Figure 4

PU.1 motifs and binding peaks are enriched among the preleukemic hypermethylated genes. (A) Enriched sequence motifs within the preleukemic hypermethylated genes. (B) Venn diagram showing the number of overlapping and non-overlapping genomic coordinates of preleukemic hypermethylated CGIs (green) and publicly available ChIP-Seq data (red). Numbers in brackets represent a more stringent analysis, namely preleukemic hypermethylated genes represented by at least two hypermethylated probes. (C) Validation of PU.1 target gene Bcor by MassARRAY. Heatmaps display methylation levels of single CpG units (columns) in different disease stages (rows). Differently colored bars to the left of the heatmaps display preleukemic (P, black), early leukemic (EL, blue), late leukemic (LL, red), wild type (W, green) and standard (S, grey). Methylation values range from 0% (light green) to 100% (dark blue). The scheme above the heatmaps displays the gene (black bar), transcription start (arrow), the PU.1 ChIP peak (PU1), the relative location of the CpG island (CGI) and the two MassARRAY amplicons (MA). The dotplots below the heatmaps show the average amplicon methylation levels of wild-type animals and different disease stages. The median methylation in a sample group is indicated by a black bar. Mann-Whitney U test was used to test for differences between wild type and stages and between stages (*P < 0.05, **P <0.01, ***P < 0.001).