Methylome-wide analysis in systemic microbial-induced experimental periodontal disease in mice with different susceptibility.

Objective: The study delved into the epigenetic factors associated with periodontal disease in two lineages of mice, namely C57bl/6 and Balb/c. Its primary objective was to elucidate alterations in the methylome of mice with distinct genetic backgrounds following systemic microbial challenge, employing high-throughput DNA methylation analysis as the investigative tool. Methods: Porphyromonas gingivalis (Pg)was orally administered to induce periodontitis in both Balb/c and C57bl/6 lineage. After euthanasia, genomic DNA from both maxilla and blood were subjected to bisulfite conversion, PCR amplification and genome-wide DNA methylation analysis using the Ovation RRBS Methyl-Seq System coupled with the Illumina Infinium Mouse Methylation BeadChip. Results: Of particular significance was the distinct methylation profile observed within the Pg-induced group of the Balb/c lineage, contrasting with both the control and Pg-induced groups of the C57bl/6 lineage. Utilizing rigorous filtering criteria, we successfully identified a substantial number of differentially methylated regions (DMRs) across various tissues and comparison groups, shedding light on the prevailing hypermethylation in non-induced cohorts and hypomethylation in induced groups. The comparison between blood and maxilla samples underscored the unique methylation patterns specific to the jaw tissue. Our comprehensive methylome analysis further unveiled statistically significant disparities, particularly within promoter regions, in several comparison groups. Conclusion: The differential DNA methylation patterns observed between C57bl/6 and Balb/c mouse lines suggest that epigenetic factors contribute to the variations in disease susceptibility. The identified differentially methylated regions associated with immune regulation and inflammatory response provide potential targets for further investigation. These findings emphasize the importance of considering epigenetic mechanisms in the development and progression of periodontitis.

Methylomes as key features for predicting recombination in some plant species.

Knowing how chromosome recombination works is essential for plant breeding. It enables the design of crosses between different varieties to combine desirable traits and create new ones. This is because the meiotic crossovers between homologous chromatids are not purely random, and various strategies have been developed to describe and predict such exchange events. Recent studies have used methylation data to predict chromosomal recombination in rice using machine learning models. This approach proved successful due to the presence of a positive correlation between the CHH context cytosine methylation and recombination rates in rice chromosomes. This paper assesses the question if methylation can be used to predict recombination in four plant species: Arabidopsis, maize, sorghum, and tomato. The results indicate a positive association between CHH context methylation and recombination rates in certain plant species, with varying degrees of strength in their relationships. The CG and CHG methylation contexts show negative correlation with recombination. Methylation data was key effectively in predicting recombination in sorghum and tomato, with a mean determination coefficient of 0.65 ± 0.11 and 0.76 ± 0.05, respectively. In addition, the mean correlation values between predicted and experimental recombination rates were 0.83 ± 0.06 for sorghum and 0.90 ± 0.05 for tomato, confirming the significance of methylomes in both monocotyledonous and dicotyledonous species. The predictions for Arabidopsis and maize were not as accurate, likely due to the comparatively weaker relationships between methylation contexts and recombination, in contrast to sorghum and tomato, where stronger associations were observed. To enhance the accuracy of predictions, further evaluations using data sets closely related to each other might prove beneficial. In general, this methylome-based method holds great potential as a reliable strategy for predicting recombination rates in various plant species, offering valuable insights to breeders in their quest to develop novel and improved varieties.

The CUT&RUN greenlist: genomic regions of consistent noise are effective normalizing factors for quantitative epigenome mapping.

