DNA Methylation changes occurring following epithelial-mesenchymal transition. (A) Box-plot for gene expression levels according to promoter DNA methylation level of genes located in partially methylated domains (PMDs) (red) and outside PMDs (blue) in human mammary epithelial cells (HMLE). The x-axis represents promoter % methylation and y-axis represents normalized expression level. *** P <0.0001, ** P <0.001, ns: non-significant. (B) Correlation of DNA methylation level of CpG sites in HMLE vector cells (Pw) (x-axis) and HMLE Twist cells (y-axis), showing dramatic changes in DNA methylation following EMT. (C) Correlation of DNA methylation level of CpG sites in HMLE vector cells transduced with two different ‘control’ vectors Pw (x-axis) and GFP (y-axis) showing no change in DNA methylation. (D) Box plots of average gene expression levels of genes with a gain, loss or no change in DNA methylation. Note that a gain of DNA methylation (increase to ≥2% DNA methylation from gene promoters which were fully unmethylated, ≤1%) was associated with 3.8-fold decrease of their expression levels, while demethylation of gene promoters leading to fully unmethylated promoters (≤1%) was associated with an increase in their gene expression levels by about 2-fold. (E) GSEA showing that genes losing gene body methylation following EMT are enriched for genes which are down-regulated in a CDH1-knockdown model of EMT (P <0.0001). The bottom graph represents the rank-ordered, non-redundant list of genes. Genes on the far right (blue) correlated most strongly with decreased gene expression in the CDH1-knockdown model of EMT. FDR, false discovery rate. (F) Box plots showing decreased expression levels of genes losing gene body methylation following Twist-induced EMT in two different models: knockdown of CDH1 and basal breast cancers compared to luminal breast cancers. y-axis represents the log2-fold change of gene expression.