LuxRep: a technical replicate-aware method for bisulfite sequencing data analysis.

BACKGROUND: DNA methylation is commonly measured using bisulfite sequencing (BS-seq). The quality of a BS-seq library is measured by its bisulfite conversion efficiency. Libraries with low conversion rates are typically excluded from analysis resulting in reduced coverage and increased costs. RESULTS: We have developed a probabilistic method and software, LuxRep, that implements a general linear model and simultaneously accounts for technical replicates (libraries from the same biological sample) from different bisulfite-converted DNA libraries. Using simulations and actual DNA methylation data, we show that including technical replicates with low bisulfite conversion rates generates more accurate estimates of methylation levels and differentially methylated sites. Moreover, using variational inference speeds up computation time necessary for whole genome analysis. CONCLUSIONS: In this work we show that taking into account technical replicates (i.e. libraries) of BS-seq data of varying bisulfite conversion rates, with their corresponding experimental parameters, improves methylation level estimation and differential methylation detection.

Early DNA methylation changes in children developing beta cell autoimmunity at a young age.

AIMS/HYPOTHESIS: Type 1 diabetes is a chronic autoimmune disease of complex aetiology, including a potential role for epigenetic regulation. Previous epigenomic studies focused mainly on clinically diagnosed individuals. The aim of the study was to assess early DNA methylation changes associated with type 1 diabetes already before the diagnosis or even before the appearance of autoantibodies. METHODS: Reduced representation bisulphite sequencing (RRBS) was applied to study DNA methylation in purified CD4+ T cell, CD8+ T cell and CD4-CD8- cell fractions of 226 peripheral blood mononuclear cell samples longitudinally collected from seven type 1 diabetes-specific autoantibody-positive individuals and control individuals matched for age, sex, HLA risk and place of birth. We also explored correlations between DNA methylation and gene expression using RNA sequencing data from the same samples. Technical validation of RRBS results was performed using pyrosequencing. RESULTS: We identified 79, 56 and 45 differentially methylated regions in CD4+ T cells, CD8+ T cells and CD4-CD8- cell fractions, respectively, between type 1 diabetes-specific autoantibody-positive individuals and control participants. The analysis of pre-seroconversion samples identified DNA methylation signatures at the very early stage of disease, including differential methylation at the promoter of IRF5 in CD4+ T cells. Further, we validated RRBS results using pyrosequencing at the following CpG sites: chr19:18118304 in the promoter of ARRDC2; chr21:47307815 in the intron of PCBP3; and chr14:81128398 in the intergenic region near TRAF3 in CD4+ T cells. CONCLUSIONS/INTERPRETATION: These preliminary results provide novel insights into cell type-specific differential epigenetic regulation of genes, which may contribute to type 1 diabetes pathogenesis at the very early stage of disease development. Should these findings be validated, they may serve as a potential signature useful for disease prediction and management.

Umbilical cord blood DNA methylation in children who later develop type 1 diabetes.

AIMS/HYPOTHESIS: Distinct DNA methylation patterns have recently been observed to precede type 1 diabetes in whole blood collected from young children. Our aim was to determine whether perinatal DNA methylation is associated with later progression to type 1 diabetes. METHODS: Reduced representation bisulphite sequencing (RRBS) analysis was performed on umbilical cord blood samples collected within the Finnish Type 1 Diabetes Prediction and Prevention (DIPP) Study. Children later diagnosed with type 1 diabetes and/or who tested positive for multiple islet autoantibodies (n = 43) were compared with control individuals (n = 79) who remained autoantibody-negative throughout the DIPP follow-up until 15 years of age. Potential confounding factors related to the pregnancy and the mother were included in the analysis. RESULTS: No differences in the umbilical cord blood methylation patterns were observed between the cases and controls at a false discovery rate <0.05. CONCLUSIONS/INTERPRETATION: Based on our results, differences between children who progress to type 1 diabetes and those who remain healthy throughout childhood are not yet present in the perinatal DNA methylome. However, we cannot exclude the possibility that such differences would be found in a larger dataset.

