Experimental manipulation of DNA methylation using cell culture shows enrichment for VMRs at <i>HOX</i> 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.