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Putative human metastable epialleles (MEs) can be identified by screening for systemic interindividual variation suggesting that stochastic DNA methylation is established before germ layer differentiation. Putative human MEs appear to be influenced but not determined by genetic variation, and are associated with particular classes of retrotransposon, reminiscent of their murine counterparts. Several putative human MEs have been linked to both environmental exposures in early development and to phenotype. ME methylation has been associated with outcomes relating to cancer, glucose metabolism, and thyroid function in later life. Putative human MEs are therefore useful for exploring how stochastic and environmental effects in early development can influence disease risk in later life via epigenetic mechanisms. The ME property of correlation across diverse tissue types allows associations with phenotypes and exposures to be studied in easily accessible tissues. First identified in isogenic mice, metastable epialleles (MEs) are loci where the extent of DNA methylation (DNAm) is variable between individuals but correlates across tissues derived from different germ layers within a given individual. This property, termed systemic interindividual variation (SIV), is attributed to stochastic methylation establishment before germ layer differentiation. Evidence suggests that some putative human MEs are sensitive to environmental exposures in early development. In this review we introduce key concepts pertaining to human MEs, describe methods used to identify MEs in humans, and review their genomic features. We also highlight studies linking DNAm at putative human MEs to early environmental exposures and postnatal (including disease) phenotypes. [ABSTRACT FROM AUTHOR]

Cleft lip with or without cleft palate (CL/P) is a common human birth defect whose etiologies remain largely unknown. Several studies have demonstrated that periconceptional supplementation of folic acid can reduce risk of CL/P in offspring. In this study, we tested the hypothesis that the preventive effect of folic acid is manifested through epigenetic modifications by determining whether DNA methylation changes are associated with CL/P. To more readily observe the potential effects of maternal folate on the offspring epigenome, we focused on births prior to mandatory dietary folate fortification in the United States (i.e. birth year 1997 or earlier). Genomic DNA methylation levels were assessed from archived newborn bloodspots in a 182-member case-control study using the Illumina® Human Beadchip 450K array. CL/P cases displayed striking epigenome-wide hypomethylation relative to controls: 63% of CpGs interrogated had lower methylation levels in case newborns, a trend which held up in racially stratified sub-groups. 28 CpG sites reached epigenome-wide significance and all were case-hypomethylated. The most significant CL/P-associated differentially methylated region encompassed the VTRNA2-1 gene, which was also hypomethylated in cases (FWER p = 0.014). This region has been previously characterized as a nutritionally-responsive, metastable epiallele and CL/P-associated methylation changes, in general, were greater at or near putative metastable epiallelic regions. Gene Set Enrichment Analysis of CL/P-associated DMRs showed an over-representation of genes involved in palate development such as WNT9B, MIR140 and LHX8. CL/P-associated DNA methylation changes may partly explain the mechanism by which orofacial clefts are responsive to maternal folate levels.

Abstract Background Transposable elements (TEs) are enriched in cytosine methylation, preventing their mobility within the genome. We previously identified a genome-wide repertoire of candidate intracisternal A particle (IAP) TEs in mice that exhibit inter-individual variability in this methylation (VM-IAPs) with implications for genome function. Results Here we validate these metastable epialleles and discover a novel class that exhibit tissue specificity (tsVM-IAPs) in addition to those with uniform methylation in all tissues (constitutive- or cVM-IAPs); both types have the potential to regulate genes in cis. Screening for variable methylation at other TEs shows that this phenomenon is largely limited to IAPs, which are amongst the youngest and most active endogenous retroviruses. We identify sequences enriched within cVM-IAPs, but determine that these are not sufficient to confer epigenetic variability. CTCF is enriched at VM-IAPs with binding inversely correlated with DNA methylation. We uncover dynamic physical interactions between cVM-IAPs with low methylation ranges and other genomic loci, suggesting that VM-IAPs have the potential for long-range regulation. Conclusion Our findings indicate that a recently evolved interplay between genetic sequence, CTCF binding, and DNA methylation at young TEs can result in inter-individual variability in transcriptional outcomes with implications for phenotypic variation.

