BACKGROUND: Previous studies have identified TET1 as a potential key regulator of genes linked to asthma. TET1 has been shown to transcriptionally respond to house dust mite extract, an allergen known to directly cause allergic asthma development, and regulate the expression of genes involved in asthma. How TET1 regulates expression of these genes, however, is unknown. TET1 is a DNA demethylase; therefore, most prior research on TET1-based gene regulation has focused on how TET1 affects methylation. However, TET1 can also interact directly with transcription factors and histone modifiers to regulate gene expression. Understanding how TET1 regulates expression to contribute to allergic responses and asthma development thus requires a comprehensive approach. To this end, we measured mRNA expression, DNA methylation, chromatin accessibility and histone modifications in control and TET1 knockdown human bronchial epithelial cells treated or untreated with house dust mite extract. RESULTS: Throughout our analyses, we detected strong similarities between the effects of TET1 knockdown alone and the effects of HDM treatment alone. One especially striking pattern was that both TET1 knockdown and HDM treatment generally led to decreased chromatin accessibility at largely the same genomic loci. Transcription factor enrichment analyses indicated that altered chromatin accessibility following the loss of TET1 may affect, or be affected by, CTCF and CEBP binding. TET1 loss also led to changes in DNA methylation, but these changes were generally in regions where accessibility was not changing. CONCLUSIONS: TET1 regulates gene expression through different mechanisms (DNA methylation and chromatin accessibility) in different parts of the genome in the airway epithelial cells, which mediates inflammatory responses to allergen. Collectively, our data suggest novel molecular mechanisms through which TET1 regulates critical pathways following allergen challenges and contributes to the development of asthma.

Exposures to diesel exhaust particles (DEP) from traffic and house dust mite (HDM) allergens significantly increase risks of airway diseases, including asthma. This negative impact of DEP and HDM may in part be mediated by epigenetic mechanisms. Beyond functioning as a mechanical barrier, airway epithelial cells provide the first line of immune defense towards DEP and HDM exposures. To understand the epigenetic responses of airway epithelial cells to these exposures, we exposed human bronchial epithelial cells to DEP and HDM and studied genome-wide 5-methyl-cytosine (5mC) and 5-hydroxy-methylcytosine (5hmC) at base resolution. We found that exposures to DEP and HDM result in elevated TET1 and DNMT1 expression, associated with 5mC and 5hmC changes. Interestingly, over 20% of CpG sites are responsive to both exposures and changes in 5mC at these sites negatively correlated with gene expression differences. These 5mC and 5hmC changes are located in genes and pathways related to oxidative stress responses, epithelial function and immune cell responses and are enriched for binding sites of transcription factors (TFs) involved in these pathways. Histone marks associated with promoters, enhancers and actively transcribed gene bodies were associated with exposure-induced DNA methylation changes. Collectively, our data suggest that exposures to DEP and HDM alter 5mC and 5hmC levels at regulatory regions bound by TFs, which coordinate with histone marks to regulate gene networks of oxidative stress responses, epithelial function and immune cell responses. These observations provide novel insights into the epigenetic mechanisms that mediate the epithelial responses to DEP and HDM in airways.

We have reported child anxiety sensitivity (Child Anxiety Sensitivity Index [CASI]) predicts chronic postsurgical pain (CPSP). Herein, we evaluated DNA methylation profiles to understand the gene-environment interactions underlying CPSP and CASI, to identify shared, enriched, genomic pathways. In 73 prospectively recruited adolescents undergoing spine fusion, preoperative CASI and pain data over 12 months after surgery were collected. DNA from the peripheral blood of evaluable subjects with (n = 16) and without CPSP (n = 40) were analyzed using MethylationEPIC arrays. We identified 637 and 2,445 differentially DNA methylated positions (DMPs) associated with CPSP and CASI, respectively (P ≤ .05). Ingenuity pathway analysis of 39 genes with DMPs for both CPSP and CASI revealed enrichment of several canonical pathways, including GABA receptor (P = .00016 for CPSP; P =.0008 for CASI) and dopamine-DARPP32 feedback in cyclic adenosine monophosphate (P = .004 for CPSP and P =.00003 for CASI) signaling. Gene-gene interaction network enrichment analysis revealed participation of pathways in cell signaling, molecular transport, metabolism, and neurologic diseases (P < 10^-8). Bioinformatic approaches to identify histone marks and transcription factor (TF) binding events underlying DMPs, showed their location in active regulatory regions in pain pathway relevant brain cells. Using Enrichr/Pinet enrichment and Library of Integrated Network-Based Cellular Signatures knockdown signatures, we identified TFs regulating genes with DMPs in association with CPSP and CASI. In conclusion, we identified epigenetically enriched pathways associated with CPSP and anxiety sensitivity in children undergoing surgery. Our findings support GABA hypofunction and the roles of the dopamine-DARPP32 pathway in emotion/reward and pain. This pilot study provides new epigenetic insights into the pathophysiology of CPSP and a basis for future studies in biomarker development and targetable interventions. PERSPECTIVE: Differential DNA methylation in regulatory genomic regions enriching shared neural pathways were associated with CPSP and CASI in adolescents undergoing spine surgery. Our findings support GABA hypofunction and the roles of the dopamine-DARPP32 pathway in emotion/reward contributing to behavioral maintenance of pain 10 to 12 months after surgery.

