Assessing DNA methylation in the developing human intestinal epithelium: potential link to inflammatory bowel disease.

DNA methylation is one of the major epigenetic mechanisms implicated in regulating cellular development and cell-type-specific gene expression. Here we performed simultaneous genome-wide DNA methylation and gene expression analysis on purified intestinal epithelial cells derived from human fetal gut, healthy pediatric biopsies, and children newly diagnosed with inflammatory bowel disease (IBD). Results were validated using pyrosequencing, real-time PCR, and immunostaining. The functional impact of DNA methylation changes on gene expression was assessed by employing in-vitro assays in intestinal cell lines. DNA methylation analyses allowed identification of 214 genes for which expression is regulated via DNA methylation, i.e. regulatory differentially methylated regions (rDMRs). Pathway and functional analysis of rDMRs suggested a critical role for DNA methylation in regulating gene expression and functional development of the human intestinal epithelium. Moreover, analysis performed on intestinal epithelium of children newly diagnosed with IBD revealed alterations in DNA methylation within genomic loci, which were found to overlap significantly with those undergoing methylation changes during intestinal development. Our study provides novel insights into the physiological role of DNA methylation in regulating functional maturation of the human intestinal epithelium. Moreover, we provide data linking developmentally acquired alterations in the DNA methylation profile to changes seen in pediatric IBD.This study was supported by funds obtained from The Evelyn Trust, Crohn’s in Childhood Research Association (CICRA) and Crohn’s and Colitis in Childhood (3Cs) charity. J.K. was funded by a PhD studentship from CICRA. Funding for E.C. was provided by the Deutsche Forschungsgemeinschaft (Grant CA226/4-3) and Interne Forschungsförderung Essen (IFORES).This is the final version of the article. It first appeared from Nature Publishing Group via http://dx.doi.org/10.1038/mi.2015.8

Genetic, environmental and stochastic factors in monozygotic twin discordance with a focus on epigenetic differences

PMCID: PMC3566971This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited

Identification of Type 1 Diabetes-Associated DNA Methylation Variable Positions That Precede Disease Diagnosis

Monozygotic (MZ) twin pair discordance for childhood-onset Type 1 Diabetes (T1D) is similar to 50%, implicating roles for genetic and non-genetic factors in the aetiology of this complex autoimmune disease. Although significant progress has been made in elucidating the genetics of T1D in recent years, the non-genetic component has remained poorly defined. We hypothesized that epigenetic variation could underlie some of the non-genetic component of T1D aetiology and, thus, performed an epigenome-wide association study (EWAS) for this disease. We generated genome-wide DNA methylation profiles of purified CD14(+) monocytes (an immune effector cell type relevant to T1D pathogenesis) from 15 T1D-discordant MZ twin pairs. This identified 132 different CpG sites at which the direction of the intra-MZ pair DNA methylation difference significantly correlated with the diabetic state, i.e. T1D-associated methylation variable positions (T1D-MVPs). We confirmed these T1D-MVPs display statistically significant intra-MZ pair DNA methylation differences in the expected direction in an independent set of T1D-discordant MZ pairs (P = 0.035). Then, to establish the temporal origins of the T1D-MVPs, we generated two further genome-wide datasets and established that, when compared with controls, T1D-MVPs are enriched in singletons both before (P = 0.001) and at (P = 0.015) disease diagnosis, and also in singletons positive for diabetes-associated autoantibodies but disease-free even after 12 years follow-up (P = 0.0023). Combined, these results suggest that T1D-MVPs arise very early in the etiological process that leads to overt T1D. Our EWAS of T1D represents an important contribution toward understanding the etiological role of epigenetic variation in type 1 diabetes, and it is also the first systematic analysis of the temporal origins of disease-associated epigenetic variation for any human complex disease

VEZF1 elements mediate protection from DNA methylation

There is growing consensus that genome organization and long-range gene regulation involves partitioning of the genome into domains of distinct epigenetic chromatin states. Chromatin insulator or barrier elements are key components of these processes as they can establish boundaries between chromatin states. The ability of elements such as the paradigm β-globin HS4 insulator to block the range of enhancers or the spread of repressive histone modifications is well established. Here we have addressed the hypothesis that a barrier element in vertebrates should be capable of defending a gene from silencing by DNA methylation. Using an established stable reporter gene system, we find that HS4 acts specifically to protect a gene promoter from de novo DNA methylation. Notably, protection from methylation can occur in the absence of histone acetylation or transcription. There is a division of labor at HS4; the sequences that mediate protection from methylation are separable from those that mediate CTCF-dependent enhancer blocking and USF-dependent histone modification recruitment. The zinc finger protein VEZF1 was purified as the factor that specifically interacts with the methylation protection elements. VEZF1 is a candidate CpG island protection factor as the G-rich sequences bound by VEZF1 are frequently found at CpG island promoters. Indeed, we show that VEZF1 elements are sufficient to mediate demethylation and protection of the APRT CpG island promoter from DNA methylation. We propose that many barrier elements in vertebrates will prevent DNA methylation in addition to blocking the propagation of repressive histone modifications, as either process is sufficient to direct the establishment of an epigenetically stable silent chromatin stat

