To define DNA demethylation changes that drive expression of AREG and EREG, sodium bisulfite conversion and whole genome sequencing was performed on genomic DNA from pancreatic cancer cells with either high (Panc1-3D7) or low (Panc1-2G6) integrin α6β4 expression. Sequencing reads were aligned to the reference genome, GRCH37, mapped to the AREG and EREG genes, and visualized using the UCSC genome browser. We found that cells with high integrin α6β4 (Fig. 3A and B; bottom panels) have reduced DNA methylation within intronic regions of both EREG (Fig. 3A) and AREG (Fig. 3B), confirming that the integrin α6β4 drives site-specific DNA demethylation, and defining the critical CpG sites of AREG and EREG that become altered downstream of integrin α6β4.Figure 3The integrin α6β4 drives both gene specific and global DNA hypomethylation. (A–C) Genomic DNA from Panc1-2G6 (β4 low; upper panels) and Panc1-3D7 (high β4; lower panels) was processed for high-resolution methyl-seq by the NextGen Sequencing Core at the Norris Comprehensive Cancer Center. Samples were analyzed bioinformatically and percent methylation shown for EREG (A), AREG (B), and AREG pseudogene (C). (D) Percent hypomethylation and hypermethylation per chromosome when comparing Panc1-3D7 vs. Panc1-2G6. (E) Defined regions of interest assessed for changes in DNA methylation (F) Location of DMLs across the genome. (G) Percent of methylation changes located in CpG islands and shores. (H) Location of DMLs associated with H3K27Ac. (H) Distance from TSSs for DMLs (I) Distances from TSSs for DMRs (J) Number of both hypomethylated and hypermethylated regions corresponding to genomic features.