Higher proportion of pathways enriched for both marks in either cell line showed intracellular co-enrichment of 5hmC and 5mC within the same expression tier on the same gene (p < 0.0001). While promoter and CGI methylation and hydroxymethylation marks governed completely different pathways, genes with intronic 5hmC marks exhibited strong 5mC and 5hmC co-enrichment for specific cellular functions in both cell lines (Fig. 4a). Furthermore, while gene-specific methylation and exonic hydroxymethylation were observed to co-regulateWe chose representative genes for Thonzonium (bromide) site Validation which were localized to hydroxymethylated regions detected by both hMeSeal-seq and hMeDIP-seq in RWPE-1, were not hydroxymethylated in 22Rv1, and overlapped RWPE-1 DHSs (Additional file 1: Table S8). Selected genes were not methylated in either cell line (Additional file 1: Figure S13). Cullin 2 (CUL2) bore methylation marks overlapping hydroxymethylation peaks in RWPE-1, but not in 22Rv1; in contrast, RasGEF domain family member 1a (RASGEF1a) and histidine triad nucleotidebinding protein 1 (HINT1) were methylated in the same region in 22Rv1 (exhibiting 5hmC “loss” in cancer while not being methylated in RWPE-1). RASGEF1a and CUL2 were located in intronic regions, while HINT1 refers to an intergenic 5hmC peak located most closely to the TSS of HINT1. Lastly, all genes chosen possessed robust and clear 5hmC enrichment peaks within the overlapping regions, with corresponding absent or low peaks in input samples (Fig. 5a). In order to validate the specificity of our sequencing data and quantify the relative 5hmC levels, we performed qPCR with standard curve protocol following hMeSeal enrichment of genomic RWPE-1 and 22Rv1 DNA. We used primers flanking the identified enrichment peaks for CUL2, RASGEF1a, and HINT1, as wellKamdar et al. Clinical Epigenetics (2016) 8:Page 9 ofabcdeFig. 4 Locus-specific co-enrichment of methylation and hydroxymethylation marks in normal prostate versus prostate cancer. a Genomic distribution of 5hmC marks exhibiting co-incidence with 5mC in normal prostate versus prostate cancer cells. Co-incident marks were defined as precise regions within the same cell line containing both 5mC and 5hmC marks which exhibited at least partial overlap. b Pathway enrichment annotations from GREAT for co-incident marks in (b) RWPE-1 exonic regions, (c) 22Rv1 intergenic regions, or intronic regions in (d) 22Rv1 or (e) RWPE-1 cells. Log p values for methylation marks (top) and hydroxymethylation marks (bottom) are shownas the negative control gene homeobox D8 (HOXD8). HOXD8 was found to be strongly methylated in RWPE1 (Additional file 1: Figure S14) and was not hydroxymethylated in either cell line. Negative control capture reactions lacking UDP-azide-glucose followed by qPCR were performed simultaneously across three technical replicates. Enrichment of HOXD8 was not detected viaqPCR, confirming the specificity of our capture reaction. CUL2, RASGEF1a, and HINT1 positive and negative control qPCR reactions PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28945807 were normalized to HOXD8 negative control using the Ct method, obtaining fold enrichment values of 2.73? 3.20? and 4.39? respectively (Fig. 5b), while enrichment was not detectable in 22Rv1 (Fig. 5c). These findings validate our hMeSeal-seqKamdar et al. Clinical Epigenetics (2016) 8:Page 10 ofabcFig. 5 Validation of differentially hydroxymethylated regions (DHMRs) via hMeSeal-qPCR. a Top: IGV Genome Browser representation of absolute hydroxymethylation peaks calle.
