In experimental animals, maternal diet during the periconceptional period influences the establishment of DNA methylation at metastable epialleles in the offspring, with permanent phenotypic ...consequences. Pronounced naturally occurring seasonal differences in the diet of rural Gambian women allowed us to test this in humans. We show that significant seasonal variations in methyl-donor nutrient intake of mothers around the time of conception influence 13 relevant plasma biomarkers. The level of several of these maternal biomarkers predicts increased/decreased methylation at metastable epialleles in DNA extracted from lymphocytes and hair follicles in infants postnatally. Our results demonstrate that maternal nutritional status during early pregnancy causes persistent and systemic epigenetic changes at human metastable epialleles.
Throughout most of the mammalian genome, genetically regulated developmental programming establishes diverse yet predictable epigenetic states across differentiated cells and tissues. At metastable ...epialleles (MEs), conversely, epigenotype is established stochastically in the early embryo then maintained in differentiated lineages, resulting in dramatic and systemic interindividual variation in epigenetic regulation. In the mouse, maternal nutrition affects this process, with permanent phenotypic consequences for the offspring. MEs have not previously been identified in humans. Here, using an innovative 2-tissue parallel epigenomic screen, we identified putative MEs in the human genome. In autopsy samples, we showed that DNA methylation at these loci is highly correlated across tissues representing all 3 embryonic germ layer lineages. Monozygotic twin pairs exhibited substantial discordance in DNA methylation at these loci, suggesting that their epigenetic state is established stochastically. We then tested for persistent epigenetic effects of periconceptional nutrition in rural Gambians, who experience dramatic seasonal fluctuations in nutritional status. DNA methylation at MEs was elevated in individuals conceived during the nutritionally challenged rainy season, providing the first evidence of a permanent, systemic effect of periconceptional environment on human epigenotype. At MEs, epigenetic regulation in internal organs and tissues varies among individuals and can be deduced from peripheral blood DNA. MEs should therefore facilitate an improved understanding of the role of interindividual epigenetic variation in human disease.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
DNA methylation is thought to be an important determinant of human phenotypic variation, but its inherent cell type specificity has impeded progress on this question. At exceptional genomic regions, ...interindividual variation in DNA methylation occurs systemically. Like genetic variants, systemic interindividual epigenetic variants are stable, can influence phenotype, and can be assessed in any easily biopsiable DNA sample. We describe an unbiased screen for human genomic regions at which interindividual variation in DNA methylation is not tissue-specific.
For each of 10 donors from the NIH Genotype-Tissue Expression (GTEx) program, CpG methylation is measured by deep whole-genome bisulfite sequencing of genomic DNA from tissues representing the three germ layer lineages: thyroid (endoderm), heart (mesoderm), and brain (ectoderm). We develop a computational algorithm to identify genomic regions at which interindividual variation in DNA methylation is consistent across all three lineages. This approach identifies 9926 correlated regions of systemic interindividual variation (CoRSIVs). These regions, comprising just 0.1% of the human genome, are inter-correlated over long genomic distances, associated with transposable elements and subtelomeric regions, conserved across diverse human ethnic groups, sensitive to periconceptional environment, and associated with genes implicated in a broad range of human disorders and phenotypes. CoRSIV methylation in one tissue can predict expression of associated genes in other tissues.
In addition to charting a previously unexplored molecular level of human individuality, this atlas of human CoRSIVs provides a resource for future population-based investigations into how interindividual epigenetic variation modulates risk of disease.
Abstract
Background
The traditional approach to studying the epigenetic mechanism CpG methylation in tissue samples is to identify regions of concordant differential methylation spanning multiple CpG ...sites (differentially methylated regions). Variation limited to single or small numbers of CpGs has been assumed to reflect stochastic processes. To test this, we developed software, Cluster-Based analysis of CpG methylation (CluBCpG), and explored variation in read-level CpG methylation patterns in whole genome bisulfite sequencing data.
Results
Analysis of both human and mouse whole genome bisulfite sequencing datasets reveals read-level signatures associated with cell type and cell type-specific biological processes. These signatures, which are mostly orthogonal to classical differentially methylated regions, are enriched at cell type-specific enhancers and allow estimation of proportional cell composition in synthetic mixtures and improved prediction of gene expression. In tandem, we developed a machine learning algorithm, Precise Read-Level Imputation of Methylation (PReLIM), to increase coverage of existing whole genome bisulfite sequencing datasets by imputing CpG methylation states on individual sequencing reads. PReLIM both improves CluBCpG coverage and performance and enables identification of novel differentially methylated regions, which we independently validate.
Conclusions
Our data indicate that, rather than stochastic variation, read-level CpG methylation patterns in tissue whole genome bisulfite sequencing libraries reflect cell type. Accordingly, these new computational tools should lead to an improved understanding of epigenetic regulation by DNA methylation.
Genetic variants can modulate phenotypic outcomes via epigenetic intermediates, for example at methylation quantitative trait loci (mQTL). We present the first large-scale assessment of mQTL at human ...genomic regions selected for interindividual variation in CpG methylation, which we call correlated regions of systemic interindividual variation (CoRSIVs). These can be assayed in blood DNA and do not reflect interindividual variation in cellular composition.