In humans, DNA methylation marks inherited from gametes are largely erased following fertilisation, prior to construction of the embryonic methylome. Exploiting a natural experiment of seasonal variation including changes in diet and nutritional status in rural Gambia, we analysed three datasets covering two independent child cohorts and identified 259 CpGs showing consistent associations between season of conception (SoC) and DNA methylation. SoC effects were most apparent in early infancy, with evidence of attenuation by mid-childhood. SoC-associated CpGs were enriched for metastable epialleles, parent-of-origin-specific methylation and germline differentially methylated regions, supporting a periconceptional environmental influence. Many SoC-associated CpGs overlapped enhancers or sites of active transcription in H1 embryonic stem cells and fetal tissues. Half were influenced but not determined by measured genetic variants that were independent of SoC. Environmental ‘hotspots’ providing a record of environmental influence at periconception constitute a valuable resource for investigating epigenetic mechanisms linking early exposures to lifelong health and disease.

Cleft lip with or without cleft palate (CL/P) is a common human birth defect whose etiologies remain largely unknown. Several studies have demonstrated that periconceptional supplementation of folic acid can reduce risk of CL/P in offspring. In this study, we tested the hypothesis that the preventive effect of folic acid is manifested through epigenetic modifications by determining whether DNA methylation changes are associated with CL/P. To more readily observe the potential effects of maternal folate on the offspring epigenome, we focused on births prior to mandatory dietary folate fortification in the United States (i.e. birth year 1997 or earlier). Genomic DNA methylation levels were assessed from archived newborn bloodspots in a 182-member case-control study using the Illumina® Human Beadchip 450K array. CL/P cases displayed striking epigenome-wide hypomethylation relative to controls: 63% of CpGs interrogated had lower methylation levels in case newborns, a trend which held up in racially stratified sub-groups. 28 CpG sites reached epigenome-wide significance and all were case-hypomethylated. The most significant CL/P-associated differentially methylated region encompassed the VTRNA2-1 gene, which was also hypomethylated in cases (FWER p = 0.014). This region has been previously characterized as a nutritionally-responsive, metastable epiallele and CL/P-associated methylation changes, in general, were greater at or near putative metastable epiallelic regions. Gene Set Enrichment Analysis of CL/P-associated DMRs showed an over-representation of genes involved in palate development such as WNT9B, MIR140 and LHX8. CL/P-associated DNA methylation changes may partly explain the mechanism by which orofacial clefts are responsive to maternal folate levels.

Background: Transposable elements (TEs) are enriched in cytosine methylation, preventing their mobility within the genome. We previously identified a genome-wide repertoire of candidate intracisternal A particle (IAP) TEs in mice that exhibit inter-individual variability in this methylation (VM-IAPs) with implications for genome function. Results: Here we validate these metastable epialleles and discover a novel class that exhibit tissue specificity (tsVM-IAPs) in addition to those with uniform methylation in all tissues (constitutive- or cVM-IAPs); both types have the potential to regulate genes in cis. Screening for variable methylation at other TEs shows that this phenomenon is largely limited to IAPs, which are amongst the youngest and most active endogenous retroviruses. We identify sequences enriched within cVM-IAPs, but determine that these are not sufficient to confer epigenetic variability. CTCF is enriched at VM-IAPs with binding inversely correlated with DNA methylation. We uncover dynamic physical interactions between cVM-IAPs with low methylation ranges and other genomic loci, suggesting that VM-IAPs have the potential for long-range regulation. Conclusion: Our findings indicate that a recently evolved interplay between genetic sequence, CTCF binding, and DNA methylation at young TEs can result in inter-individual variability in transcriptional outcomes with implications for phenotypic variation. [ABSTRACT FROM AUTHOR]

