Fig 1.
(A) We studied population variability of DNA methylation in five different purified cell types derived from blood, skin and brain. (B) Utilizing a 1kb sliding window we identified Variably Methylated Regions (VMRs), representing clusters of ≥3 probes within the top 5% of population variability within each cell type. (C) An example VMR identified at the promoter region of EDARADD in fibroblasts. As indicated by the accompanying UCSC Genome Browser tracks, ENCODE data identifies this locus as being bound by several different TFs. Dashed red lines represent DNA methylation profiles for each of the 90 cell lines from the GenCord population, showing extreme epigenetic variability at this locus in the normal population.
Fig 2.
Epigenetic variation in different cell types.
(A) While some VMRs are common to multiple different cell types, in contrast, other VMRs identified in one cell type show minimal epigenetic variation in other tissues. (B) Venn diagram showing the degree of overlap for VMRs found in B-cells, T-cells, Fibroblasts, Neurons and Glia.
Fig 3.
Heat map of pair wise correlation values between all CpGs located within VMRs defined in fibroblasts.
CpGs on both axes are ordered by genomic position, revealing the presence of multiple VMRs located on different chromosomes that show highly correlated methylation levels in trans. Black bars (right and top) show the location of the HOXA (chr7), HOXB (chr17), HOXC (chr12) and HOXD (chr2) gene clusters, which correspond to some of the strongest regions of correlated methylation in both cis and trans. This observation suggests coordinated epigenetic regulation among loci distributed genome-wide.
Fig 4.
Cis and trans co-regulation of VMRs located at functionally related networks of genes that govern key developmental pathways.
(A) After selecting one CpG per VMR with the highest variance, we applied WGCNA to identify networks of significantly co-regulated VMRs. The Circos plot shows a representation of one of the largest co-regulated VMR modules identified in fibroblasts, which comprises 34 independent VMRs located on four different chromosomes, comprising all four clusters of HOX genes (outer circle). CpGs within VMRs in the co-regulated module are represented by blue tick marks (inner grey circle), with black lines joining VMRs that have methylation levels with pair wise absolute correlation values R≥0.7 (highlighted in yellow). Green bars show locations of genes at each locus. Blue bars show the location of transcription factor binding sites for SUZ12, EZH2 and CTBP2, all of which are significantly enriched within this co-regulated module. (B) Analysis of transcription factor binding sites defined using ChIP-seq [64] showed that VMRs within the co-regulated HOX gene module shown in (A) are significantly enriched for SUZ12, EZH2 and CTBP2 binding compared to all VMRs defined in fibroblasts (Bonferroni corrected p = 5.3x10-11, p = 1.5x10-9 and p = 5x10-5, respectively). Thus, binding of these TFs represents a potential mechanism by which epigenetic variation could be coordinated at multiple independent loci in trans. (C) Results of Gene Ontology (GO) analysis of genes associated with VMRs in the most significant co-regulated module identified in fibroblasts. We identified highly significant enrichments for multiple biological processes, including body patterning, growth and morphogenesis (S5 Table).
Table 1.
The top three Gene Ontology terms associated with co-regulated VMR modules found in each cell type.
Table 2.
Top 5 transcription factor binding sites overlapping with VMRs in various WGCNA modules in 5 cell types.
Fig 5.
Methylation levels at VMRs are influenced by heritable and non-heritable factors.
(A) Cell count corrected heritability [17] for VMRs in five cell types. Shared VMRs found in >1 cell type show significantly higher heritability, suggesting these are mostly under genetic control. (B) Methylation differences found within 426 pairs of monozygotic twins. CpGs that lie within VMRs show significantly increased MZ twin divergence compared to other CpGs, which is consistent with an environmental influence on methylation levels at VMRs.
Fig 6.
Experimental manipulation of DNA methylation using cell culture shows enrichment for VMRs at HOX genes and imprinted loci.
(A) To directly assess the effect of varying environmental conditions on epigenetic state, we grew genetically-identical fibroblasts under conditions of varying cell density and culture media replenishment. Cells from a single human fibroblast line were seeded in parallel at low density in ten culture flasks, and allowed to grow continuously for up to 10 days, either with or without regular change of media. Every 48 hours one flask was harvested and genome-wide DNA methylation patterns profiled. (B) Applying a sliding window approach identified 135 VMRs where methylation levels showed robust changes with varying culture conditions, including loci at HOX genes and multiple imprinted loci. Gene ontology analysis of VMRs induced by cell culture showed enrichments for fundamental control of growth, including similar GO categories to the co-methylated network identified in fibroblasts from the Gencord cohort (S9 Table). (C) Environmentally-responsive VMRs induced by cell culture showed a 35-fold enrichment for probes within the differentially methylated regions associated with seven different imprinted genes (p = 5.6x10-79). The left plot shows methylation profiles at the imprinted region of GNAS, which was also identified as a VMR in cultured fibroblasts. Each line shows the methylation profile at a different time point, with lighter shades of grey with increasing time. The right plot shows the change in methylation level with time at a single CpG (cg09885502) within the GNAS VMR.