T was also seen within this tier in cancer, although most genes involved were expressed at the same level in both normal and PCa cells. Perhaps most intriguingly, intergenic regions exhibited pathway enrichment within the lowest tier of expression unique to 22Rv1 cells. Strikingly, many of these pathways were associated with tumorigenic functions (Fig. 3e), suggesting a putative role for intergenic hydroxymethylation in the downregulation of inflammation and cellular adhesion and the upregulation of epithelial cell proliferation. Of particular interest, however, was the presence of 5hmC enrichment in pathways governing the negative regulation of androgen signaling. Furthermore, differentially hydroxymethylated genes within this pathway, including the PCa-inhibitory genes secreted fizzled-related protein 1 (SFRP1) and Dab, mitogen-responsive phosphoprotein homolog 2 PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/27693494 (DAB2), exhibited significantly lower expression in cancer than in the normal cell line. In sharp contrast, Chloroquine (diphosphate) chemical information RWPE-1 showed few significant functional annotations for low-tier intergenic hydroxymethylation marks (Additional file 1: Figure S12). Overall, exonic and intergenic regions showed the most striking differential pathway enrichment between normal and cancer cells for both 5mC and 5hmC marks. Differentially hydroxymethylated genes from high-level exonic pathways in RWPE-1 and low-tier intergenic pathways in 22Rv1 exhibited large differences in expression between cell lines, potentially indicating a critical role for aberrations in exonic and intergenic patterning of 5mC and 5hmC marks in cancer.Locus-specific 5mC and 5hmC marks share a novel cooperative role in the regulation of functional pathwayspathways related to basic cellular functionalities such as ATP binding and hydrolase activity in RWPE-1 (Fig. 4b), this co-regulation was entirely lost in 22Rv1, mirroring the loss of exonic 5hmC marks in cancer. Intriguingly, co-occurrence of 5mC with intergenic 5hmC epigenetic marks was observed in 22Rv1, but not in RWPE-1, coinciding with high-level functional annotations in the lowest tier of expression including signaling, regulation of cellular proliferation and cAMP biosynthesis regulation in cancer (Fig. 4c). Additionally, we found that most hydroxymethylated intronic pathways showing concomitant 5mC enrichment in cancer were related to basic cellular functions, such as binding, protein localization, and cytoskeletal organization, within each expression tier (Fig. 4d). This observation contrasts sharply with hydroxymethylated intronic co-enrichment pathways in RWPE-1, where basic processes predominated in the middle and lowest expression tiers (Fig. 4e). However, annotations for genes exhibiting both marks within the highest tier of expression were enriched for multiple highly specific processes, including pathways related to the electron transport chain as well as binding of both NF-B and p53. This data provides novel evidence for the highly locus- and gene-specific dysregulation of 5hmC in governing oncogenesis.Genes involved in key differential biological processes are detectable via hMeSeal-qPCRWe compared biological pathway annotations for genes enriched in both 5mC and 5hmC within each cell line by expression tier and genomic feature in order to determine whether or not intercellular differences observed in the type of epigenetic modifications resulted in significant differences in pathway regulation (Additional file 1). Surprisingly, we found that a significantly.