Cleavage Under Targets and Release Using Nuclease (CUT&RUN) is a recent development for epigenome mapping, but its unique methodology can hamper proper quantitative analyses. As traditional normalization approaches have been shown to be inaccurate, we sought to determine endogenous normalization factors based on the human genome regions of constant nonspecific signal. This constancy was determined by applying Shannon's information entropy, and the set of normalizer regions, which we named the 'Greenlist', was extensively validated using publicly available datasets. We demonstrate here that the greenlist normalization outperforms the current top standards, and remains consistent across different experimental setups, cell lines and antibodies; the approach can even be applied to different species or to CUT&Tag. Requiring no additional experimental steps and no added cost, this approach can be universally applied to CUT&RUN experiments to greatly minimize the interference of technical variation over the biological epigenome changes of interest.

An enriched maternal environment and stereotypies of sows differentially affect the neuro-epigenome of brain regions related to emotionality in their piglets.

Epigenetic mechanisms are important modulators of neurodevelopmental outcomes in the offspring of animals challenged during pregnancy. Pregnant sows living in a confined environment are challenged with stress and lack of stimulation which may result in the expression of stereotypies (repetitive behaviours without an apparent function). Little attention has been devoted to the postnatal effects of maternal stereotypies in the offspring. We investigated how the environment and stereotypies of pregnant sows affected the neuro-epigenome of their piglets. We focused on the amygdala, frontal cortex, and hippocampus, brain regions related to emotionality, learning, memory, and stress response. Differentially methylated regions (DMRs) were investigated in these brain regions of male piglets born from sows kept in an enriched vs a barren environment. Within the latter group of piglets, we compared the brain methylomes of piglets born from sows expressing stereotypies vs sows not expressing stereotypies. DMRs emerged in each comparison. While the epigenome of the hippocampus and frontal cortex of piglets is mainly affected by the maternal environment, the epigenome of the amygdala is mainly affected by maternal stereotypies. The molecular pathways and mechanisms triggered in the brains of piglets by maternal environment or stereotypies are different, which is reflected on the differential gene function associated to the DMRs found in each piglets' brain region . The present study is the first to investigate the neuro-epigenomic effects of maternal enrichment in pigs' offspring and the first to investigate the neuro-epigenomic effects of maternal stereotypies in the offspring of a mammal.

Regulation and Immunotherapeutic Targeting of the Epigenome in Exhausted CD8 T Cell Responses.

Exhaustion is a state of CD8 T cell differentiation that occurs in settings of chronic Ag such as tumors, chronic viral infection, and autoimmunity. Cellular differentiation is driven by a series of environmental signals that promote epigenetic landscapes that set transcriptomes needed for function. For CD8 T cells, the epigenome that underlies exhaustion is distinct from effector and memory cell differentiation, suggesting that signals early on set in motion a process where the epigenome is modified to promote a trajectory toward a dysfunctional state. Although we know many signals that promote exhaustion, putting this in the context of the epigenetic changes that occur during differentiation has been less clear. In this review, we aim to summarize the epigenetic changes associated with exhaustion in the context of signals that promote it, highlighting immunotherapeutic studies that support these observations or areas for future therapeutic opportunities.

Targeting the epigenome of cancer stem cells in pediatric nervous system tumors.

Medulloblastoma, neuroblastoma, and pediatric glioma account for almost 30% of all cases of pediatric cancers. Recent evidence indicates that pediatric nervous system tumors originate from stem or progenitor cells and present a subpopulation of cells with highly tumorigenic and stem cell-like features. These cancer stem cells play a role in initiation, progression, and resistance to treatment of pediatric nervous system tumors. Histone modification, DNA methylation, chromatin remodeling, and microRNA regulation display a range of regulatory activities involved in cancer origin and progression, and cellular identity, especially those associated with stem cell features, such as self-renewal and pluripotent differentiation potential. Here, we review the contribution of different epigenetic mechanisms in pediatric nervous system tumor cancer stem cells. The choice between a differentiated and undifferentiated state can be modulated by alterations in the epigenome through the regulation of stemness genes such as CD133, SOX2, and BMI1 and the activation neuronal of differentiation markers, RBFOX3, GFAP, and S100B. Additionally, we highlighted the stage of development of epigenetic drugs and the clinical benefits and efficacy of epigenetic modulators in pediatric nervous system tumors.