We use target-capture bisulfite sequencing to assess DNA methylation at 4086 CoRSIVs in multiple tissues from each of 188 donors in the NIH Gene-Tissue Expression (GTEx) program. At CoRSIVs, DNA methylation in peripheral blood correlates with methylation and gene expression in internal organs. We also discover unprecedented mQTL at these regions. Genetic influences on CoRSIV methylation are extremely strong (median R
=0.76), cumulatively comprising over 70-fold more human mQTL than detected in the most powerful previous study. Moreover, mQTL beta coefficients at CoRSIVs are highly skewed (i.e., the major allele predicts higher methylation). Both surprising findings are independently validated in a cohort of 47 non-GTEx individuals. Genomic regions flanking CoRSIVs show long-range enrichments for LINE-1 and LTR transposable elements; the skewed beta coefficients may therefore reflect evolutionary selection of genetic variants that promote their methylation and silencing. Analyses of GWAS summary statistics show that mQTL polymorphisms at CoRSIVs are associated with metabolic and other classes of disease.
A focus on systemic interindividual epigenetic variants, clearly enhanced in mQTL content, should likewise benefit studies attempting to link human epigenetic variation to the risk of disease.
Background: DNA methylation is an epigenetic mechanism central to development and maintenance of complex mammalian tissues, but our understanding of its role in intestinal development is limited. ...Results: We use whole genome bisulfite sequencing, and find that differentiation of mouse colonic intestinal stem cells to intestinal epithelium is not associated with major changes in DNA methylation. However, we detect extensive dynamic epigenetic changes in intestinal stem cells and their progeny during the suckling period, suggesting postnatal epigenetic development in this stem cell population. We find that postnatal DNA methylation increases at 3' CpG islands (CGIs) correlate with transcriptional activation of glycosylation genes responsible for intestinal maturation. To directly test whether 3' CGI methylation regulates transcription, we conditionally disrupted two major DNA methyltransferases, Dnmt1 or Dnmt3a, in fetal and adult intestine. Deficiency of Dnmt1 causes severe intestinal abnormalities in neonates and disrupts crypt homeostasis in adults, whereas Dnmt3a loss was compatible with intestinal development. These studies reveal that 3' CGI methylation is functionally involved in the regulation of transcriptional activation in vivo, and that Dnmt1 is a critical regulator of postnatal epigenetic changes in intestinal stem cells. Finally, we show that postnatal 3' CGI methylation and associated gene activation in intestinal epithelial cells are significantly altered by germ-free conditions. Conclusions: Our results demonstrate that the suckling period is critical for epigenetic development of intestinal stem cells, with potential important implications for lifelong gut health, and that the gut microbiome guides and/or facilitates these postnatal epigenetic processes.
Monozygotic twins have long been studied to estimate heritability and explore epigenetic influences on phenotypic variation. The phenotypic and epigenetic similarities of monozygotic twins have been ...assumed to be largely due to their genetic identity.
Here, by analyzing data from a genome-scale study of DNA methylation in monozygotic and dizygotic twins, we identified genomic regions at which the epigenetic similarity of monozygotic twins is substantially greater than can be explained by their genetic identity. This "epigenetic supersimilarity" apparently results from locus-specific establishment of epigenotype prior to embryo cleavage during twinning. Epigenetically supersimilar loci exhibit systemic interindividual epigenetic variation and plasticity to periconceptional environment and are enriched in sub-telomeric regions. In case-control studies nested in a prospective cohort, blood DNA methylation at these loci years before diagnosis is associated with risk of developing several types of cancer.
These results establish a link between early embryonic epigenetic development and adult disease. More broadly, epigenetic supersimilarity is a previously unrecognized phenomenon that may contribute to the phenotypic similarity of monozygotic twins.
DNA methylation regulates cell type-specific gene expression. Here, in a transgenic mouse model, we show that deletion of the gene encoding DNA methyltransferase Dnmt3a in hypothalamic AgRP neurons ...causes a sedentary phenotype characterized by reduced voluntary exercise and increased adiposity. Whole-genome bisulfite sequencing (WGBS) and transcriptional profiling in neuronal nuclei from the arcuate nucleus of the hypothalamus (ARH) reveal differentially methylated genomic regions and reduced expression of AgRP neuron-associated genes in knockout mice. We use read-level analysis of WGBS data to infer putative ARH neural cell types affected by the knockout, and to localize promoter hypomethylation and increased expression of the growth factor Bmp7 to AgRP neurons, suggesting a role for aberrant TGF-β signaling in the development of this phenotype. Together, these data demonstrate that DNA methylation in AgRP neurons is required for their normal epigenetic development and neuron-specific gene expression profiles, and regulates voluntary exercise behavior.
Coupling bisulfite conversion with next-generation sequencing (Bisulfite-seq) enables genome-wide measurement of DNA methylation, but poses unique challenges for mapping. However, despite a ...proliferation of Bisulfite-seq mapping tools, no systematic comparison of their genomic coverage and quantitative accuracy has been reported. We sequenced bisulfite-converted DNA from two tissues from each of two healthy human adults and systematically compared five widely used Bisulfite-seq mapping algorithms: Bismark, BSMAP, Pash, BatMeth and BS Seeker. We evaluated their computational speed and genomic coverage and verified their percentage methylation estimates. With the exception of BatMeth, all mappers covered >70% of CpG sites genome-wide and yielded highly concordant estimates of percentage methylation (r(2) ≥ 0.95). Fourfold variation in mapping time was found between BSMAP (fastest) and Pash (slowest). In each library, 8-12% of genomic regions covered by Bismark and Pash were not covered by BSMAP. An experiment using simulated reads confirmed that Pash has an exceptional ability to uniquely map reads in genomic regions of structural variation. Independent verification by bisulfite pyrosequencing generally confirmed the percentage methylation estimates by the mappers. Of these algorithms, Bismark provides an attractive combination of processing speed, genomic coverage and quantitative accuracy, whereas Pash offers considerably higher genomic coverage.
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