Frequency of natural regulatory T cells specific for factor VIII in the peripheral blood of healthy donors.

Tolerance to self-proteins involves multiple mechanisms, including conventional CD4+ T-cell (Tconv) deletion in the thymus and the recruitment of natural regulatory T cells (nTregs). The significant incidence of autoantibodies specific for the blood coagulation factor VIII (FVIII) in healthy donors illustrates that tolerance to self-proteins is not always complete. In contrast to FVIII-specific Tconvs, FVIII-specific nTregs have never been revealed and characterized. To determine the frequency of FVIII-specific Tregs in human peripheral blood, we assessed the specificity of in vitro expanded Tregs by the membrane expression of the CD137 activation marker. Amplified Tregs maintain high levels of FOXP3 expression and exhibit almost complete demethylation of the FOXP3 Treg-specific demethylated region. The cells retained FOXP3 expression after long-term culture in vitro, strongly suggesting that FVIII-specific Tregs are derived from the thymus. From eleven healthy donors, we estimated the frequencies of FVIII-specific Tregs at 0.17 cells per million, which is about 10-fold lower than the frequency of FVIII-specific CD4+ T cells we previously published. Our results shed light on the mechanisms of FVIII tolerance by a renewed approach that could be extended to other self- or non-self-antigens.

Whole-Genome Bisulfite Sequencing Protocol for the Analysis of Genome-Wide DNA Methylation and Hydroxymethylation Patterns at Single-Nucleotide Resolution.

The analysis of genome-wide epigenomic alterations including DNA methylation and hydroxymethylation has become a subject of intensive research for many biological and clinical questions. DNA methylation analysis bears the particular promise to supplement or replace biochemical and imaging-based tests for the next generation of personalized medicine. Whole-genome bisulfite sequencing (WGBS) using next-generation sequencing technologies is currently considered the gold standard for a comprehensive and quantitative analysis of DNA methylation throughout the genome. However, bisulfite conversion does not allow distinguishing between cytosine methylation and hydroxymethylation requiring an additional chemical or enzymatic step to identify hydroxymethylated cytosines. Here, we provide a detailed protocol based on a commercial kit for the preparation of sequencing libraries for the comprehensive whole-genome analysis of DNA methylation and/or hydroxymethylation. The protocol is based on the construction of sequencing libraries from limited amounts of input DNA by ligation of methylated adaptors to the fragmented DNA prior to bisulfite conversion. For analyses requiring a quantitative distinction between 5-methylcytosine and 5-hydroxymethylcytosines levels, an oxidation step is included in the same workflow to perform oxidative bisulfite sequencing (OxBs-Seq). In this case, two sequencing libraries will be generated and sequenced: a classic methylome following bisulfite conversion and analyzing modified cytosines (not distinguishing between methylated and hydroxymethylated cytosines) and a methylome analyzing only methylated cytosines, respectively. Hydroxymethylation levels are deduced from the differences between the two reactions. We also provide a step-by-step description of the data analysis using publicly available bioinformatic tools. The described protocol has been successfully applied to different human and plant samples and yields robust and reproducible results.

Placental DNA methylation signatures of prenatal air pollution exposure and potential effects on birth outcomes: an analysis of three prospective cohorts.

BACKGROUND: Pregnancy air pollution exposure (PAPE) has been linked to a wide range of adverse birth and childhood outcomes, but there is a paucity of data on its influence on the placental epigenome, which can regulate the programming of physiological functions and affect child development. This study aimed to investigate the association between prenatal air pollutant exposure concentrations and changes in placental DNA methylation patterns, and to explore the potential windows of susceptibility and sex-specific alterations. METHODS: This multi-site study used three prospective population-based mother-child cohorts: EDEN, PELAGIE, and SEPAGES, originating from four French geographical regions (Nancy, Poitiers, Brittany, and Grenoble). Pregnant women were included between 2003 and 2006 for EDEN and PELAGIE, and between 2014 and 2017 for SEPAGES. The main eligibility criteria were: being older than 18 years, having a singleton pregnancy, and living and planning to deliver in one of the maternity clinics in one of the study areas. A total of 1539 mother-child pairs were analysed, measuring placental DNA methylation using Illumina BeadChips. We used validated spatiotemporally resolved models to estimate PM2·5, PM10, and NO2 exposure over each trimester of pregnancy at the maternal residential address. We conducted a pooled adjusted epigenome-wide association study to identify differentially methylated 5'-C-phosphate-G-3' (CpG) sites and regions (assessed using the Infinium HumanMethylationEPIC BeadChip array, n=871), including sex-specific and sex-linked alterations, and independently validated our results (assessed using the Infinium HumanMethylation450 BeadChip array, n=668). FINDINGS: We identified four CpGs and 28 regions associated with PAPE in the total population, 469 CpGs and 87 regions in male infants, and 150 CpGs and 66 regions in female infants. We validated 35% of the CpGs available. More than 30% of the identified CpGs were related to one (or more) birth outcome and most significant alterations were enriched for neural development, immunity, and metabolism related genes. The 28 regions identified for both sexes overlapped with imprinted genes (four genes), and were associated with neurodevelopment (nine genes), immune system (seven genes), and metabolism (five genes). Most associations were observed for the third trimester for female infants (134 of 150 CpGs), and throughout pregnancy (281 of 469 CpGs) and the first trimester (237 of 469 CpGs) for male infants. INTERPRETATION: These findings highlight the molecular pathways through which PAPE might affect child health in a widespread and sex-specific manner, identifying the genes involved in the major physiological functions of a developing child. Further studies are needed to elucidate whether these epigenetic changes persist and affect health later in life. FUNDING: French Agency for National Research, Fondation pour la Recherche Médicale, Fondation de France, and the Plan Cancer.