Integrating Methylome and Transcriptome Signatures Expands the Molecular Classification of the Pituitary Tumors.

OBJECTIVE: To explore pituitary tumors by methylome and transcriptome signatures in a heterogeneous ethnic population. METHODS: In this retrospective cross-sectional study, clinicopathological features, methylome, and transcriptome were evaluated in pituitary tumors from 77 patients (61% women, age 12-72 years) followed due to functioning (FPT: GH-secreting n = 18, ACTH-secreting n = 14) and nonfunctioning pituitary tumors (NFPT, n = 45) at Ribeirao Preto Medical School, University of São Paulo. RESULTS: Unsupervised hierarchical clustering analysis (UHCA) of methylome (n = 77) and transcriptome (n = 65 out of 77) revealed 3 clusters each: one enriched by FPT, one by NFPT, and a third by ACTH-secreting and NFPT. Comparison between each omics-derived clusters identified 3568 and 5994 differentially methylated and expressed genes, respectively, which were associated with each other, with tumor clinical presentation, and with 2017 and 2022 WHO classifications. UHCA considering 11 transcripts related to pituitary development/differentiation also supported 3 clusters: POU1F1-driven somatotroph, TBX19-driven corticotroph, and NR5A1-driven gonadotroph adenomas, with rare exceptions (NR5A1 expressed in few GH-secreting and corticotroph silent adenomas; POU1F1 in few ACTH-secreting adenomas; and TBX19 in few NFPTs). CONCLUSION: This large heterogenic ethnic Brazilian cohort confirms that integrated methylome and transcriptome signatures classify FPT and NFPT, which are associated with clinical presentation and tumor invasiveness. Moreover, the cluster NFPT/ACTH-secreting adenomas raises interest regarding tumor heterogeneity, supporting the challenge raised by the 2017 and 2022 WHO definition regarding the discrepancy, in rare cases, between clinical presentation and pituitary lineage markers. Finally, making our data publicly available enables further studies to validate genes/pathways involved in pituitary tumor pathogenesis and prognosis.

Mecanismos epigenéticos nos espermatozoides e o impacto sobre a fertilidade masculina / Epigenetic mechanisms on sperm cells and the impact on male fertility

Por ser uma célula altamente especializada, o espermatozoide apresenta diferentes mecanismos epigenéticos, sendo os principais as metilações do DNA, o código de histonas, os ncRNAs (RNAs não codificadores), e a alta condensação da cromatina pela presença das protaminas. Estes mecanismos interagem entre si, contribuindo para a formação do epigenoma espermático, que modela a carga molecular espermática, que, por sua vez, pode impactar sobre as características do desenvolvimento embrionário e da progênie. Dessa forma, atualmente é consenso que o papel do espermatozoide ultrapassa a entrega de DNA de qualidade para o oócito no momento da fecundação. Pesquisas recentes de diversos grupos, incluindo o nosso, mostram que além da contribuição com DNA de qualidade, o espermatozoide entrega moléculas ao oócito no momento da fecundação que influenciam o desenvolvimento do embrião. Recentemente, essas moléculas de origem espermática (Em inglês: sperm-borne) também são associadas com alterações metabólicas e cognitivas da progênie. Embora ainda pouco se entenda como esses mecanismos podem persistir mesmo com o ciclo de reprogramação celular que ocorre logo após a fecundação, é evidente que estes podem impactar as características da progênie. Nesta revisão abordaremos sobre a modulação do epigenoma espermático e seus efeitos no desenvolvimento embrionário.(AU)

