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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 diseaseassociated loci, thus providing a novel route to aid unravelling common complex diseases. © 2010 Bell et al.

Understanding the origin of phenotypic variation remains one of the principle challenges of contemporary biology. Recent genome-wide association studies have identified association between common genetic variants and complex phenotype; however, the minimal effect sizes observed in such studies highlight the potential for other causal factors to be involved in phenotypic variation. The epigenetic state of an organism (or ‘epigenome’) incorporates a landscape of complex and plastic molecular events that may underlie the ‘missing link’ that integrates genotype with phenotype. The nature of these processes has been the subject of intense scientific study over the recent years, and characterisation of epigenetic variation, in the form of ‘epialleles’, is providing fascinating insight into how the genome functions within a range of developmental processes, environments, and in states of health and disease. This review will discuss how and when mammalian epialleles may be generated and their interaction with genetic and environmental factors. We will outline how an epiallele has a variable relationship with phenotype, and how new technologies may be used for their detection and to facilitate an understanding of their contribution to phenotype. Finally, we will consider epialleles in population variation and their teleological role in evolution. variation and their teleological role in evolution. Environ. Mol. Mutagen. 52:1–11, 2011. © 2010 Wiley-Liss, Inc.

The genome of extraembryonic tissue, such as the placenta, is hypomethylated relative to that in somatic tissues. However, the origin and role of this hypomethylation remains unclear. The DNA methyltransferases DNMT1, -3A, and -3B are the primary mediators of the establishment and maintenance of DNA methylation in mammals. In this study, we investigated promoter methylation-mediated epigenetic down-regulation of DNMT genes as a potential regulator of global methylation levels in placental tissue. Although DNMT3A and -3B promoters lack methylation in all somatic and extraembryonic tissues tested, we found specific hypermethylation of the maintenance DNA methyltransferase (DNMT1) gene and found hypomethylation of the DNMT3L gene in full term and first trimester placental tissues. Bisulfite DNA sequencing revealed monoallelic methylation of DNMT1, with no evidence of imprinting (parent of origin effect). In vitro reporter experiments confirmed that DNMT1 promoter methylation attenuates transcriptional activity in trophoblast cells. However, global hypomethylation in the absence of DNMT1 down-regulation is apparent in non-primate placentas and in vitro derived human cytotrophoblast stem cells, suggesting that DNMT1 down-regulation is not an absolute requirement for genomic hypomethylation in all instances. These data represent the first demonstration of methylation-mediated regulation of the DNMT1 gene in any system and demonstrate that the unique epigenome of the human placenta includes down-regulation of DNMT1 with concomitant hypomethylation of the DNMT3L gene. This strongly implicates epigenetic regulation of the DNMT gene family in the establishment of the unique epigenetic profile of extraembryonic tissue in humans.

The folding of chromatin into topologically constrained loop domains is essential for genomic function. We have identified genomic anchors that define the organization of chromatin loop domains across the human major histocompatibility complex (MHC). This locus contains critical genes for immunity and is associated with more diseases than any other region of the genome. Classical MHC genes are expressed in a cell type-specific pattern and can be induced by cytokines such as interferon-gamma (IFNG). Transcriptional activation of the MHC was associated with a reconfiguration of chromatin architecture resulting from the formation of additional genomic anchors. These findings suggest that the dynamic arrangement of genomic anchors and loops plays a role in transcriptional regulation.

