chromatin-based molecular response to a chemically-defined VDR ligand, 1,25(OH)2D3, relevant to stromal differentiation and preparation for decidualization. HighlightsO_LIFirst evidence suggesting the direct impact of active vitamin D, 1,25-dihydroxyvitamin D3, 1,25(OH)2D3, enhanced the signal intensity of chromatin accessibility in human endometrial stromal cells C_LIO_LIMost accessible chromatin regions were shared between vehicle and ligand-treated human endometrial stromal cells C_LIO_LI1,25(OH)2D3-responsive transcription occurs largely within pre-accessible chromatin in human endometrial stromal cells C_LIO_LIAssay for transposase-accessible chromatin sequencing (ATAC-seq) defines a chromatin-level pharmacologic response to a chemically defined VDR ligand in human endometrial stromal cells C_LI

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.

INTRODUCTION: MethylCog is a 29-CpG blood DNA methylation (DNAm) proxy for general cognitive ability (g). Its incremental association with blood biomarkers of Alzheimer's disease and related dementias (ADRD) and prospective cognitive ability remains unclear. METHODS: In the held-out test set from the original MethylCog study, we tested whether MethylCog explained baseline g beyond four ADRD blood biomarkers, and whether it predicted six-year follow-up g beyond baseline g and biomarkers. RESULTS: MethylCog showed a stronger age-adjusted association with baseline g than individual biomarkers (r=.368 vs absolute r=.083-.162). MethylCog added 10.0% variance beyond all four biomarkers cross-sectionally (p<.001) and predicted six-year follow-up g in the biomarker-adjusted model (beta=.108, p=.002). No individual ADRD biomarker independently predicted follow-up g. DISCUSSION: MethylCog may provide cognition-related DNAm information complementary to blood-based ADRD biomarkers.

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.

Cartilage and bone that comprise craniofacial structures as well as neurons and glia of the peripheral nervous system are derived from a multipotent population of cranial neural crest cells, that respond to both cell intrinsic and extrinsic cues to differentiate into precise cell states. Both a genetic and epigenetic regulatory network are required for each step in the differentiation process, involving transcription factors, histone modifiers and chromatin remodelers. Here, we examined the direct transcriptional targets of two histone methyltransferases, Prdm3 and Prdm16 in zebrafish neural crest cells at 48 hours post fertilization in zebrafish. Using CUT&RUN, we examined both direct DNA binding and nucleosome association. At this stage of development, CUT&RUN fragment size analysis indicated that Prdm3 and Prdm16 are largely associated with nucleosomes. We further analyzed these nucleosome peak sets to identify 6 clusters where differential binding of Prdm3 and Prdm16 and differential enrichment of gene ontology terms for target genes was observed. We validated gene expression in each cluster by in situ hybridization chain reaction (HCR) at 48 hpf demonstrating that prdm3 and prdm16 mutants exhibit corresponding changes in gene expression of the putative gene targets identified. Finally, we performed CUT&RUN-qPCR in prdm3 and prdm16 mutant zebrafish embryos and demonstrated reduced binding at putative target loci. Together these data suggest that Prdm3 and Prdm16 regulate their transcriptional targets primarily by binding nucleosomes around their putative target loci to control downstream gene expression. HighlightsPrdm3 and Prdm16 associate with nucleosomes for regulation of gene expression Gene targets are altered in prdm3 and prdm16 mutant zebrafish Reduced binding is observed in respective mutants

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.

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.

Background & AimsGastric epithelial cells maintain homeostasis through dynamic self-renewal mechanisms involving stem and progenitor cells; however, identifying them has been challenging. This study aims to identify stem cells of healthy gastric epithelium and cell type-specific regulators defining gastric epithelial homeostasis via single-nucleus multiome analysis. MethodsTen unique gastric samples were collected from 8-12 week old wildtype mice. Isolated nuclei were subjected to simultaneous profiling of gene expression and chromatin accessibility. After quality control, 31,598 cells were analyzed with Seurat and Signac using weighted-nearest neighbors analysis for joint RNA and ATAC clustering. Furthermore, SCENIC+, MultiVelo, EpiCHAOS and Cell plasticity score were used to uncover gene regulatory networks, cell state dynamics and lineage trajectories. ResultsOur analyses were validated by the identification of known regulators of stem-cell differentiation into mature cell types. More importantly, it revealed previously uncharacterized regulatory networks comprising novel transcription factor combinations that define cell identities, including Ppara, Pparg, Arid5b and Sox5 as candidate regulators of parietal, foveolar, chief and neck cells, respectively. Further, our data support the identity of isthmus cells as stem-like cells of healthy gastric epithelium, as evidenced by epigenetic plasticity that simultaneously contains open chromatin states of all differentiated cell types in the absence of transcriptional reprogramming. ConclusionConsistent with Waddingtons epigenetic landscape hypothesis, gastric epithelial homeostasis is controlled by orchestrated epigenetic and transcriptional programs. Contrary to the prevailing hypothesis, stem cells can be defined not by a separate epigenetic state but by epigenetic superposition of differentiated cell states. Future work is needed to define the universality of these results.