Since it is a highly specialized cell, the spermatozoa display different epigenetic mechanisms; the main ones are DNA methylation, histone code, ncRNAs (non-coding RNAs), and high chromatin condensation by the presence of protamines. These mechanisms act in synergy contributing to forming the sperm epigenome, which modulates the spermatic molecular cargo, and, may impact embryo and offspring development features. Thus, it is currently a consensus that the role of spermatozoa goes beyond delivering quality DNA to the oocyte at fertilization. Relevant findings from several research groups, including ours, have shown that sperm delivers several molecules to the oocyte at fertilization, beyond the contribution to DNA, which influences the development of the embryo. Recently, these sperm-borne molecules have also been associated with metabolic and cognitive changes in the offspring. Although the mechanism by which these changes can persist even after embryo reprogramming is not completely understood, evidence shows that sperm cell molecular content impacts embryo and offspring development. This review will mainly focus on the modulation of the sperm epigenome and its effects on embryo development.(AU)

Single-base resolution methylomes of somatic embryogenesis in Theobroma cacao L. reveal epigenome modifications associated with somatic embryo abnormalities.

Propagation by somatic embryogenesis in Theobroma cacao has some issues to be solved, as many morphologically abnormal somatic embryos that do not germinate into plants are frequently observed, thus hampering plant production on a commercial scale. For the first time the methylome landscape of T. cacao somatic embryogenesis was examined, using whole-genome bisulfite sequencing technique, with the aim to understand the epigenetic basis of somatic embryo abnormalities. We identified 873 differentially methylated genes (DMGs) in the CpG context between zygotic embryos, normal and abnormal somatic embryos, with important roles in development, programmed cell death, oxidative stress, and hypoxia induction, which can help to explain the morphological abnormalities of somatic embryos. We also identified the role of ethylene and its precursor 1-aminocyclopropane-1-carboxylate in several biological processes, such as hypoxia induction, cell differentiation and cell polarity, that could be associated to the development of abnormal somatic embryos. The biological processes and the hypothesis of ethylene and its precursor involvement in the somatic embryo abnormalities in cacao are discussed.

Epigenome-wide association study and epigenetic age acceleration associated with cigarette smoking among Costa Rican adults.

Smoking-associated DNA methylation (DNAm) signatures are reproducible among studies of mostly European descent, with mixed evidence if smoking accelerates epigenetic aging and its relationship to longevity. We evaluated smoking-associated DNAm signatures in the Costa Rican Study on Longevity and Healthy Aging (CRELES), including participants from the high longevity region of Nicoya. We measured genome-wide DNAm in leukocytes, tested Epigenetic Age Acceleration (EAA) from five clocks and estimates of telomere length (DNAmTL), and examined effect modification by the high longevity region. 489 participants had a mean (SD) age of 79.4 (10.8) years, and 18% were from Nicoya. Overall, 7.6% reported currently smoking, 35% were former smokers, and 57.4% never smoked. 46 CpGs and five regions (e.g. AHRR, SCARNA6/SNORD39, SNORA20, and F2RL3) were differentially methylated for current smokers. Former smokers had increased Horvath's EAA (1.69-years; 95% CI 0.72, 2.67), Hannum's EAA (0.77-years; 95% CI 0.01, 1.52), GrimAge (2.34-years; 95% CI1.66, 3.02), extrinsic EAA (1.27-years; 95% CI 0.34, 2.21), intrinsic EAA (1.03-years; 95% CI 0.12, 1.94) and shorter DNAmTL (- 0.04-kb; 95% CI - 0.08, - 0.01) relative to non-smokers. There was no evidence of effect modification among residents of Nicoya. Our findings recapitulate previously reported and novel smoking-associated DNAm changes in a Latino cohort.

Low levels of sulfur and cobalt during the pre- and periconceptional periods affect the oocyte yield of donors and the DNA methylome of preimplantation bovine embryos.