We report a novel resource (methylation profiles of DNA, or mPod) for human genome-wide tissue-specific DNA methylation profiles. mPod consists of three fully integrated parts, genome-wide DNA methylation reference profiles of 13 normal somatic tissues, placenta, sperm, and an immortalized cell line, a visualization tool that has been integrated with the Ensembl genome browser and a new algorithm for the analysis of immunoprecipitation-based DNA methylation profiles. We demonstrate the utility of our resource by identifying the first comprehensive genome-wide set of tissue-specific differentially methylated regions (tDMRs) that may play a role in cellular identity and the regulation of tissue-specific genome function. We also discuss the implications of our findings with respect to the regulatory potential of regions with varied CpG density, gene expression, transcription factor motifs, gene ontology, and correlation with other epigenetic marks such as histone modifications. ©2008 by Cold Spring Harbor Laboratory Press. Fil: Rakyan, Vardhman K.. Barts And The London School Of Medicine And Dentistry; Fil: Down, Thomas A.. University of Cambridge; Estados Unidos Fil: Thorne, Natalie P.. University of Cambridge; Estados Unidos Fil: Flicek, Paul. European Bioinformatics Institute; Fil: Kulesha, Eugene. European Bioinformatics Institute; Fil: Gräf, Stefan. European Bioinformatics Institute; Fil: Tomazou, Eleni M.. Wellcome Trust Sanger Institute; Fil: Bäckdahl, Liselotte. Ucl Cancer Institute; Fil: Johnson, Nathan. European Bioinformatics Institute; Fil: Herberth, Marlis. University of Cambridge; Estados Unidos Fil: Howe, Kevin L.. University of Cambridge; Estados Unidos Fil: Jackson, David K.. Wellcome Trust Sanger Institute; Fil: Miretti, Marcos Mateo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Biología Subtropical. Instituto de Biología Subtropical - Nodo Posadas | Universidad Nacional de Misiones. Instituto de Biología Subtropical. Instituto de Biología Subtropical - Nodo Posadas; Argentina Fil: Fiegler, Heike. Roche Diagnostics Ltd.; . Wellcome Trust Sanger Institute; Fil: Marioni, John C.. University of Cambridge; Estados Unidos Fil: Birney, Ewan. European Bioinformatics Institute; Fil: Hubbard, Tim J. P.. Wellcome Trust Sanger Institute; Fil: Carter, Nigel P.. Wellcome Trust Sanger Institute; Fil: Tavaré, Simon. University of Cambridge; Estados Unidos Fil: Beck, Stephan G.. Ucl Cancer Institute

Human placentation displays many similarities with tumourigenesis, including rapid cell division, migration and invasion, overlapping gene expression profiles and escape from immune detection. Recent data have identified promoter methylation in the Ras association factor and adenomatous polyposis coli tumour suppressor genes as part of this process. However, the extent of tumour-associated methylation in the placenta remains unclear. Using whole genome methylation data as a starting point, we have examined this phenomenon in placental tissue. We found no evidence for methylation of the majority of common tumour suppressor genes in term placentas, but identified methylation in several genes previously described in some human tumours. Notably, promoter methylation of four independent negative regulators of Wnt signalling has now been identified in human placental tissue and purified trophoblasts. Methylation is present in baboon, but not in mouse placentas. This supports a role for elevated Wnt signalling in primate trophoblast invasiveness and placentation. Examination of invasive choriocarcinoma cell lines revealed altered methylation patterns consistent with a role of methylation change in gestational trophoblastic disease. This distinct pattern of tumour-associated methylation implicates a coordinated series of epigenetic silencing events, similar to those associated with some tumours, in the distinct features of normal human placental invasion and function.