Psychosocial stress is a significant risk factor for mental and physical illness, and emerging evidence suggests that altered oral microRNAs (miRNAs) and microbiome may act as biomarkers or mediators of stress responses. This study investigated stress-associated molecular changes in saliva from 113 male police officers. Based on repeated administrations of the Karasek Demand/Control and Effort/Reward Imbalance questionnaires, subjects were stratified by perceived stress response (SR) to homogeneous occupational stressors into low, intermediate, or high responders. Salivary miRNA profiles were analyzed using small RNA sequencing, and microbiome composition was assessed through shotgun metagenomics. Eighteen miRNAs were significantly differentially expressed between high- and low-SR groups, with four miRNAs with increasing (miR-10400-5p, miR-1290, miR-6074-5p, and miR-9902) and fourteen with decreasing (including miR-21-5p and mirR-142-3p) levels in the high SR group (adj.p<0.05). The identified salivary miRNAs showed a progressive alteration from low- to high-SR groups. Functional enrichment analysis indicated that dysregulated miRNA targets are involved in apoptosis, cellular stress responses, and metabolic regulation. Distinct salivary microbial communities were also observed across SR groups. Several taxa displayed progressive abundance shifts, with Prevotella baroniae and Schaalia odontolytica increasing and Actinomyces naeslundii and Capnocytophaga ochracea decreasing in the high SR group. Functional predictions revealed, in this group, a significant enrichment of inositol degradation pathways, paralleled by a reduction in bacteria involved in L-tryptophan and thiamine biosynthesis. These findings suggest that salivary miRNAs and microbiota profiles may serve as non-invasive biomarkers of psychosocial stress and provide insight into molecular mechanisms linking chronic stress to physiological and behavioral outcomes.

Stressful life events impact individual's functionality and contribute to disease outcomes, yet the biological pathways underlying life stress remain unclear. We characterized the metabolomic profiles of stressful life events using data from 3,264 participants (5,163 observations) of the Dutch NESDA cohort. 98 metabolites were identified, with upregulated metabolites overrepresented in phosphatidylethanolamine and downregulated metabolites overrepresented in fatty acid metabolism. 92 of these metabolites were available in the Dutch NEO cohort (N=599): 11 were significantly replicated including six lipids (e.g., three bile acids (glycochenodeoxycholate 3-sulfate)), one carbohydrate, and one xenobiotic. 21 overlapping metabolites were additionally available in the Chinese GBCS cohort (N=200): 10-undecenoate (11:1n1) (fatty acid) and glycochenodeoxycholate 3-sulfate (bile acid) showed consistent associations across both Dutch and Chinese cohorts. Stressful life events are associated with metabolic dysregulation, particularly involving fatty acid and bile acid pathways, highlighting promising biological targets to reduce the impact of stress on mental and somatic health.

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.

Background Preterm birth (PTB) rates among Hispanic/Latina individuals in the United States have risen over the past decade. Data suggests this rise may be driven in part by psychosocial stress. Leukocyte telomere length (LTL), a marker of cumulative cellular aging that shortens under chronic stress, may capture stress-related biological vulnerability, but has not been examined as a potential population-level contributor to PTB in Hispanic/Latina pregnancies. Objective To examine the association between mid-pregnancy maternal LTL and PTB in a population-based Hispanic/Latina cohort. Methods In a case-control study nested within a California singleton birth cohort (n = 436 Hispanic/Latina individuals; 215 PTB, 221 term births), LTL was measured by quantitative PCR from biobank specimens collected from 15 to 20 weeks of gestation. Covariates from linked birth certificate and hospital discharge records were included. Logistic regression estimated ORs and 95% CIs of PTB by LTL examined continuously and by percentile category (<=10th, 11th-89th, >=90th) with and without adjustment for covariates. Results Mean and median LTL did not differ between PTB and term births. LTL at or below the 10th percentile was associated with elevated odds of PTB relative to full-term birth (12.6% versus 4.3%; ORc = 3.2, 95% CI 1.3-7.9), persisting after partial (ORadj1 = 3.2, 95% CI 1.3-8.3) and full covariate adjustment (ORadj2 = 3.4, 95% CI 1.3-9.3). Subgroup analyses showed consistent directional patterns across PTB subgroups and for early term birth (ORadj2 = 5.1, 95% CI 1.5-17.0). Conclusions Mid-pregnancy maternal LTL <=10th percentile was associated with more than three times the odds of PTB, with risk concentrated at the extreme low tail of the distribution. Consistent with a cumulative allostatic load model, markedly short LTL at mid-gestation may reflect elevated stress-related biological risk for preterm delivery. These findings support upstream investment in stress reduction and prospective LTL research in high-burden populations.