Maternal nutrition is critical in mammalian development, influencing the epigenetic reprogramming of gametes, embryos, and fetal programming. We evaluated the effects of different levels of sulfur (S) and cobalt (Co) in the maternal diet throughout the pre- and periconceptional periods on the biochemical and reproductive parameters of the donors and the DNA methylome of the progeny in Bos indicus cattle. The low-S/Co group differed from the control with respect to homocysteine, folic acid, B12, insulin growth factor 1, and glucose. The oocyte yield was lower in heifers from the low S/Co group than that in the control heifers. Embryos from the low-S/Co group exhibited 2320 differentially methylated regions (DMRs) across the genome compared with the control embryos. We also characterized candidate DMRs linked to the DNMT1 and DNMT3B genes in the blood and sperm cells of the adult progeny. A DMR located in DNMT1 that was identified in embryos remained differentially methylated in the sperm of the progeny from the low-S/Co group. Therefore, we associated changes in specific compounds in the maternal diet with DNA methylation modifications in the progeny. Our results help to elucidate the impact of maternal nutrition on epigenetic reprogramming in livestock, opening new avenues of research to study the effect of disturbed epigenetic patterns in early life on health and fertility in adulthood. Considering that cattle are physiologically similar to humans with respect to gestational length, our study may serve as a model for studies related to the developmental origin of health and disease in humans.

Nuclear and Mitochondrial Genome, Epigenome and Gut Microbiome: Emerging Molecular Biomarkers for Parkinson's Disease.

BACKGROUND: Parkinson's disease (PD) is currently the second most common neurodegenerative disorder, burdening about 10 million elderly individuals worldwide. The multifactorial nature of PD poses a difficult obstacle for understanding the mechanisms involved in its onset and progression. Currently, diagnosis depends on the appearance of clinical signs, some of which are shared among various neurologic disorders, hindering early diagnosis. There are no effective tools to prevent PD onset, detect the disease in early stages or accurately report the risk of disease progression. Hence, there is an increasing demand for biomarkers that may identify disease onset and progression, as treatment-based medicine may not be the best approach for PD. Over the last few decades, the search for molecular markers to predict susceptibility, aid in accurate diagnosis and evaluate the progress of PD have intensified, but strategies aimed to improve individualized patient care have not yet been established. CONCLUSIONS: Genomic variation, regulation by epigenomic mechanisms, as well as the influence of the host gut microbiome seem to have a crucial role in the onset and progress of PD, thus are considered potential biomarkers. As such, the human nuclear and mitochondrial genome, epigenome, and the host gut microbiome might be the key elements to the rise of personalized medicine for PD patients.

Blood lead levels in Peruvian adults are associated with proximity to mining and DNA methylation.

BACKGROUND: Inorganic lead (Pb) is common in the environment, and is toxic to neurological, renal, and cardiovascular systems. Pb exposure influences the epigenome with documented effects on DNA methylation (DNAm). We assessed the impact of low levels of Pb exposure on DNAm among non-miner individuals from two locations in Peru: Lima, the capital, and Cerro de Pasco, a highland mining town, to study the effects of Pb exposure on physiological outcomes and DNAm. METHODS: Pb levels were measured in whole blood (n = 305). Blood leukocyte DNAm was determined for 90 DNA samples using the Illumina MethylationEPIC chip. An epigenome-wide association study was performed to assess the relationship between Pb and DNAm. RESULTS: Individuals from Cerro de Pasco had higher Pb than individuals from Lima (p-value = 2.00E-16). Males had higher Pb than females (p-value = 2.36E-04). Pb was positively associated with hemoglobin (p-value = 8.60E-04). In Cerro de Pasco, blood Pb decreased with the distance from the mine (p-value = 0.04), and association with soil Pb was approaching significance (p-value = 0.08). We identified differentially methylated positions (DMPs) associated with genes SOX18, ZMIZ1, and KDM1A linked to neurological function. We also found 45 differentially methylated regions (DMRs), seven of which were associated with genes involved in metal ion binding and nine to neurological function and development. CONCLUSIONS: Our results demonstrate that even low levels of Pb can have a significant impact on the body including changes to DNAm. We report associations between Pb and hemoglobin, Pb and distance from mining, and between blood and soil Pb. We also report associations between loci- and region-specific DNAm and Pb.