Background: Overlap of pathways enriched by single nucleotide polymorphisms and DNA-methylation underlying chronic postsurgical pain (CPSP), prompted pilot study of CPSP-associated methylation quantitative trait loci (meQTL). Materials & methods: Children undergoing spine-fusion were recruited prospectively. Logistic-regression for genome- and epigenome-wide CPSP association and DNA-methylation-single nucleotide polymorphism association/mediation analyses to identify meQTLs were followed by functional genomics analyses. Results: CPSP (n = 20/58) and non-CPSP groups differed in pain-measures. Of 2753 meQTLs, DNA-methylation at 127 cytosine-guanine dinucleotides mediated association of 470 meQTLs with CPSP (p < 0.05). At PARK16 locus, CPSP risk meQTLs were associated with decreased DNA-methylation at RAB7L1 and increased DNA-methylation at PM20D1. Corresponding RAB7L1/PM20D1 blood eQTLs (GTEx) and cytosine-guanine dinucleotide-loci enrichment for histone marks, transcription factor binding sites and ATAC-seq peaks suggest altered transcription factor-binding. Conclusion: CPSP-associated meQTLs indicate epigenetic mechanisms mediate genetic risk. Clinical trial registration: NCT01839461, NCT01731873 (ClinicalTrials.gov).

AIM:We aim to study DNA methylation (DNAm) variations associated with childhood asthma. METHODS:Nasal DNAm was compared between sibling pairs discordant for asthma, 29 sib pairs for genome-wide association studies and 54 sib pairs for verification by pyrosequencing. Associations of methylation with asthma symptoms, allergy and environmental exposures were evaluated. In vitro experiments and functional genomic analyses were performed to explore biologic relevance. RESULTS:Three CpGs were associated with asthma. cg14830002 was associated with allergies in nonasthmatics. cg23602092 was associated with asthma symptoms. cg14830002 and cg23602092 were associated with traffic-related air pollution exposure. Nearby genes were transcriptionally regulated by diesel exhaust, house dust mite and 5-aza-2'-deoxycytidine. Active chromatin marks and transcription factor binding were found around these sites. CONCLUSION:We identified novel DNAm variations associated with childhood asthma and suggested new disease-contributing epigenetic mechanisms.

Previous studies have suggested a role for Tet1 in the pathogenesis of childhood asthma. However, how Tet1 contributes to asthma remains unknown. Here we used mice deficient for Tet1 in a well-established model of allergic airway inflammation and demonstrated that loss of Tet1 increased disease severity including airway hyperresponsiveness and lung eosinophilia. Increased expression of Muc5ac, Il13, Il33, Il17a, Egfr, and Tff2 were observed in HDM-challenged Tet1-deficient mice compared to Tet1^+/+ littermates. Further, transcriptomic analysis of lung RNA followed by pathway and protein network analysis showed that the IFN signaling pathway was significantly upregulated and the aryl hydrocarbon receptor (AhR) pathway was significantly downregulated in HDM-challenged Tet1^-/- mice. This transcriptional regulation of the IFN and AhR pathways by Tet1 was also present in human bronchial epithelial cells at base line and following HDM challenges. Genes in these pathways were further associated with changes in DNA methylation, predicted binding of transcriptional factors with relevant functions in their promoters, and the presence of histone marks generated by histone enzymes that are known to interact with Tet1. Collectively, our data suggest that Tet1 inhibits HDM-induced allergic airway inflammation by direct regulation of the IFN and AhR pathways.

Background

Asthma is highly heterogeneous, and severity evaluation is key to asthma management. DNA methylation (DNAm) contributes to asthma pathogenesis. This study aimed to identify nasal epithelial DNAm differences between severe and nonsevere asthmatic children and evaluate the impact of environmental exposures.

Methods

Thirty-three nonsevere and 22 severe asthmatic African American children were included in an epigenome-wide association study. Genome-wide nasal epithelial DNAm and gene expression were measured. CpG sites associated with asthma severity and environmental exposures and predictive of severe asthma were identified. DNAm was correlated with gene expression. Enrichment for transcription factor (TF) binding sites or histone modifications surrounding DNAm differences were determined.

Results

We identified 816 differentially methylated CpG positions (DMPs) and 10 differentially methylated regions (DMRs) associated with asthma severity. Three DMPs exhibited discriminatory ability for severe asthma. Intriguingly, six DMPs were simultaneously associated with asthma, allergic asthma, total IgE, environmental IgE, and FeNO in an independent cohort of children. Twenty-seven DMPs were associated with traffic-related air pollution or secondhand smoke. DNAm at 22 DMPs was altered by diesel particles or allergen in human bronchial epithelial cells. DNAm levels at 39 DMPs were correlated with mRNA expression. Proximal to 816 DMPs, three histone marks and several TFs involved in asthma pathogenesis were enriched.

