The major histocompatibility complex (MHC) is essential for human immunity and is highly associated with common diseases, including cancer. While the genetics of the MHC has been studied intensively for many decades, very little is known about the epigenetics of this most polymorphic and disease-associated region of the genome.

As sex determines mammalian development, understanding the nature and developmental dynamics of the sexually dimorphic transcriptome is important. To explore this, we generated 76 genome-wide RNA-seq profiles from mouse eight-cell embryos, late gestation and adult livers, together with 4 ground-state pluripotent embryonic (ES) cell lines from which we generated both RNA-seq and multiple ChIP-seq profiles. We complemented this with previously published data to yield 5 snap-shots of pre-implantation development, late-gestation placenta and somatic tissue and multiple adult tissues for integrative analysis.

Fetal growth is determined by the feto-placental genome interacting with the maternal in utero environment. Failure of this interplay leads to poor placental development and fetal growth restriction (FGR), which is associated with future metabolic disease. We investigated whether whole genome methylation differences existed in umbilical cord blood and placenta, between gestational-matched, FGR, and appropriately grown (AGA) neonates. Using the Infinium HumanMethylation450 BeadChip®, we found that DNA from umbilical cord blood of FGR born at term (n = 19) had 839 differentially methylated positions (DMPs) that reached genome-wide significance compared with AGA (n = 18). Using gestational age as a continuous variable, we identified 76,249 DMPs in cord blood (adj. P < 0.05) of which 121 DMPs were common to the 839 DMPs and were still evident when comparing 12 FGR with 12 AGA [39.9 ± 1.2 vs. 40.0 ± 1.0 weeks (mean ± SD), respectively]. A total of 53 DMPs had a β methylation difference >10% and 25 genes were co-methylated more than twice within 1000 base pairs. Gene Ontology (GO) analysis of DMPs supported their involvement in gene regulation and transcription pathways related to organ development and metabolic function. A similar profile of DMPs was found across different cell types in the cord blood. At term, no DMPs between FGR and AGA placentae reached genome-wide significance, validated with an external dataset. GO analysis of 284 pre-term, placental DMPs associated with autophagy, oxidative stress and hormonal responses. Growth restricted neonates have distinct DNA methylation profiles in pre-term placenta and in cord blood at birth, which may predispose to future adult disease.

Cellular senescence is a stable arrest of proliferation and is considered a key component of processes associated with carcinogenesis and other ageing-related phenotypes. Here, we perform methylome analysis of actively dividing and deeply senescent normal human epithelial cells.

Methylated DNA immunoprecipitation followed by high-throughput sequencing (MeDIP-seq) has the potential to identify changes in DNA methylation important in cancer development. In order to understand the role of epigenetic modulation in the development of acute myeloid leukemia (AML) we have applied MeDIP-seq to the DNA of 12 AML patients and 4 normal bone marrows. This analysis revealed leukemia-associated differentially methylated regions that included gene promoters, gene bodies, CpG islands and CpG island shores. Two genes (SPHKAP and DPP6) with significantly methylated promoters were of interest and further analysis of their expression showed them to be repressed in AML. We also demonstrated considerable cytogenetic subtype specificity in the methylomes affecting different genomic features. Significantly distinct patterns of hypomethylation of certain interspersed repeat elements were associated with cytogenetic subtypes. The methylation patterns of members of the SINE family tightly clustered all leukemic patients with an enrichment of Alu repeats with a high CpG density (P<0.0001). We were able to demonstrate significant inverse correlation between intragenic interspersed repeat sequence methylation and gene expression with SINEs showing the strongest inverse correlation (R(2) = 0.7). We conclude that the alterations in DNA methylation that accompany the development of AML affect not only the promoters, but also the non-promoter genomic features, with significant demethylation of certain interspersed repeat DNA elements being associated with AML cytogenetic subtypes. MeDIP-seq data were validated using bisulfite pyrosequencing and the Infinium array.

Environmental influences fluctuate throughout the life course of an organism. It is therefore important to understand how the timing of exposure impacts molecular responses. Herein, we examine the responses of two key molecular markers of dietary stress, namely variant-specific methylation at ribosomal DNA (rDNA) and small RNA distribution, including tRNA fragments, in a mouse model of protein restriction (PR) with exposure at pre- and/or post-weaning.

In mammals, DNA methylation profiles vary substantially between tissues. Recent genome-scale studies report that blood displays a highly distinctive methylomic profile from other somatic tissues. In this study, we sought to understand why blood DNA methylation state is so different to the one found in other tissues. We found that whole blood contains approximately twice as many tissue-specific differentially methylated positions (tDMPs) than any other somatic tissue examined. Furthermore, a large subset of blood tDMPs showed much lower levels of methylation than tDMPs for other tissues. Surprisingly, these regions of low methylation in blood show no difference regarding genomic location, genomic content, evolutionary rates, or histone marks when compared to other tDMPs. Our results reveal why blood displays a distinctive methylation profile relative to other somatic tissues. In the future, it will be important to study how these blood specific tDMPs are mechanistically involved in blood-specific functions.

Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative treatment for many hematological conditions. Acute graft-versus-host disease (aGVHD) is a prevalent immune-mediated complication following HSCT. Current diagnostic biomarkers that correlate with aGVHD severity, progression, and therapy response in graft recipients are insufficient. Here, we investigated whether epigenetic marks measured in peripheral blood of healthy graft donors stratify aGVHD severity in human leukocyte antigen (HLA)-matched sibling recipients prior to T cell-depleted HSCT.

Recent multi-dimensional approaches to the study of complex disease have revealed powerful insights into how genetic and epigenetic factors may underlie their aetiopathogenesis. We examined genotype-epigenotype interactions in the context of Type 2 Diabetes (T2D), focussing on known regions of genomic susceptibility. We assayed DNA methylation in 60 females, stratified according to disease susceptibility haplotype using previously identified association loci. CpG methylation was assessed using methylated DNA immunoprecipitation on a targeted array (MeDIP-chip) and absolute methylation values were estimated using a Bayesian algorithm (BATMAN). Absolute methylation levels were quantified across LD blocks, and we identified increased DNA methylation on the FTO obesity susceptibility haplotype, tagged by the rs8050136 risk allele A (p = 9.40×10(-4), permutation p = 1.0×10(-3)). Further analysis across the 46 kb LD block using sliding windows localised the most significant difference to be within a 7.7 kb region (p = 1.13×10(-7)). Sequence level analysis, followed by pyrosequencing validation, revealed that the methylation difference was driven by the co-ordinated phase of CpG-creating SNPs across the risk haplotype. This 7.7 kb region of haplotype-specific methylation (HSM), encapsulates a Highly Conserved Non-Coding Element (HCNE) that has previously been validated as a long-range enhancer, supported by the histone H3K4me1 enhancer signature. This study demonstrates that integration of Genome-Wide Association (GWA) SNP and epigenomic DNA methylation data can identify potential novel genotype-epigenotype interactions within disease-associated loci, thus providing a novel route to aid unravelling common complex diseases.

Monozygotic (MZ) twin pair discordance for childhood-onset Type 1 Diabetes (T1D) is ∼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.

Inter-individual epigenetic variation, due to genetic, environmental or random influences, is observed in many eukaryotic species. In mammals, however, the molecular nature of epiallelic variation has been poorly defined, partly due to the restricted focus on DNA methylation. Here we report the first genome-scale investigation of mammalian epialleles that integrates genomic, methylomic, transcriptomic and histone state information.

Variation in cancer risk among somatic tissues has been attributed to variations in the underlying rate of stem cell division. For a given tissue type, variable cancer risk between individuals is thought to be influenced by extrinsic factors which modulate this rate of stem cell division. To date, no molecular mitotic clock has been developed to approximate the number of stem cell divisions in a tissue of an individual and which is correlated with cancer risk.

The incidence of type 1 diabetes (T1D) has substantially increased over the past decade, suggesting a role for non-genetic factors such as epigenetic mechanisms in disease development. Here we present an epigenome-wide association study across 406,365 CpGs in 52 monozygotic twin pairs discordant for T1D in three immune effector cell types. We observe a substantial enrichment of differentially variable CpG positions (DVPs) in T1D twins when compared with their healthy co-twins and when compared with healthy, unrelated individuals. These T1D-associated DVPs are found to be temporally stable and enriched at gene regulatory elements. Integration with cell type-specific gene regulatory circuits highlight pathways involved in immune cell metabolism and the cell cycle, including mTOR signalling. Evidence from cord blood of newborns who progress to overt T1D suggests that the DVPs likely emerge after birth. Our findings, based on 772 methylomes, implicate epigenetic changes that could contribute to disease pathogenesis in T1D.

The Human Epigenome Project aims to identify, catalogue, and interpret genome-wide DNA methylation phenomena. Occurring naturally on cytosine bases at cytosine-guanine dinucleotides, DNA methylation is intimately involved in diverse biological processes and the aetiology of many diseases. Differentially methylated cytosines give rise to distinct profiles, thought to be specific for gene activity, tissue type, and disease state. The identification of such methylation variable positions will significantly improve our understanding of genome biology and our ability to diagnose disease. Here, we report the results of the pilot study for the Human Epigenome Project entailing the methylation analysis of the human major histocompatibility complex. This study involved the development of an integrated pipeline for high-throughput methylation analysis using bisulphite DNA sequencing, discovery of methylation variable positions, epigenotyping by matrix-assisted laser desorption/ionisation mass spectrometry, and development of an integrated public database available at http://www.epigenome.org. Our analysis of DNA methylation levels within the major histocompatibility complex, including regulatory exonic and intronic regions associated with 90 genes in multiple tissues and individuals, reveals a bimodal distribution of methylation profiles (i.e., the vast majority of the analysed regions were either hypo- or hypermethylated), tissue specificity, inter-individual variation, and correlation with independent gene expression data.