ZNF718, HOXA4, and ZFP57 are differentially methylated in periodontitis in comparison with periodontal health: Epigenome-wide DNA methylation pilot study.

OBJECTIVE: To investigate the differences in the epigenomic patterns of DNA methylation in peripheral leukocytes between patients with periodontitis and gingivally healthy controls evaluating its functional meaning by functional enrichment analysis. BACKGROUND: The DNA methylation profiling of peripheral leukocytes as immune-related tissue potentially relevant as a source of biomarkers between periodontitis patients and gingivally healthy subjects has not been investigated. METHODS: A DNA methylation epigenome-wide study of peripheral leukocytes was conducted using the Illumina MethylationEPIC platform in sixteen subjects, eight diagnosed with periodontitis patients and eight age-matched and sex-matched periodontally healthy controls. A trained periodontist performed the clinical evaluation. Global DNA methylation was estimated using methylation-sensitive high-resolution melting in LINE1. Routine cell count cytometry and metabolic laboratory tests were also performed. The analysis of differentially methylated positions (DMPs) and differentially methylated regions (DMRs) was made using R/Bioconductor environment considering leukocyte populations assessed in both routine cell counts and using the FlowSorted.Blood.EPIC package. Finally, a DMP and DMR intersection analysis was performed. Functional enrichment analysis was carried out with the differentially methylated genes found in DMP. RESULTS: DMP analysis identified 81 differentially hypermethylated genes and 21 differentially hypomethylated genes. Importantly, the intersection analysis showed that zinc finger protein 718 (ZNF718) and homeobox A4 (HOXA4) were differentially hypermethylated and zinc finger protein 57 (ZFP57) was differentially hypomethylated in periodontitis. The functional enrichment analysis found clearly immune-related ontologies such as "detection of bacterium" and "antigen processing and presentation." CONCLUSION: The results of this study propose three new periodontitis-related genes: ZNF718, HOXA4, and ZFP57 but also evidence the suitability and relevance of studying leukocytes' DNA methylome for biological interpretation of systemic immune-related epigenetic patterns in periodontitis.

Aging and rejuvenation - a modular epigenome model.

The view of aging has evolved in parallel with the advances in biomedical sciences. Long considered as an irreversible process where interventions were only aimed at slowing down its progression, breakthrough discoveries like animal cloning and cell reprogramming have deeply changed our understanding of postnatal development, giving rise to the emerging view that the epigenome is the driver of aging. The idea was significantly strengthened by the converging discovery that DNA methylation (DNAm) at specific CpG sites could be used as a highly accurate biomarker of age defined by an algorithm known as the Horvath clock. It was at this point where epigenetic rejuvenation came into play as a strategy to reveal to what extent biological age can be set back by making the clock tick backwards. Initial evidence suggests that when the clock is forced to tick backwards in vivo, it is only able to drag the phenotype to a partially rejuvenated condition. In order to explain the results, a bimodular epigenome is proposed, where module A represents the DNAm clock component and module B the remainder of the epigenome. Epigenetic rejuvenation seems to hold the key to arresting or even reversing organismal aging.

Transcriptome and methylome analysis reveals three cellular origins of pituitary tumors.