Conclusions

Significant differences in nasal epithelial DNAm were observed between nonsevere and severe asthma in African American children, a subset of which may be useful to predict disease severity. These CpG sites are subjected to the influences of environmental exposures and may regulate gene expression.

INTRODUCTION:The perioperative pain experience shows great interindividual variability and is difficult to predict. The mu-1 opioid receptor gene (OPRM1) is known to play an important role in opioid-pain pathways. Since deoxyribonucleic acid (DNA) methylation is a potent repressor of gene expression, DNA methylation was evaluated at the OPRM1 promoter, as a predictor of preoperative, acute, and chronic postsurgical pain (CPSP). METHODS:A prospective observational cohort study was conducted in 133 adolescents with idiopathic scoliosis undergoing spine fusion under standard protocols. Data regarding pain, opioid consumption, anxiety, and catastrophizing (using validated questionnaires) were collected before and 2-3 months postsurgery. Outcomes evaluated were preoperative pain, acute postoperative pain (area under curve [AUC] for pain scores over 48 hours), and CPSP (numerical rating scale >3/10 at 2-3 months postsurgery). Blood samples collected preoperatively were analyzed for DNA methylation by pyrosequencing of 22 CpG sites at the OPRM1 gene promoter. The association of each pain outcome with the methylation percentage of each CpG site was assessed using multivariable regression, adjusting for significant (P<0.05) nongenetic variables. RESULTS:Majority (83%) of the patients reported no pain preoperatively, while CPSP occurred in 36% of the subjects (44/121). Regression on dichotomized preoperative pain outcome showed association with methylation at six CpG sites (1, 3, 4, 9, 11, and 17) (P<0.05). Methylation at CpG sites 4, 17, and 18 was associated with higher AUC after adjusting for opioid consumption and preoperative pain score (P<0.05). After adjusting for postoperative opioid consumption and preoperative pain score, methylation at CpG sites 13 and 22 was associated with CPSP (P<0.05). DISCUSSION:Novel CPSP biomarkers were identified in an active regulatory region of the OPRM1 gene that binds multiple transcription factors. Inhibition of binding by DNA methylation potentially decreases the OPRM1 gene expression, leading to a decreased response to endogenous and exogenous opioids, and an increased pain experience.

Background

Treatment response to systemic corticosteroid in asthmatic children is heterogeneous and may be mediated by epigenetic mechanism(s). We aim to identify DNA methylation (DNAm) changes responsive to steroid, and DNAm biomarkers that distinguish treatment response.

Materials and methods

We followed 33 children (ages 5-18) presenting to the Emergency Department (ED) for asthma exacerbation. Based on whether they met discharge criteria in ≤24 hours, participants were grouped into good and poor responders to steroid treatment. Nasal samples were collected upon presentation to the ED (T0) and 18-24 hours later (T1). Genome-wide DNAm was measured for both time points in 20 subjects, and compared between T0 and T1 in good and poor responders respectively. DNAm at T1 was also compared between two responder groups. DNAm of selected CpGs was verified in the complete cohort, and expression of associated genes was examined. Interactions between DNAm, common single nucleotide polymorphism (SNP) located at the CpG sites and treatment responses were assessed.

Results

Three CpGs located in the OTX2 promoter showed responder-specific DNAm changes from T0 to T1, in which DNAm decreased in good but not in poor responders. Good and poor responders showed differential DNAm at T1 in 127 CpGs without and 182 CpGs with common SNP co-localization. Negative correlations between DNAm and gene expression were observed at CpGs located within the LDHC promoter, suggesting an impact of DNAm on gene regulation. Interactions between SNPs, DNAm and treatment response were detected.

Conclusion

Acute systemic steroid treatment modifies nasal DNAm in good responders. Nasal DNAm, dependent or independent of SNPs, can differentiate response to treatment in acute asthmatic children.

Background

Dendritic cells (DCs) are important mediators of innate and adaptive immune responses, but the gene networks governing their lineage differentiation and maturation are poorly understood. To gain insight into the mechanisms that promote human DC differentiation and contribute to the acquisition of their functional phenotypes, we performed genome-wide base-resolution mapping of 5-methylcytosine in purified monocytes and in monocyte-derived immature and mature DCs.

Results

DC development and maturation were associated with a great loss of DNA methylation across many regions, most of which occurs at predicted enhancers and binding sites for known transcription factors affiliated with DC lineage specification and response to immune stimuli. In addition, we discovered novel genes that may contribute to DC differentiation and maturation. Interestingly, many genes close to demethylated CG sites were upregulated in expression. We observed dynamic changes in the expression of TET2, DNMT1, DNMT3A and DNMT3B coupled with temporal locus-specific demethylation, providing possible mechanisms accounting for the dramatic loss in DNA methylation.

Conclusions

Our study is the first to map DNA methylation changes during human DC differentiation and maturation in purified cell populations and will greatly enhance the understanding of DC development and maturation and aid in the development of more efficacious DC-based therapeutic strategies.