Global chromatin state analysis reveals lineage-specific enhancers during the initiation of human T helper 1 and T helper 2 cell polarization.

Naive CD4⁺ T cells can differentiate into specific helper and regulatory T cell lineages in order to combat infection and disease. The correct response to cytokines and a controlled balance of these populations is critical for the immune system and the avoidance of autoimmune disorders. To investigate how early cell-fate commitment is regulated, we generated the first human genome-wide maps of histone modifications that reveal enhancer elements after 72 hr of in vitro polarization toward T helper 1 (Th1) and T helper 2 (Th2) cell lineages. Our analysis indicated that even at this very early time point, cell-specific gene regulation and enhancers were at work directing lineage commitment. Further examination of lineage-specific enhancers identified transcription factors (TFs) with known and unknown T cell roles as putative drivers of lineage-specific gene expression. Lastly, an integrative analysis of immunopathogenic-associated SNPs suggests a role for distal regulatory elements in disease etiology.

Evaluation of tools for identifying large copy number variations from ultra-low-coverage whole-genome sequencing data.

BACKGROUND: Detection of copy number variations (CNVs) from high-throughput next-generation whole-genome sequencing (WGS) data has become a widely used research method during the recent years. However, only a little is known about the applicability of the developed algorithms to ultra-low-coverage (0.0005-0.8×) data that is used in various research and clinical applications, such as digital karyotyping and single-cell CNV detection. RESULT: Here, the performance of six popular read-depth based CNV detection algorithms (BIC-seq2, Canvas, CNVnator, FREEC, HMMcopy, and QDNAseq) was studied using ultra-low-coverage WGS data. Real-world array- and karyotyping kit-based validation were used as a benchmark in the evaluation. Additionally, ultra-low-coverage WGS data was simulated to investigate the ability of the algorithms to identify CNVs in the sex chromosomes and the theoretical minimum coverage at which these tools can accurately function. Our results suggest that while all the methods were able to detect large CNVs, many methods were susceptible to producing false positives when smaller CNVs (< 2 Mbp) were detected. There was also significant variability in their ability to identify CNVs in the sex chromosomes. Overall, BIC-seq2 was found to be the best method in terms of statistical performance. However, its significant drawback was by far the slowest runtime among the methods (> 3 h) compared with FREEC (~ 3 min), which we considered the second-best method. CONCLUSIONS: Our comparative analysis demonstrates that CNV detection from ultra-low-coverage WGS data can be a highly accurate method for the detection of large copy number variations when their length is in millions of base pairs. These findings facilitate applications that utilize ultra-low-coverage CNV detection.

Genome-wide profiling of interleukin-4 and STAT6 transcription factor regulation of human Th2 cell programming.

Dissecting the molecular mechanisms by which T helper (Th) cells differentiate to effector Th2 cells is important for understanding the pathogenesis of immune-mediated diseases, such as asthma and allergy. Because the STAT6 transcription factor is an upstream mediator required for interleukin-4 (IL-4)-induced Th2 cell differentiation, its targets include genes important for this process. Using primary human CD4(+) T cells, and by blocking STAT6 with RNAi, we identified a number of direct and indirect targets of STAT6 with ChIP sequencing. The integration of these data sets with detailed kinetics of IL-4-driven transcriptional changes showed that STAT6 was predominantly needed for the activation of transcription leading to the Th2 cell phenotype. This integrated genome-wide data on IL-4- and STAT6-mediated transcription provide a unique resource for studies on Th cell differentiation and, in particular, for designing interventions of human Th2 cell responses.

The impact of probiotic supplementation during pregnancy on DNA methylation of obesity-related genes in mothers and their children.