An integrated omics approach highlights how epigenetic events can explain and predict response to neoadjuvant chemotherapy and bevacizumab in breast cancer.

Treatment with the anti-angiogenic drug bevacizumab in addition to chemotherapy has shown efficacy for breast cancer in some clinical trials, but better biomarkers are needed to optimally select patients for treatment. Here, we present an omics approach where DNA methylation profiles are integrated with gene expression and results from proteomic data in breast cancer patients to predict response to therapy and pinpoint response-related epigenetic events. Fresh-frozen tumor biopsies taken before, during, and after treatment from human epidermal growth factor receptor 2 negative non-metastatic patients receiving neoadjuvant chemotherapy with or without bevacizumab were subjected to molecular profiling. Here, we report that DNA methylation at enhancer CpGs related to cell cycle regulation can predict response to chemotherapy and bevacizumab for the estrogen receptor positive subset of patients (AUC = 0.874), and we validated this observation in an independent patient cohort with a similar treatment regimen (AUC = 0.762). Combining the DNA methylation scores with the scores from a previously published protein signature resulted in a slight increase in the prediction performance (AUC = 0.784). We also show that tumors receiving the combination treatment underwent more extensive epigenetic alterations. Finally, we performed an integrative expression-methylation quantitative trait loci analysis on alterations in DNA methylation and gene expression levels, showing that the epigenetic alterations that occur during treatment are different between responders and non-responders and that these differences may be explained by the proliferation-epithelial-to-mesenchymal transition axis through the activity of grainyhead like transcription factor 2. Using tumor purity computed from copy number data, we developed a method for estimating cancer cell-specific methylation to confirm that the association to response reflects DNA methylation in cancer cells. Taken together, these results support the potential for clinical benefit of the addition of bevacizumab to chemotherapy when administered to the correct patients.

Epigenetic footprints: Investigating placental DNA methylation in the context of prenatal exposure to phenols and phthalates.

BACKGROUND: Endocrine disrupting compounds (EDCs) such as phthalates and phenols can affect placental functioning and fetal health, potentially via epigenetic modifications. We investigated the associations between pregnancy exposure to synthetic phenols and phthalates estimated from repeated urine sampling and genome wide placental DNA methylation. METHODS: The study is based on 387 women with placental DNA methylation assessed with Infinium MethylationEPIC arrays and with 7 phenols, 13 phthalates, and two non-phthalate plasticizer metabolites measured in pools of urine samples collected twice during pregnancy. We conducted an exploratory analysis on individual CpGs (EWAS) and differentially methylated regions (DMRs) as well as a candidate analysis focusing on 20 previously identified CpGs. Sex-stratified analyses were also performed. RESULTS: In the exploratory analysis, when both sexes were studied together no association was observed in the EWAS. In the sex-stratified analysis, 114 individual CpGs (68 in males, 46 in females) were differentially methylated, encompassing 74 genes (36 for males and 38 for females). We additionally identified 28 DMRs in the entire cohort, 40 for females and 42 for males. Associations were mostly positive (for DMRs: 93% positive associations in the entire cohort, 60% in the sex-stratified analysis), with the exception of several associations for bisphenols and DINCH metabolites that were negative. Biomarkers associated with most DMRs were parabens, DEHP, and DiNP metabolite concentrations. Some DMRs encompassed imprinted genes including APC (associated with parabens and DiNP metabolites), GNAS (bisphenols), ZIM2;PEG3;MIMT1 (parabens, monoethyl phthalate), and SGCE;PEG10 (parabens, DINCH metabolites). Terms related to adiposity, lipid and glucose metabolism, and cardiovascular function were among the enriched phenotypes associated with differentially methylated CpGs. The candidate analysis identified one CpG mapping to imprinted LGALS8 gene, negatively associated with ethylparaben. CONCLUSIONS: By combining improved exposure assessment and extensive placental epigenome coverage, we identified several novel genes associated with the exposure, possibly in a sex-specific manner.

Tunable DNMT1 degradation reveals DNMT1/DNMT3B synergy in DNA methylation and genome organization.

DNA methylation (DNAme) is a key epigenetic mark that regulates critical biological processes maintaining overall genome stability. Given its pleiotropic function, studies of DNAme dynamics are crucial, but currently available tools to interfere with DNAme have limitations and major cytotoxic side effects. Here, we present cell models that allow inducible and reversible DNAme modulation through DNMT1 depletion. By dynamically assessing whole genome and locus-specific effects of induced passive demethylation through cell divisions, we reveal a cooperative activity between DNMT1 and DNMT3B, but not of DNMT3A, to maintain and control DNAme. We show that gradual loss of DNAme is accompanied by progressive and reversible changes in heterochromatin, compartmentalization, and peripheral localization. DNA methylation loss coincides with a gradual reduction of cell fitness due to G1 arrest, with minor levels of mitotic failure. Altogether, this system allows DNMTs and DNA methylation studies with fine temporal resolution, which may help to reveal the etiologic link between DNAme dysfunction and human disease.