Enroth et al. BMC Cancer 2011, 11:450 http://www.biomedcentral.com/1471-2407/11/450 transitions in Axin, PP2A, b-catenin and in many down- stream factors along the WNT-signalling pathway affect- ing transcription, cell cycle and CRC development, especially within the TCF/LEF family [41,42] (Figure 4B). This suggests a repression on the b-catenin degra- dation complex and an up regulation of b-catenin dependent gene expression with downstream effects on the cell cycle [43]. Recently, a dual role of APC in the WNT-signalling pathway has been suggested with both activating and repressing mechanisms [44] which could explain the patterns found in patient 2 where APC has gained activating markers (Additional file 2, Figure S4B) In addition, patient 1 shows an activation of several known repressors of the WNT-pathway. CtBP represses E-cadherin which in turn is a tumor repressor restrict- ing tumor cell motility and invasion [45]. The ubiquitin ligase Cul1 is part of a mechanism targeting proteins for degradation and thus an important factor in cell cycle control. Specifically, Cul1 acts as an oncogene that forms complexes that specifically target the cell cycle inhibitor p27/Kip1 for degradation [46]. Global expression changes of H3K4me3 and H3K27me3 targets in CRC Although epigenetic transitions within category iii) are expected to have important functional consequences, few examples are detected in a given CRC patient. Therefore, these examples are less likely to have large- scale changes in gene expression. More global relation- ships between chromatin state and gene expression were analyzed using all epigenetic transitions involving more than 100 genes i.e. maintenance of H3K4me3 or H3K27me3, tumor loss of H3K4me3 or H3K27me3 as well as tumor gain of H3K4me3 or H3K27me3. Micro- array expression data for paired normal and tumor sam- ples from 24 CRC patients [23] were used, and the expression changes between normal and cancer samples for the genes included in each transition category (patient specific) was compared (Figure 5 and Additional file 2, Figures S1-S3). Genes in transitions where H3K4me3 was maintained or specifically gained in tumors, showed significantly higher expression levels in cancer samples (Figure 5A and Additional file 2, Figure S2). Opposite expression shifts were observed for genes that kept, gained or lost H3K27me3 in the tumor samples (Figure 5B, Additional file 2, Figure S2). The H3K4me3 and H3K27me3 gene groups also differed in the baseline expression in normal samples as H3K4me3 genes were highly expressed, while most H3K27me3 genes showed low expression levels. Furthermore, genes in H3K4me3 transition groups are typically already activated in normal tissue and become “hyperactivated” in tumors, while genes in H3K27me3 transition groups are lowly expressed in normal colon Page 7 of 13 and become “hypersilenced” in cancer samples. The generality of the observations is supported by (i) the similar expression shifts obtained when considering the epigenetic transition groups from either patient 1 and patient 2 (Additional file 2, Figure S2A); and (ii) from a second expression data set with paired normal and tumor samples from 9 CRC obtained in a different array platform [47] (Additional file 2, Figure S2 B-C). The significantly up- or down- regulated genes in can- cer samples in the two expression microarray data sets described above assuming a paired sample design was analyzed further. For both patients and microarray data sets (Figure 5C and Additional file 2, Figure S3) genes keeping or gaining H3K4me3 in tumors were signifi- cantly over-represented among up-regulated genes and significantly under-represented among down-regulated genes. Opposite patterns were observed for genes that kept, gained or lost H3K27me3 in tumor samples (Fig- ure 5C and, Additional file 2, Figure S3). These results indicate that at least for a fraction of genes the reported expression changes may be of large magnitude and occur in most patients. We gathered microarray expres- sion data for HT29 [23] and considered the same epige- netic transitions as described above, and can report that the tendencies towards hyperactivation or hypersilencing of H3K4me3 and H3K27me3 gene groups became even more pronounced in HT29 cells (Figure 5D-E and Addi- tional file 2, Figure S4). Hypersilencing of some H3K27me3 genes may involve loss of H3K27me3 and DNA hypermethylation The expression levels between normal colon, tumor samples and HT29 cell line for all H3K4me3 and H3K27me3 enriched genes were compared in the three samples types and for those genes that were shared or unique to any of them. The overall H3K4me3 or H3K27me3 gene groups showed progressive hyperacti- vation and hypersilencing in tumor and HT29 cells compared to normal colon, respectively (Figure 6A,B). This was also true for the genes with shared histone modification states, but less obvious in some of the unique groups. For the H3K27me3 unique groups, the hypersilencing pattern was still found for those genes carrying that histone mark only in normal colon sam- ples (Figure 6B). It has been recently reported [12] that genes enriched in H3K27me3 in normal prostate frequently become DNA hypermethylated and lose H3K27me3 in prostate cancer cell lines. Furthermore, DNA hypermethylation seems to be exacerbated by in vitro culture [14]. In order to test whether genes with the H3K27me3 mark in normal colon overlapped more with genes hyper- methylated in CRC than genes that carried the histone mark in tumor samples, the lists of H3K27me3 genes