PURPOSE: Dietary supplementation with probiotics during pregnancy has been suggested to decrease the risk for obesity in women after delivery and to minimize excessive weight gain in their children. Epigenetic DNA methylation has been proposed to impact on gene activity, thereby providing a plausible molecular mechanism for a broad range of biological processes and diseases. This pilot study aimed to evaluate whether probiotic supplementation during pregnancy could modify the DNA methylation status of the promoters of obesity and weight gain-related genes in mothers and their children. METHODS: A sample of 15 pregnant women was taken from a prospective, randomized mother and infant nutrition and probiotic study. Seven women received the probiotic supplementation and eight served as controls. The women's and their children's DNA methylation status of obesity (623 genes) and weight gain-related (433) gene promoters were analyzed from blood samples at the mean of 9.8 months (range 6.1-12.7 months) postpartum. RESULTS: Probiotic supplementation led to significantly decreased levels of DNA methylation in 37 gene promoters and increased levels of DNA methylation in one gene promoter in women. In their children, 68 gene promoters were significantly affected consistently with a lower level of DNA methylation in the probiotic group. CONCLUSIONS: On the basis of our pilot study, we suggest that probiotic supplementation during pregnancy may affect the DNA methylation status of certain promoters of obesity and weight gain-related genes both in mothers and their children, thereby providing a potential mechanism for long-lasting health effects.

Genetic and epigenetic stability of human pluripotent stem cells.

Studies using high-resolution genome-wide approaches have recently reported that genomic and epigenomic alterations frequently accumulate in human pluripotent cells. Detailed characterization of these changes is crucial for understanding the impact of these alterations on self-renewal and proliferation, and particularly on the developmental and malignant potential of the cells. Such knowledge is required for the optimized and safe use of pluripotent cells for therapeutic purposes, such as regenerative cellular therapies using differentiated derivatives of pluripotent cells.In this Review, we summarize the current knowledge of the genomic and epigenomic stability of pluripotent human cells and the implications for stem cell research.

Activation of Plasmacytoid Dendritic Cells in Colon-Draining Lymph Nodes during Citrobacter rodentium Infection Involves Pathogen-Sensing and Inflammatory Pathways Distinct from Conventional Dendritic Cells.

Dendritic cells (DCs) bear the main responsibility for initiation of adaptive immune responses necessary for antimicrobial immunity. In the small intestine, afferent lymphatics convey Ags and microbial signals to mesenteric lymph nodes (LNs) to induce adaptive immune responses against microbes and food Ags derived from the small intestine. Whether the large intestine is covered by the same lymphatic system or represents its own lymphoid compartment has not been studied until very recently. We identified three small mesenteric LNs, distinct from small intestinal LNs, which drain lymph specifically from the colon, and studied DC responses to the attaching and effacing pathogen Citrobacter rodentium in these. Transcriptional profiling of conventional (CD11c(high)CD103(high)) DC and plasmacytoid (plasmacytoid DC Ag-1(high)B220(+)CD11c(int)) DC (pDC) populations during steady-state conditions revealed activity of distinct sets of genes in these two DC subsets, both in small intestinal and colon-draining LNs. C. rodentium activated DC especially in colon-draining LNs, and gene expression changed in pDC more profoundly than in conventional DC. Among the genes most upregulated in pDC were C-type lectin receptor CLEC4E, IL-1Rs (IL-1R1 and -2), proinflammatory cytokines (IL-1a and IL-6), and TLR6. Our results indicate that colon immune surveillance is distinct from that of the small intestine in terms of draining LNs, and identify pDC as active sentinels of colonic inflammation and/or microbial dysbiosis.

Notch signaling regulates the differentiation of neural crest from human pluripotent stem cells.

