Additional file 3 of Bivalent promoter hypermethylation in cancer is linked to the H327me3/H3K4me3 ratio in embryonic stem cells

Figure S3. Differential H3K27me3 to H3K4me3 ChIPseq ratios at different genomic resolutions. (a) In Fig. 2d we show differential H3K27me3:H3K4me3 ChIPseq ratios over a genomic window of 4 kb. To test whether this result was due to the differing breadth of H3K27me3 and H3K4me3 ChIPseq peaks we repeated the analysis at 4 kb including two greater resolutions: 0.2 and 1 kb. hiBiv and loBiv were compared using Pearson’s correlation indicating statistically different H3K27me3:H3K4me3 ChIPseq ratios independent of window size. (b) To statistically test if H3K27me3 depletion is linked to transcription start site proximity, we analysed H3K27me3 depletion at three different resolutions. This showed that the greatest difference in H3K27me3 depletion is at the highest genomic distance (+/− 5 kb from peak centre) by boxplot analysis of mean J1:TKO H3K27me3 (rpkm) ratio per locus and non-equal variance two sample Student’s t-Test. Further, largest TKO H3K27me3 alterations occur at the highest DNA methylation level resolution (+/− 5 kb). Over shorter genomic ranges (0.2-1 kb) hiBiv and loBiv differential sensitivity is skewed towards loBiv regions based on median values at lower significance. This is likely due to the marked reduction in Polycomb presence at hiBiv regions, which are relatively dominated by H3K4me3 occupancy over these narrow windows. Mapping of average DNA methylation over the three genomic resolutions indicates that DNA methylation is most abundant over the 10 kb range, implying that DNA methylation is unlikely to play a prominent role in determining Polycomb localisation at the 0.2 and 1 kb ranges.