Background: 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. Methods: To facilitate comprehensive epigenetic analyses of this region, we have generated a genomic tiling array of 2 Kb resolution covering the entire 4 Mb MHC region. The array has been designed to be compatible with chromatin immunoprecipitation (ChIP), methylated DNA immunoprecipitation (MeDIP), array comparative genomic hybridization (aCGH) and expression profiling, including of non-coding RNAs. The array comprises 7832 features, consisting of two replicates of both forward and reverse strands of MHC amplicons and appropriate controls. Results: Using MeDIP, we demonstrate the application of the MHC array for DNA methylation profiling and the identification of tissue-specific differentially methylated regions (tDMRs). Based on the analysis of two tissues and two cell types, we identified 90 tDMRs within the MHC and describe their characterisation. Conclusion: A tiling array covering the MHC region was developed and validated. Its successful application for DNA methylation profiling indicates that this array represents a useful tool for molecular analyses of the MHC in the context of medical genomics. Fil: Tomazou, Eleni M. Wellcome Trust Sanger Institute; Reino Unido Fil: Rakyan, Vardhman K. Institute of Cell and Molecular Science; Reino Unido Fil: Lefebvre, Gregory. Wellcome Trust Sanger Institute; Reino Unido Fil: Andrews, Robert. Wellcome Trust Sanger Institute; Reino Unido Fil: Ellis, Peter. Wellcome Trust Sanger Institute; Reino Unido Fil: Jackson, David K. Wellcome Trust Sanger Institute; Reino Unido Fil: Langford, Cordelia. Wellcome Trust Sanger Institute; Reino Unido Fil: Francis, Matthew D. Wellcome Trust Sanger Institute; Reino Unido Fil: Bäckdahl, Liselotte. University College London; Reino Unido Fil: Miretti, Marcos Mateo. Wellcome Trust Sanger Institute; Reino Unido. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Biología Subtropical. Instituto de Biología Subtropical - Nodo Posadas | Universidad Nacional de Misiones. Instituto de Biología Subtropical. Instituto de Biología Subtropical - Nodo Posadas; Argentina Fil: Coggill, Penny. Wellcome Trust Sanger Institute; Reino Unido Fil: Ottaviani, Diego. London Research Institute; Reino Unido. London School of Medicine and Dentistry; Reino Unido Fil: Sheer, Denise. London School of Medicine and Dentistry; Reino Unido Fil: Murrell, Adele. Cambridge Research Institute; Reino Unido Fil: Beck, Stephan G.. University College London; Reino Unido

Phenotypic variation that cannot be explained by genetic or environmental heterogeneity has intrigued geneticists for decades. The molecular basis of this phenomenon, however, is largely a mystery. Axin-fused ( Axin Fu ), first identified in 1937, is a classic example of a mammalian allele displaying extremely variable expression states. Here we demonstrate that the presence or absence of its characteristic phenotype, a kinked tail, correlates with differential DNA methylation at a retrotransposon within Axin Fu and identify mutant transcripts arising adjacent to the retrotransposon LTR that are likely to be causative of the phenotype. Furthermore, the epigenetic state at Axin Fu can be inherited transgenerationally after both maternal and paternal transmission. This is in contrast to epigenetic inheritance at the murine agouti-viable yellow ( A vy ) allele, which occurs through the female only. Unlike the egg, the sperm contributes very little (if any) cytoplasm to the zygote, and therefore paternal inheritance at Axin Fu argues against the possibility that the effects are due to cytoplasmic or metabolic influences. Consistent with the idea of transgenerational inheritance of epigenetic marks, we find that the methylation state of Axin Fu in mature sperm reflects the methylation state of the allele in the somatic tissue of the animal, suggesting that it does not undergo epigenetic reprogramming during gametogenesis. Finally, we show that epigenetic inheritance is influenced by strain background. These findings enable us to propose a model for transgenerational epigenetic inheritance in mammals.