Neural crest cells are specified at the border between the neural plate and the epiderm. They are capable of differentiating into various somatic cell types, including craniofacial and peripheral nerve tissues. Notch signaling plays important roles during neurogenesis; however, its function during human neural crest development is poorly understood. Here, we generated self-renewing premigratory neural-crest-like cells (pNCCs) from human pluripotent stem cells (hPSCs) and investigated the roles of Notch signaling during neural crest differentiation. pNCCs expressed various neural-crest-specifier genes, including SLUG (also known as SNAI2), SOX10 and TWIST1, and were able to differentiate into most neural crest derivatives. Blocking Notch signaling during the pNCC differentiation suppressed the expression of neural-crest-specifier genes. By contrast, ectopic expression of activated Notch1 intracellular domain (NICD1) augmented the expression of neural-crest-specifier genes, and NICD1 was found to bind to their promoter regions. Notch activity was also required for the maintenance of the premigratory neural crest state, and the suppression of Notch signaling led to the generation of neural-crest-derived neurons. Taken together, we provide a protocol for the generation of pNCCs and show that Notch signaling regulates the formation, migration and differentiation of neural crest from hPSCs.

RNA-binding protein L1TD1 interacts with LIN28 via RNA and is required for human embryonic stem cell self-renewal and cancer cell proliferation.

Human embryonic stem cells (hESC) have a unique capacity to self-renew and differentiate into all the cell types found in human body. Although the transcriptional regulators of pluripotency are well studied, the role of cytoplasmic regulators is still poorly characterized. Here, we report a new stem cell-specific RNA-binding protein L1TD1 (ECAT11, FLJ10884) required for hESC self-renewal and cancer cell proliferation. Depletion of L1TD1 results in immediate downregulation of OCT4 and NANOG. Furthermore, we demonstrate that OCT4, SOX2, and NANOG all bind to the promoter of L1TD1. Moreover, L1TD1 is highly expressed in seminomas, and depletion of L1TD1 in these cancer cells influences self-renewal and proliferation. We show that L1TD1 colocalizes and interacts with LIN28 via RNA and directly with RNA helicase A (RHA). LIN28 has been reported to regulate translation of OCT4 in complex with RHA. Thus, we hypothesize that L1TD1 is part of the L1TD1-RHA-LIN28 complex that could influence levels of OCT4. Our results strongly suggest that L1TD1 has an important role in the regulation of stemness.

From maternal glucocorticoid and thyroid hormones to epigenetic regulation of offspring gene expression: An experimental study in a wild bird species.

Offspring phenotype at birth is determined by its genotype and the prenatal environment including exposure to maternal hormones. Variation in both maternal glucocorticoids and thyroid hormones can affect offspring phenotype, but the underlying molecular mechanisms, especially those contributing to long-lasting effects, remain unclear. Epigenetic changes (such as DNA methylation) have been postulated as mediators of long-lasting effects of early-life environment. In this study, we determined the effects of elevated prenatal glucocorticoid and thyroid hormones on handling stress response (breath rate) as well as DNA methylation and gene expression of glucocorticoid receptor (GR) and thyroid hormone receptor (THR) in great tits (Parus major). Eggs were injected before incubation onset with corticosterone (the main avian glucocorticoid) and/or thyroid hormones (thyroxine and triiodothyronine) to simulate variation in maternal hormone deposition. Breath rate during handling and gene expression of GR and THR were evaluated 14 days after hatching. Methylation status of GR and THR genes was analyzed from the longitudinal blood cells sampled 7 and 14 days after hatching, as well as the following autumn. Elevated prenatal corticosterone level significantly increased the breath rate during handling, indicating an enhanced metabolic stress response. Prenatal corticosterone manipulation had CpG-site-specific effects on DNA methylation at the GR putative promoter region, while it did not significantly affect GR gene expression. GR expression was negatively associated with earlier hatching date and chick size. THR methylation or expression did not exhibit any significant relationship with the hormonal treatments or the examined covariates, suggesting that TH signaling may be more robust due to its crucial role in development. This study provides some support to the hypothesis suggesting that maternal corticosterone may influence offspring metabolic stress response via epigenetic alterations, yet their possible adaptive role in optimizing offspring phenotype to the prevailing conditions, context-dependency, and the underlying molecular interplay needs further research.