Human genetic studies have emphasised the dominant contribution of pancreatic islet dysfunction to development of Type 2 Diabetes (T2D). However, limited annotation of the islet epigenome has constrained efforts to define the molecular mechanisms mediating the, largely regulatory, signals revealed by Genome-Wide Association Studies (GWAS). We characterised patterns of chromatin accessibility (ATAC-seq, n = 17) and DNA methylation (whole-genome bisulphite sequencing, n = 10) in human islets, generating high-resolution chromatin state maps through integration with established ChIP-seq marks. We found enrichment of GWAS signals for T2D and fasting glucose was concentrated in subsets of islet enhancers characterised by open chromatin and hypomethylation, with the former annotation predominant. At several loci (including CDC123, ADCY5, KLHDC5) the combination of fine-mapping genetic data and chromatin state enrichment maps, supplemented by allelic imbalance in chromatin accessibility pinpointed likely causal variants. The combination of increasingly-precise genetic and islet epigenomic information accelerates definition of causal mechanisms implicated in T2D pathogenesis.

Mammalian species exhibit a wide range of lifespans. To date, a robust and dynamic molecular readout of these lifespan differences has not yet been identified. Recent studies have established the existence of ageing-associated differentially methylated positions (aDMPs) in human and mouse. These are CpG sites at which DNA methylation dynamics show significant correlations with age. We hypothesise that aDMPs are pan-mammalian and are a dynamic molecular readout of lifespan variation among different mammalian species.

A major concern in common disease epigenomics is distinguishing causal from consequential epigenetic variation. One means of addressing this issue is to identify the temporal origins of epigenetic variants via longitudinal analyses. However, prospective birth-cohort studies are expensive and time consuming. Here, we report DNA methylomics of archived Guthrie cards for the retrospective longitudinal analyses of in-utero-derived DNA methylation variation. We first validate two methodologies for generating comprehensive DNA methylomes from Guthrie cards. Then, using an integrated epigenomic/genomic analysis of Guthrie cards and follow-up samplings, we identify interindividual DNA methylation variation that is present both at birth and 3 yr later. These findings suggest that disease-relevant epigenetic variation could be detected at birth, i.e., before overt clinical disease. Guthrie card methylomics offers a potentially powerful and cost-effective strategy for studying the dynamics of interindividual epigenomic variation in a range of common human diseases.

Identifying loci with parental differences in DNA methylation is key to unraveling parent-of-origin phenotypes. By conducting a MeDIP-Seq screen in maternal-methylation free postimplantation mouse embryos (Dnmt3L-/+), we demonstrate that maternal-specific methylation exists very scarcely at midgestation. We reveal two forms of oocyte-specific methylation inheritance: limited to preimplantation, or with longer duration, i.e. maternally imprinted loci. Transient and imprinted maternal germline DMRs (gDMRs) are indistinguishable in gametes and preimplantation embryos, however, de novo methylation of paternal alleles at implantation delineates their fates and acts as a major leveling factor of parent-inherited differences. We characterize two new imprinted gDMRs, at the Cdh15 and AK008011 loci, with tissue-specific imprinting loss, again by paternal methylation gain. Protection against demethylation after fertilization has been emphasized as instrumental in maintaining parent-of-origin methylation inherited from the gametes. Here we provide evidence that protection against de novo methylation acts as an equal major pivot, at implantation and throughout life.

The naked mole-rat, Heterocephalus glaber (NMR), the longest-lived rodent, is of significance and interest in the study of biomarkers for ageing. Recent breakthroughs in this field have revealed 'epigenetic clocks' that are based on the temporal accumulation of DNA methylation at specific genomic sites. Here, we validate the hypothesis of an epigenetic clock in NMRs based on changes in methylation of targeted CpG sites. We initially analysed 51 CpGs in NMR livers spanning an age range of 39-1,144 weeks and found 23 to be significantly associated with age (p<0.05). We then built a predictor of age using these sites. To test the accuracy of this model, we analysed an additional set of liver samples, and were successfully able to predict their age with a root mean squared error of 166 weeks. We also profiled skin samples with the same age range, finding a striking correlation between their predicted age versus their actual age (R=0.93), but which was lower when compared to the liver, suggesting that skin ages slower than the liver in NMRs. Our model will enable the prediction of age in wild-caught and captive NMRs of unknown age, and will be invaluable for further mechanistic studies of mammalian ageing.

Ribosomal DNA (rDNA) displays substantial inter-individual genetic variation in human and mouse. A systematic analysis of how this variation impacts epigenetic states and expression of the rDNA has thus far not been performed.