5fC is associated with active gene transcription. (a) Relationship between gene expression levels and cytosine modifications. The first three green bars show the gene expression levels of all genes (All), genes with CGI promoter (+ CpG) and genes with non-CGI promoters (- CpG). The subsequent bars are labeled with the notation 'x > y' (for example, mC > hmC) to indicate genes whose promoter CGI is relatively more enriched in one modification (for example, mC) than in the other (for example, hmC). The number below each label is the number of genes belonging to each category. See Table s5 in Additional file 1 for significance of difference between groups. The × symbols show the group means. In all the boxplots in this figure the whiskers extend up to 1.5 times the interquartile range and data points beyond this range have been omitted for clarity of presentation. (b) Genes are categorized into low (0 to 25%, white), medium (25 to 75%, grey) and high (75 to 100%, blue) expressed genes and correlated with 5fC levels of two biological replicates (normalized read counts). (c) Relationship between 5fC, 5hmC, 5mC and H3K4me3 and H3K27me3 at promoters. (d) Enrichments of cytosine modifications in the transcription factor binding sites of CTCF, p300 and Pol II over the input. RPKM, reads per kilobase per million mapped reads.