Permutation-based significance analysis reduces the type 1 error rate in bisulphite sequencing data analysis of human umbilical cord blood samples.

DNA methylation patterns are largely established in-utero and might mediate the impacts of in-utero conditions on later health outcomes. Associations between perinatal DNA methylation marks and pregnancy-related variables, such as maternal age and gestational weight gain, have been earlier studied with methylation microarrays, which typically cover less than 2% of human CpG sites. To detect such associations outside these regions, we chose the bisulphite sequencing approach. We collected and curated clinical data on 200 newborn infants; whose umbilical cord blood samples were analysed with the reduced representation bisulphite sequencing (RRBS) method. A generalized linear mixed-effects model was fit for each high coverage CpG site, followed by spatial and multiple testing adjustment of P values to identify differentially methylated cytosines (DMCs) and regions (DMRs) associated with clinical variables, such as maternal age, mode of delivery, and birth weight. Type 1 error rate was then evaluated with a permutation analysis. We discovered a strong inflation of spatially adjusted P values through the permutation analysis, which we then applied for empirical type 1 error control. The inflation of P values was caused by a common method for spatial adjustment and DMR detection, implemented in tools comb-p and RADMeth. Based on empirically estimated significance thresholds, very little differential methylation was associated with any of the studied clinical variables, other than sex. With this analysis workflow, the sex-associated differentially methylated regions were highly reproducible across studies, technologies, and statistical models.

Placental DNA methylation marks are associated with maternal depressive symptoms during early pregnancy.

Maternal depressive symptoms during pregnancy are a significant risk factor for adverse developmental and health outcomes of the offspring. The molecular mechanisms mediating the long-term effects of this exposure are not well understood. Previous studies have found association between prenatal exposure to maternal psychological distress and placental DNA methylation of candidate genes, which can influence placental barrier function and development of the fetus. Our objective in this study was to determine epigenome wide association of maternal depressive symptoms in early pregnancy with the placental DNA methylation. For this purpose we examined DNA methylomes of 92 placental samples by using reduced representation bisulfite sequencing. The placental samples were collected after deliveries of 39 girls and 59 boys, whose mothers had Edinburgh Postnatal Depression Score ranging from 0 to 19 at gestational week 14. According to our results maternal depressive symptoms are associated with DNA methylation of 2833 CpG sites, which are particularly over-represented in genic enhancers. The genes overlapping or nearest to these sites are functionally enriched for development of neurons and show expression enrichment in several regions of developing brain. The genomic regions harboring the DNA methylation marks are enriched for single nucleotide polymorphisms associated with mental disease trait class. Potential cellular signaling cascades mediating the effects include inflammatory and hormonal pathways. As a conclusion our results suggest that maternal depressive symptoms during early pregnancy are associated with DNA methylation marks in placenta in genes, which are important for the development and long-term health of the brain. Whether similar marks can be detected in exposed children remains to be elucidated in further studies.

Analysis of H3K4me3 and H3K27me3 bivalent promotors in HER2+ breast cancer cell lines reveals variations depending on estrogen receptor status and significantly correlates with gene expression.