Most transposable elements (TEs) in the mouse genome are heavily modified by DNA methylation and repressive histone modifications. However, a subset of TEs exhibit variable methylation levels in genetically identical individuals, and this is associated with epigenetically conferred phenotypic differences, environmental adaptability, and transgenerational epigenetic inheritance. The evolutionary origins and molecular mechanisms underlying interindividual epigenetic variability remain unknown. Using a repertoire of murine variably methylated intracisternal A-particle (VM-IAP) epialleles as a model, we demonstrate that variable DNA methylation states at TEs are highly susceptible to genetic background effects. Taking a classical genetics approach coupled with genome-wide analysis, we harness these effects and identify a cluster of KRAB zinc finger protein (KZFP) genes that modifies VM-IAPs in trans in a sequence-specific manner. Deletion of the cluster results in decreased DNA methylation levels and altered histone modifications at the targeted VM-IAPs. In some cases, these effects are accompanied by dysregulation of neighboring genes. We find that VM-IAPs cluster together phylogenetically and that this is linked to differential KZFP binding, suggestive of an ongoing evolutionary arms race between TEs and this large family of epigenetic regulators. These findings indicate that KZFP divergence and concomitant evolution of DNA binding capabilities are mechanistically linked to methylation variability in mammals, with implications for phenotypic variation and putative paradigms of mammalian epigenetic inheritance. [ABSTRACT FROM AUTHOR]

Background: Phenotypic variability among inbred littermates reared in controlled environments remains poorly understood. Metastable epialleles refer to loci that intrinsically behave in this way and a few examples have been described. They display differential methylation in association with differential expression. For example, inbred mice carrying the agouti viable yellow (Avy) allele show a range of coat colours associated with different DNA methylation states at the locus. The availability of next-generation sequencing, in particular whole genome sequencing of bisulphite converted DNA, allows us, for the first time, to search for metastable epialleles at base pair resolution. Results: Using whole genome bisulphite sequencing of DNA from the livers of five mice from the Avy colony, we searched for sites at which DNA methylation differed among the mice. A small number of loci, 356, were detected and we call these inter-individual Differentially Methylated Regions, iiDMRs, 55 of which overlap with endogenous retroviral elements (ERVs). Whole genome resequencing of two mice from the colony identified very few differences and these did not occur at or near the eiders. Further work suggested that the majority of ERV iiDMRs are metastable epialleles; the level of methylation was maintained in tissue from other germ layers and the level of mRNA from the neighbouring gene inversely correlated with methylation state. Most iiDMRs that were not overlapping ERV insertions occurred at tissue-specific DMRs and it cannot be ruled out that these are driven by changes in the ratio of cell types in the tissues analysed. Conclusions: Using the most thorough genome-wide profiling technologies for differentially methylated regions, we find very few intrinsically epigenetically variable regions that we term iiDMRs. The most robust of these are at retroviral elements and appear to be metastable epialleles. The non-ERV iiDMRs cannot be described as metastable epialleles at this stage but provide a novel class of variably methylated elements for further study. [ABSTRACT FROM AUTHOR]

Aims & methods: The aim of this study was to characterize the methylation level of a polymorphically imprinted gene, VTRNA2-1 / nc886 , in human populations and somatic tissues.48 datasets, consisting of more than 30 tissues and >30,000 individuals, were used. Results: nc886 methylation status is associated with twin status and ethnic background, but the variation between populations is limited. Monozygotic twin pairs present concordant methylation, whereas ∼30% of dizygotic twin pairs present discordant methylation in the nc886 locus. The methylation levels of nc886 are uniform across somatic tissues, except in cerebellum and skeletal muscle. Conclusion: The nc886 imprint may be established in the oocyte, and, after implantation, the methylation status is stable, excluding a few specific tissues.

In humans, DNA methylation marks inherited from gametes are largely erased following fertilisation, prior to construction of the embryonic methylome. Exploiting a natural experiment of seasonal variation including changes in diet and nutritional status in rural Gambia, we analysed three datasets covering two independent child cohorts and identified 259 CpGs showing consistent associations between season of conception (SoC) and DNA methylation. SoC effects were most apparent in early infancy, with evidence of attenuation by mid-childhood. SoC-associated CpGs were enriched for metastable epialleles, parent-of-origin-specific methylation and germline differentially methylated regions, supporting a periconceptional environmental influence. Many SoC-associated CpGs overlapped enhancers or sites of active transcription in H1 embryonic stem cells and fetal tissues. Half were influenced but not determined by measured genetic variants that were independent of SoC. Environmental 'hotspots' providing a record of environmental influence at periconception constitute a valuable resource for investigating epigenetic mechanisms linking early exposures to lifelong health and disease., Competing Interests: MS, AS, NK, GC, CF, PI, AD, MB, SM, MR, ZH, CC, MD, PJ, DM, AP No competing interests declared, (© 2022, Silver et al.)