What is it? If you thought all heritable traits are written in the DNA sequence, think again! We have come to realize that, at some alleles, the epigenetic state of the DNA can also be inherited. This is indicated by analysis of alleles where the establishment of the epigenetic state is stochastic. As epigenetic modifications influence transcriptional activity, these alleles are variably expressed in a population of individuals. It has been shown that the epigenetic state, and associated phenotype, can be inherited transgenerationally.What is the physical nature of epigenetic modifications? They include DNA methylation and/or covalent modifications of histone proteins. Epigenetic modifications influence gene expression but do not change the DNA sequence, and are mitotically relatively stable.What is variable expressivity? In general, the establishment of epigenetic modifications at mammalian alleles during early development is a predictable process. At some alleles, however, the establishment is stochastic. Once established, the epigenetic state is maintained throughout the life of the individual. So identical alleles can be variably expressed in a population and, importantly, this is not due to genetic or environmental heterogeneity. Such alleles are referred to as ‘metastable epialleles’.How does epigenetic inheritance occur? There is genome-wide epigenetic reprogramming during gametogenesis and early embryogenesis. It was thought this would erase epigenetic modifications acquired during the life of the animal, in order to restore the totipotency of the fertilized egg. But it turns out that sometimes epigenetic modifications at variably expressed alleles are not completely erased, resulting in some memory of the epigenetic state persisting in the next generation.How about an example? The coats of isogenic mice carrying the agouti viable-yellow (Avy) allele vary from completely yellow to the wild-type agouti. Avy has a retrotransposon upstream of the agouti gene. An unmethylated retrotransposon promoter overrides the agouti promoter, resulting in ectopic agouti expression and a yellow coat. A methylated retrotransposon cannot do this, resulting in a wild-type agouti coat. Yellow mothers produce more yellow offspring than agouti mothers, even when all the mice are genetically identical – transgenerational epigenetic inheritance. Transgenerational inheritance of epigenetic states has also been demonstrated in plants, yeast, Drosophila and in mice after transgenesis and nuclear transfer.Isn't parental imprinting also epigenetic inheritance? No! Parental imprinting is the epigenetic silencing of one allele of a gene based on the parent of origin, resulting in monoallelic expression in somatic cells. Epigenetic differences between the two alleles of parentally imprinted genes are completely erased through meiosis and then re-established according to the sex of the animal. For paternally expressed genes, for example, males transmit active alleles, whereas females transmit inactive alleles.What is the difference between transgenerational and mitotic epigenetic inheritance? Epigenetic modifications are propagated through mitotic cell divisions and this is sometimes called epigenetic inheritance. It is important to distinguish mitotic epigenetic inheritance from transgenerational inheritance of epigenetic states through meiotic cell divisions.What is the take-home message? Genotype and environment are not the only determinants of phenotype and DNA is not the sole unit of heredity. It is hard to estimate how widespread epigenetic inheritance is, but such effects seem to be associated with retrotransposon insertions. It is worth noting that almost 9% of the human genome is thought to be of retroviral origin. Studies of transgenerational epigenetic inheritance in humans have been confounded by our extreme genetic and environmental heterogeneity, but it is not inconceivable that some heritable characteristics in humans may in fact have an epigenetic basis.

There are some mammalian alleles that display the unusual characteristic of variable expressivity in the absence of genetic heterogeneity. It has recently become evident that this is because the activity of these alleles is dependent on their epigenetic state. Interestingly, the epigenetic state is somewhat labile, resulting in phenotypic mosaicism between cells (variegation) and also between individuals (variable expressivity). The establishment of the epigenetic state occurs during early embryogenesis and is a probabilistic event that is influenced by whether the allele is carried on the paternal or maternal alleles. In addition, the epigenetic state determines whether these alleles are dominant. We propose that mammalian alleles with such characteristics should be termed metastable epialleles to distinguish them from traditional alleles. At this stage, it is unclear how common these alleles are, but an appreciation of their existence will aid in their identification.

Recent epigenome-wide association studies have indicated a potential role for epigenetic variation in the etiology of complex human diseases. However, one major challenge is to distinguish true epigenetic variation from changes caused by differences in cellular composition between the disease and non-disease state, a problem that is particularly relevant when analyzing whole blood. For studies with large numbers of samples, it can be expensive and very time consuming to perform cell sorting, and it is often not clear which is the correct cell type to profile. Two recently published papers have attempted to address this confounding issue using bioinformatics.

Priming of NB4 promyelocytic cells with all-trans retinoic acid, followed by extracellular ATP in the presence of a phosphodiesterase inhibitor, elevated cAMP and activated protein kinase A. The order of potency for cAMP production was ATP (EC50 = 95 +/- 13 micromol/L)ADPAMP = adenosine. The order of potency of ATP analogues was 2'- and 3'-O-(4-benzoylbenzoyl)-ATP (EC50 = 54 +/- 15 micromol/L) = adenosine 5'-O-(3-thio) triphosphate (EC50 = 66 +/- 4 micromol/L)ATPbeta,gamma-methylene ATP (EC50 = 200 +/- 55 micromol/L). Adenosine 5'-O-thiomonophosphate and adenosine 5'-O-(2-thio) diphosphate inhibited ATP-induced cAMP production. Differentiation also occurred as measured by increased expression of CD11b and N-formyl peptide receptor and changes in cell morphology. UTP did not elevate cAMP or induce differentiation, indicating that P2Y2, P2Y4, and P2Y6 receptors were not involved. The P2Y11 receptor, a cAMP-linked receptor on promyelocytic HL-60 cells, was detected in NB4 cells by reverse transcription-polymerase chain reaction and northern blotting. This receptor has the same order of potency with respect to cAMP production as that observed in HL-60 cells.