BACKGROUND: The role of histone modifications is poorly characterized in breast cancer, especially within the major subtypes. While epigenetic modifications may enhance the adaptability of a cell to both therapy and the surrounding environment, the mechanisms by which this is accomplished remains unclear. In this study we focus on the HER2 subtype and investigate two histone trimethylations that occur on the histone 3; the trimethylation located at lysine 4 (H3K4me3) found in active promoters and the trimethylation located at lysine 27 (H3K27me3) that correlates with gene repression. A bivalency state is the result of the co-presence of these two marks at the same promoter. METHODS: In this study we investigated the relationship between these histone modifications in promoter regions and their proximal gene expression in HER2+ breast cancer cell lines. In addition, we assessed these patterns with respect to the presence or absence of the estrogen receptor (ER). To do this, we utilized ChIP-seq and matching RNA-seq from publicly available data for the AU565, SKBR3, MB361 and UACC812 cell lines. In order to visualize these relationships, we used KEGG pathway enrichment analysis, and Kaplan-Meyer plots. RESULTS: We found that the correlation between the three types of promoter trimethylation statuses (H3K4me3, H3K27me3 or both) and the expression of the proximal genes was highly significant overall, while roughly a third of all genes are regulated by this phenomenon. We also show that there are several pathways related to cancer progression and invasion that are associated with the bivalent status of the gene promoters, and that there are specific differences between ER+ and ER- HER2+ breast cancer cell lines. These specific differences that are differentially trimethylated are also shown to be differentially expressed in patient samples. One of these genes, HIF1AN, significantly correlates with patient outcome. CONCLUSIONS: This study highlights the importance of looking at epigenetic markings at a subtype specific level by characterizing the relationship between the bivalent promoters and gene expression. This provides a deeper insight into a mechanism that could lead to future targets for treatment and prognosis, along with oncogenesis and response to therapy of HER2+ breast cancer patients.

Single cell characterization of B-lymphoid differentiation and leukemic cell states during chemotherapy in ETV6-RUNX1-positive pediatric leukemia identifies drug-targetable transcription factor activities.

BACKGROUND: Tight regulatory loops orchestrate commitment to B cell fate within bone marrow. Genetic lesions in this gene regulatory network underlie the emergence of the most common childhood cancer, acute lymphoblastic leukemia (ALL). The initial genetic hits, including the common translocation that fuses ETV6 and RUNX1 genes, lead to arrested cell differentiation. Here, we aimed to characterize transcription factor activities along the B-lineage differentiation trajectory as a reference to characterize the aberrant cell states present in leukemic bone marrow, and to identify those transcription factors that maintain cancer-specific cell states for more precise therapeutic intervention. METHODS: We compared normal B-lineage differentiation and in vivo leukemic cell states using single cell RNA-sequencing (scRNA-seq) and several complementary genomics profiles. Based on statistical tools for scRNA-seq, we benchmarked a workflow to resolve transcription factor activities and gene expression distribution changes in healthy bone marrow lymphoid cell states. We compared these to ALL bone marrow at diagnosis and in vivo during chemotherapy, focusing on leukemias carrying the ETV6-RUNX1 fusion. RESULTS: We show that lymphoid cell transcription factor activities uncovered from bone marrow scRNA-seq have high correspondence with independent ATAC- and ChIP-seq data. Using this comprehensive reference for regulatory factors coordinating B-lineage differentiation, our analysis of ETV6-RUNX1-positive ALL cases revealed elevated activity of multiple ETS-transcription factors in leukemic cells states, including the leukemia genome-wide association study hit ELK3. The accompanying gene expression changes associated with natural killer cell inactivation and depletion in the leukemic immune microenvironment. Moreover, our results suggest that the abundance of G1 cell cycle state at diagnosis and lack of differentiation-associated regulatory network changes during induction chemotherapy represent features of chemoresistance. To target the leukemic regulatory program and thereby overcome treatment resistance, we show that inhibition of ETS-transcription factors reduced cell viability and resolved pathways contributing to this using scRNA-seq. CONCLUSIONS: Our data provide a detailed picture of the transcription factor activities characterizing both normal B-lineage differentiation and those acquired in leukemic bone marrow and provide a rational basis for new treatment strategies targeting the immune microenvironment and the active regulatory network in leukemia.

Peripheral blood DNA methylation differences in twin pairs discordant for Alzheimer's disease.