Pituitary adenomas (PA) are the second most common intracranial tumors. These neoplasms are classified according to the hormone they produce. The majority of PA occur sporadically, and their molecular pathogenesis is incompletely understood. The present transcriptomic and methylomic analysis of PA revealed that they segregate into three molecular clusters according to the transcription factor driving their terminal differentiation. First cluster, driven by NR5A1, consists of clinically non-functioning PA (CNFPA), comprising gonadotrophinomas and null cell; the second cluster consists of clinically evident ACTH adenomas and silent corticotroph adenomas, driven by TBX19; and the third, POU1F1-driven TSH-, PRL- and GH-adenomas, segregated together. Genes such as CACNA2D4, EPHA4 and SLIT1, were upregulated in each of these three clusters, respectively. Pathway enrichment analysis revealed specific alterations of these clusters: calcium signaling pathway in CNFPA; renin-angiotensin system for ACTH-adenomas and fatty acid metabolism for the TSH-, PRL-, GH-cluster. Non-tumoral pituitary scRNAseq data confirmed that this clustering also occurs in normal cytodifferentiation. Deconvolution analysis identify potential mononuclear cell infiltrate in PA consists of dendritic, NK and mast cells. Our results are consistent with a divergent origin of PA, which segregate into three clusters that depend on the specific transcription factors driving late pituitary cytodifferentiation.

Highly accurate skin-specific methylome analysis algorithm as a platform to screen and validate therapeutics for healthy aging.

BACKGROUND: DNA methylation (DNAm) age constitutes a powerful tool to assess the molecular age and overall health status of biological samples. Recently, it has been shown that tissue-specific DNAm age predictors may present superior performance compared to the pan- or multi-tissue counterparts. The skin is the largest organ in the body and bears important roles, such as body temperature control, barrier function, and protection from external insults. As a consequence of the constant and intimate interaction between the skin and the environment, current DNAm estimators, routinely trained using internal tissues which are influenced by other stimuli, are mostly inadequate to accurately predict skin DNAm age. RESULTS: In the present study, we developed a highly accurate skin-specific DNAm age predictor, using DNAm data obtained from 508 human skin samples. Based on the analysis of 2,266 CpG sites, we accurately calculated the DNAm age of cultured skin cells and human skin biopsies. Age estimation was sensitive to the biological age of the donor, cell passage, skin disease status, as well as treatment with senotherapeutic drugs. CONCLUSIONS: This highly accurate skin-specific DNAm age predictor constitutes a holistic tool that will be of great use in the analysis of human skin health status/molecular aging, as well as in the analysis of the potential of established and novel compounds to alter DNAm age.

Expression Quantitative Trait Methylation Analysis Reveals Methylomic Associations With Gene Expression in Childhood Asthma.

BACKGROUND: Nasal (airway) epithelial methylation profiles have been associated with asthma, but the effects of such profiles on expression of distant cis-genes are largely unknown. RESEARCH QUESTION: To identify genes whose expression is associated with proximal and distal CpG probes (within 1 Mb), and to assess whether and how such genes are differentially expressed in atopic asthma. STUDY DESIGN AND METHODS: Genome-wide expression quantitative trait methylation (eQTM) analysis in nasal epithelium from Puerto Rican subjects (aged 9-20 years) with (n = 219) and without (n = 236) asthma. After the eQTM analysis, a Gene Ontology Enrichment analysis was conducted for the top 500 eQTM genes, and mediation analyses were performed to identify paths from DNA methylation to atopic asthma through gene expression. Asthma was defined as physician-diagnosed asthma and wheeze in the previous year, and atopy was defined as at least one positive IgE to allergens. Atopic asthma was defined as the presence of both atopy and asthma. RESULTS: We identified 16,867 significant methylation-gene expression pairs (false-discovery rate-adjusted P < .01) in nasal epithelium from study participants. Most eQTM methylation probes were distant (average distance, ∼378 kb) from their target genes, and also more likely to be located in enhancer regions of their target genes in lung tissue than control probes. The top 500 eQTM genes were enriched in pathways for immune processes and epithelial integrity and were more likely to have been previously identified as differentially expressed in atopic asthma. In a mediation analysis, we identified 5,934 paths through which methylation markers could affect atopic asthma through gene expression in nasal epithelium. INTERPRETATION: Previous epigenome-wide association studies of asthma have estimated the effects of DNA methylation markers on expression of nearby genes in airway epithelium. Our findings suggest that distant epigenetic regulation of gene expression in airway epithelium plays a role in atopic asthma.