Promoters marked by 5hmC in normal colon resist DNA methylation gain in tumours. (a) DNA methylation changes in adenocarcinoma (n = 17) relative to matched normal tissues (n = 17) (Infinium arrays). Each dot represents a CpG (grey dots are changes with P <0.01). (b) 5hmC read content measured in windows around the Infinium probes (black bars). CpG island (CpGi) as orange bar. (c) Overlay of 5hmC high or 5hmC low promoters on the methylation states. (d) Left panel: 5hmC content around the Infinium probes of promoters with a significant change in methylation. High 5hmC promoters are prone to loss of DNA methylation in tumours whereas low 5hmC promoters are prone to methylation gain in tumours (limma geneSetTest). Middle panel: 5hmC content in normal and levels of DNA methylation in normal to show that methylation gain or loss occurs across a range of methylation levels in normal (P values from a Wilcox test). Right panel: 5hmC content in normal and expression levels in normal. DNA methylation prone genes (5hmC low) have low expression in the normal tissue (P values from a Wilcox test). (e) Heatmap comparing 5hmC and 5mC levels in normal to the 5mC changes in tumours at selected loci.