BACKGROUND: Alzheimer's disease results from a neurodegenerative process that starts well before the diagnosis can be made. New prognostic or diagnostic markers enabling early intervention into the disease process would be highly valuable. Environmental and lifestyle factors largely modulate the disease risk and may influence the pathogenesis through epigenetic mechanisms, such as DNA methylation. As environmental and lifestyle factors may affect multiple tissues of the body, we hypothesized that the disease-associated DNA methylation signatures are detectable in the peripheral blood of discordant twin pairs. RESULTS: Comparison of 23 disease discordant Finnish twin pairs with reduced representation bisulfite sequencing revealed peripheral blood DNA methylation differences in 11 genomic regions with at least 15.0% median methylation difference and FDR adjusted p value ≤ 0.05. Several of the affected genes are primarily associated with neuronal functions and pathologies and do not display disease-associated differences in gene expression in blood. The DNA methylation mark in ADARB2 gene was found to be differentially methylated also in the anterior hippocampus, including entorhinal cortex, of non-twin cases and controls. Targeted bisulfite pyrosequencing of the DNA methylation mark in ADARB2 gene in 62 Finnish and Swedish twin pairs revealed that, in addition to the disease status, DNA methylation of this region is influenced by gender, age, zygosity, APOE genotype, and smoking. Further analysis of 120 Swedish twin pairs indicated that this specific DNA methylation mark is not predictive for Alzheimer's disease and becomes differentially methylated after disease onset. CONCLUSIONS: DNA methylation differences can be detected in the peripheral blood of twin pairs discordant for Alzheimer's disease. These DNA methylation signatures may have value as disease markers and provide insights into the molecular mechanisms of pathogenesis. We found no evidence that the DNA methylation marks would be associated with gene expression in blood. Further studies are needed to elucidate the potential importance of the associated genes in neuronal functions and to validate the prognostic or diagnostic value of the individual marks or marker panels.

Early Detection of Peripheral Blood Cell Signature in Children Developing ß-Cell Autoimmunity at a Young Age.

The appearance of type 1 diabetes (T1D)-associated autoantibodies is the first and only measurable parameter to predict progression toward T1D in genetically susceptible individuals. However, autoantibodies indicate an active autoimmune reaction, wherein the immune tolerance is already broken. Therefore, there is a clear and urgent need for new biomarkers that predict the onset of the autoimmune reaction preceding autoantibody positivity or reflect progressive ß-cell destruction. Here we report the mRNA sequencing-based analysis of 306 samples including fractionated samples of CD4+ and CD8+ T cells as well as CD4-CD8- cell fractions and unfractionated peripheral blood mononuclear cell samples longitudinally collected from seven children who developed ß-cell autoimmunity (case subjects) at a young age and matched control subjects. We identified transcripts, including interleukin 32 (IL32), that were upregulated before T1D-associated autoantibodies appeared. Single-cell RNA sequencing studies revealed that high IL32 in case samples was contributed mainly by activated T cells and NK cells. Further, we showed that IL32 expression can be induced by a virus and cytokines in pancreatic islets and ß-cells, respectively. The results provide a basis for early detection of aberrations in the immune system function before T1D and suggest a potential role for IL32 in the pathogenesis of T1D.

Epigenomic Landscapes of hESC-Derived Neural Rosettes: Modeling Neural Tube Formation and Diseases.

We currently lack a comprehensive understanding of the mechanisms underlying neural tube formation and their contributions to neural tube defects (NTDs). Developing a model to study such a complex morphogenetic process, especially one that models human-specific aspects, is critical. Three-dimensional, human embryonic stem cell (hESC)-derived neural rosettes (NRs) provide a powerful resource for in vitro modeling of human neural tube formation. Epigenomic maps reveal enhancer elements unique to NRs relative to 2D systems. A master regulatory network illustrates that key NR properties are related to their epigenomic landscapes. We found that folate-associated DNA methylation changes were enriched within NR regulatory elements near genes involved in neural tube formation and metabolism. Our comprehensive regulatory maps offer insights into the mechanisms by which folate may prevent NTDs. Lastly, our distal regulatory maps provide a better understanding of the potential role of neurological-disorder-associated SNPs.