Correlation of Global and Gene-Specific DNA Methylation in Maternal-Infant Pairs

The inheritance of DNA methylation patterns is a popular theory to explain the influence of parental genetic and environmental factors on the phenotype of their offspring but few studies have examined this relationship in humans. Using 120 paired maternal-umbilical cord blood samples randomly selected from a prospective birth cohort in Bangladesh, we quantified DNA methylation by pyrosequencing seven CpG positions in the promoter region of p16, four CpG positions in the promoter region of p53, LINE-1 and Alu. Positive correlations were observed between maternal and umbilical cord blood at p16, LINE-1, and Alu but not p53. Multiple linear regression models observed a significant association between maternal and umbilical cord blood at LINE-1 and Alu (LINE-1: β = 0.63, p<0.0001; Alu: β = 0.28, p = 0.009). After adjusting for multiple comparisons, maternal methylation of p16 at position 4 significantly predicted methylation at the same position in umbilical cord blood (β = 0.43, p = <0.0001). These models explained 48%, 5% and 16% of the observed variability in umbilical cord %5mC for LINE-1, Alu and p16 at position 4, respectively. These results suggest that DNA methylation in maternal blood was correlated with her offspring at LINE-1, Alu, and p16 but not p53. Additional studies are needed to confirm whether these observed associations were due to the inheritance of epigenetic events or the shared environment between mother and fetus. Future studies should also use a multi-generational family-based design that would quantify both maternal and paternal contributions to DNA methylation in offspring across more than one generation

An integrative epi-transcriptomic approach identifies the human cartilage chitinase 3-like protein 2 (CHI3L2) as a potential mediator of B12 deficiency in adipocytes

Vitamin B12 has multiple biochemical functions including in the one-carbon cycle generating a methyl group for DNA methylation, and metabolism of fatty acids and amino acids to generate energy via the citric acid cycle. The aim of our study was to use a combined epigenomic and transcriptomic approach to identify novel genes mediating the effect of B12 on adipogenesis. Human pre-adipocytes (CHUB-S7) were treated with a range of B12 (0–500 nM) concentrations from the day of cell seeding until harvesting in discovery and validation experiments prior to genome-wide methylation analysis using the Illumina HumanMethylation 450Beadchip. For transcriptomic analysis, RNA-seq libraries were run on the Illumina HiSeq 2500. To further investigate the expression of any genes on human adipogenesis, a second human preadipocyte strain was studied (SGBS) by real-time quantitative PCR (qRT-PCR). A combined epigenetic and transcriptomic approach in differentiated human pre-adipocyte cell line, CHUB-S7, identified that the Human cartilage chitinase 3-like protein 2 (CHI3L2) gene was hypo-methylated and had increased expression in low B12 conditions. Furthermore, there was an approximately 1000-fold increase in CHI3L2 expression in the early days of adipocyte differentiation, which paralleled an increase of lipid droplets in differentiated SGBS cells and an increased expression level of markers of mature adipocytes. In summary, we have identified a potential role of the human cartilage chitinase 3-like protein 2 (CHI3L2) in adipocyte function in the presence of low B12 levels

Can we apply the Mendelian randomization methodology without considering epigenetic effects?

<p>Abstract</p> <p>Introduction</p> <p>Instrumental variable (IV) methods have been used in econometrics for several decades now, but have only recently been introduced into the epidemiologic research frameworks. Similarly, Mendelian randomization studies, which use the IV methodology for analysis and inference in epidemiology, were introduced into the epidemiologist's toolbox only in the last decade.</p> <p>Analysis</p> <p>Mendelian randomization studies using instrumental variables (IVs) have the potential to avoid some of the limitations of observational epidemiology (confounding, reverse causality, regression dilution bias) for making causal inferences. Certain limitations of randomized controlled trials, such as problems with generalizability, feasibility and ethics for some exposures, and high costs, also make the use of Mendelian randomization in observational studies attractive. Unlike conventional randomized controlled trials (RCTs), Mendelian randomization studies can be conducted in a representative sample without imposing any exclusion criteria or requiring volunteers to be amenable to random treatment allocation.</p> <p>Within the last decade, epigenetics has gained recognition as an independent field of study, and appears to be the new direction for future research into the genetics of complex diseases. Although previous articles have addressed some of the limitations of Mendelian randomization (such as the lack of suitable genetic variants, unreliable associations, population stratification, linkage disequilibrium (LD), pleiotropy, developmental canalization, the need for large sample sizes and some potential problems with binary outcomes), none has directly characterized the impact of epigenetics on Mendelian randomization. The possibility of epigenetic effects (non-Mendelian, heritable changes in gene expression not accompanied by alterations in DNA sequence) could alter the core instrumental variable assumptions of Mendelian randomization.</p> <p>This paper applies conceptual considerations, algebraic derivations and data simulations to question the appropriateness of Mendelian randomization methods when epigenetic modifications are present.</p> <p>Conclusion</p> <p>Given an inheritance of gene expression from parents, Mendelian randomization studies not only need to assume a random distribution of alleles in the offspring, but also a random distribution of epigenetic changes (e.g. gene expression) at conception, in order for the core assumptions of the Mendelian randomization methodology to remain valid. As an increasing number of epidemiologists employ Mendelian randomization methods in their research, caution is therefore needed in drawing conclusions from these studies if these assumptions are not met.</p

Comparative epigenetic analysis of tumour initiating cells and syngeneic EPSC-derived neural stem cells in glioblastoma

Epigenetic mechanisms which play an essential role in normal developmental processes, such as self-renewal and fate specification of neural stem cells (NSC) are also responsible for some of the changes in the glioblastoma (GBM) genome. Here we develop a strategy to compare the epigenetic and transcriptional make-up of primary GBM cells (GIC) with patient-matched expanded potential stem cell (EPSC)-derived NSC (iNSC). Using a comparative analysis of the transcriptome of syngeneic GIC/iNSC pairs, we identify a glycosaminoglycan (GAG)-mediated mechanism of recruitment of regulatory T cells (Tregs) in GBM. Integrated analysis of the transcriptome and DNA methylome of GBM cells identifies druggable target genes and patient-specific prediction of drug response in primary GIC cultures, which is validated in 3D and in vivo models. Taken together, we provide a proof of principle that this experimental pipeline has the potential to identify patient-specific disease mechanisms and druggable targets in GBM