Linear positive correlation with gene XR9576MedChemExpress Tariquidar expression in normal prostate (similar to 5mC), this correlation was strongly altered in prostate cancer, where the presence of hydroxymethylation in these features showed nonlinear correlation with expression in the median tier. This trend persisted despite the significant decrease of 5hmC in cancer observed in exonic and DHS-proximal regions PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28461567 and may indicate altered functionality of 5hmC marks in prostate cancer. The same correlation was also performed for RWPE-1 hMeDIP-seq data (Additional file 1: Table S5), where intergenic, genic, and DHS-proximal trends from hMeSeal-seq were strongly corroborated (Additional file 1: Figure S8).Absence, gain, and retention of 5hmC differs significantly by genomic region between normal and cancer cell linesIn order to determine the effect of the observed differential epigenetic mark distribution in 22Rv1 on gene regulation, we performed integrative analysis correlating (hydroxy)methylation-enriched genes stratified by genomic feature to gene expression levels obtained from publicly available microarray data for RWPE-1 [Gene Expression Omnibus (GEO) Accession: GSM375783] (n = 1) and 22Rv1 [GEO Accession: GSE36135] (n = 3). We divided the expression data into three equal tiers representing low, median, and high relative expression and correlated genes significantly differentially enriched for either 5mC or 5hmC marks to expression within each tier (Additional file 1: Table S3 and S4). We found gene expression to exhibit negative correlation with methylation enrichment in CGIs (both within and outside core promoter regions) and positive correlation with methylation in genic regions and regions within 5 kbp of UCSC RWPE-1 DHSs in both cell lines, as expected (Fig. 2c, d). Correlations between 5mC and expression tended to be more robust in the cancer cell line, with strong negative correlation to expression also observed for intergenic and promoter methylation alone in 22Rv1.We examined absolute peak regions where 5hmC marks detected in RWPE-1 either overlapped with peak regions detected as having 5mC marks in 22Rv1 (5hmC “absence”) or 5hmC marks in 22Rv1 (5hmC “retention”), as well as peaks where 5mC marks in RWPE-1 overlapped with 5hmC marks in 22Rv1 (5hmC “gain”). We then tested these peak regions, stratified by genomic location, to determine genomic features where the frequency of occurrence of these overlapping peaks could not be explained by either random change in RWPE-1 epigenetic marks or by random distribution of 22Rv1 marks within each genomic feature (Additional file 1: Table S6). Peaks located within exonic, promoter, or RWPE-1 DHS genomic features exhibited greater 5hmC depletion than expected from the proportion of global RWPE-1 5hmC or 22Rv1 5mC; however, they did not significantly differ from expected values for random 5hmC retention or gain in cancer (Table 1). In other words, peaks within these features were far more likely to exhibit absence of 5hmC in cancer than to gain or retain it (Additional file 1: Table S7), suggesting that loss of 5hmC within these specificKamdar et al. Clinical Epigenetics (2016) 8:Page 6 ofTable 1 Locus-specific hydroxymethylation changes in cancer. 5hmC changes were described by the overlap of 5mC and/or 5hmC marks between cell lines. Overlapping 5mC or 5hmC marks were compared to the overall distribution of each mark within each cell line to determine significant differencesDifferential 5mC/5hmC status in PCa 5hmC “Abs.
