Metastable epialleles in humans.

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.

Tissue- and ethnicity-independent hypervariable DNA methylation states show evidence of establishment in the early human embryo.

We analysed DNA methylation data from 30 datasets comprising 3474 individuals, 19 tissues and 8 ethnicities at CpGs covered by the Illumina450K array. We identified 4143 hypervariable CpGs ('hvCpGs') with methylation in the top 5% most variable sites across multiple tissues and ethnicities. hvCpG methylation was influenced but not determined by genetic variation, and was not linked to probe reliability, epigenetic drift, age, sex or cell heterogeneity effects. hvCpG methylation tended to covary across tissues derived from different germ-layers and hvCpGs were enriched for proximity to ERV1 and ERVK retrovirus elements. hvCpGs were also enriched for loci previously associated with periconceptional environment, parent-of-origin-specific methylation, and distinctive methylation signatures in monozygotic twins. Together, these properties position hvCpGs as strong candidates for studying how stochastic and/or environmentally influenced DNA methylation states which are established in the early embryo and maintained stably thereafter can influence life-long health and disease.

DNA methylation at the suppressor of cytokine signaling 3 (SOCS3) gene influences height in childhood.

Human height is strongly influenced by genetics but the contribution of modifiable epigenetic factors is under-explored, particularly in low and middle-income countries (LMIC). We investigate links between blood DNA methylation and child height in four LMIC cohorts (n = 1927) and identify a robust association at three CpGs in the suppressor of cytokine signaling 3 (SOCS3) gene which replicates in a high-income country cohort (n = 879). SOCS3 methylation (SOCS3m)-height associations are independent of genetic effects. Mendelian randomization analysis confirms a causal effect of SOCS3m on height. In longitudinal analysis, SOCS3m explains a maximum 9.5% of height variance in mid-childhood while the variance explained by height polygenic risk score increases from birth to 21 years. Children's SOCS3m is associated with prenatal maternal folate and socio-economic status. In-vitro characterization confirms a regulatory effect of SOCS3m on gene expression. Our findings suggest epigenetic modifications may play an important role in driving child height in LMIC.

Environmentally sensitive hotspots in the methylome of the early human embryo.

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.

Extracellular DNA, cell surface proteins and c-di-GMP promote biofilm formation in Clostridioides difficile.

Clostridioides difficile is the leading cause of nosocomial antibiotic-associated diarrhoea worldwide, yet there is little insight into intestinal tract colonisation and relapse. In many bacterial species, the secondary messenger cyclic-di-GMP mediates switching between planktonic phase, sessile growth and biofilm formation. We demonstrate that c-di-GMP promotes early biofilm formation in C. difficile and that four cell surface proteins contribute to biofilm formation, including two c-di-GMP regulated; CD2831 and CD3246, and two c-di-GMP-independent; CD3392 and CD0183. We demonstrate that C. difficile biofilms are composed of extracellular DNA (eDNA), cell surface and intracellular proteins, which form a protective matrix around C. difficile vegetative cells and spores, as shown by a protective effect against the antibiotic vancomycin. We demonstrate a positive correlation between biofilm biomass, sporulation frequency and eDNA abundance in all five C. difficile lineages. Strains 630 (RT012), CD305 (RT023) and M120 (RT078) contain significantly more eDNA in their biofilm matrix than strains R20291 (RT027) and M68 (RT017). DNase has a profound effect on biofilm integrity, resulting in complete disassembly of the biofilm matrix, inhibition of biofilm formation and reduced spore germination. The addition of exogenous DNase could be exploited in treatment of C. difficile infection and relapse, to improve antibiotic efficacy.