BACKGROUND: 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. RESULTS: First, in a small sample set, we demonstrate that non-genetically determined inter-individual differentially methylated regions (iiDMRs) can be temporally stable over at least 2 years. Then, we show that iiDMRs are associated with changes in chromatin state as measured by inter-individual differences in histone variant H2A.Z levels. However, the correlation of promoter iiDMRs with gene expression is negligible and not improved by integrating H2A.Z information. We find that most promoter epialleles, whether genetically or non-genetically determined, are associated with low levels of transcriptional activity, depleted for housekeeping genes, and either depleted for H3K4me3/enriched for H3K27me3 or lacking both these marks in human embryonic stem cells. The preferential enrichment of iiDMRs at regions of relative transcriptional inactivity validates in a larger independent cohort, and is reminiscent of observations previously made for promoters that undergo hypermethylation in various cancers, in vitro cell culture and ageing. CONCLUSIONS: Our work identifies potential key features of epiallelic variation in humans, including temporal stability of non-genetically determined epialleles, and concomitant perturbations of chromatin state. Furthermore, our work suggests a novel mechanistic link among inter-individual epialleles observed in the context of normal variation, cancer and ageing.

Background Diabetic nephropathy is a serious complication of diabetes mellitus and is associated with considerable morbidity and high mortality. There is increasing evidence to suggest that dysregulation of the epigenome is involved in diabetic nephropathy. We assessed whether epigenetic modification of DNA methylation is associated with diabetic nephropathy in a case-control study of 192 Irish patients with type 1 diabetes mellitus (T1D). Cases had T1D and nephropathy whereas controls had T1D but no evidence of renal disease. Methods We performed DNA methylation profiling in bisulphite converted DNA from cases and controls using the recently developed Illumina Infinium® HumanMethylation27 BeadChip, that enables the direct investigation of 27,578 individual cytosines at CpG loci throughout the genome, which are focused on the promoter regions of 14,495 genes. Results Singular Value Decomposition (SVD) analysis indicated that significant components of DNA methylation variation correlated with patient age, time to onset of diabetic nephropathy, and sex. Adjusting for confounding factors using multivariate Cox-regression analyses, and with a false discovery rate (FDR) of 0.05, we observed 19 CpG sites that demonstrated correlations with time to development of diabetic nephropathy. Of note, this included one CpG site located 18 bp upstream of the transcription start site of UNC13B, a gene in which the first intronic SNP rs13293564 has recently been reported to be associated with diabetic nephropathy. Conclusion This high throughput platform was able to successfully interrogate the methylation state of individual cytosines and identified 19 prospective CpG sites associated with risk of diabetic nephropathy. These differences in DNA methylation are worthy of further follow-up in replication studies using larger cohorts of diabetic patients with and without nephropathy.

Background 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. Results We identify senescence-associated differentially methylated positions (senDMPs) from multiple experiments using cells from one donor. We find that human senDMP epigenetic signatures are positively and significantly correlated with both cancer and ageing-associated methylation dynamics. We also identify germline genetic variants, including those associated with the p16INK4A locus, which are associated with the presence of in vivo senDMP signatures. Importantly, we also demonstrate that a single senDMP signature can be effectively reversed in a newly-developed protocol of transient senescence reversal. Conclusions The senDMP signature has significant potential for understanding some of the key (epi)genetic etiological factors that may lead to cancer and age-related diseases in humans. Electronic supplementary material The online version of this article (doi:10.1186/s13059-015-0748-4) contains supplementary material, which is available to authorized users.