Figure 2: The DGKA DMR shows gene enhancer characteristics.
From: Epigenetic regulation of diacylglycerol kinase alpha promotes radiation-induced fibrosis
(a) DGKA mRNA expression in primary human dermal fibroblasts on exposure to increasing doses of gamma irradiation. Data depict mean±s.e.m. from duplicate experiments in three different normal human dermal fibroblast strains derived from healthy donors. (b) Radiation-inducible and constitutive DGKA mRNA expression correlated with DNA methylation at the intragenic DGKA DMR in dermal fibroblasts (n=16). (c) Chromatin immunoprecipitation in differentially methylated patient-derived fibroblasts (n=8) and hypermethylated control cell lines (BJ, IMR90, HCT116) for enhancer histone marks (H3K4me1 and H3K27ac), as well as promoter marks (H3K4me3) at the DGKA DMR. Data depict mean±s.e.m. of four independent immunoprecipitations (triplicates for mRNA and DNA methylation measurements) per fibroblast strain. (d) Map of the interrogated DGKA 5′ UTR with CpG islands (green), DGKA transcripts (blue), ChIP-quantitative PCR amplicons (red) and luciferase reporter inserts (black). (e) Luciferase assay in pGL 4.10-based reporters measuring promoter activity in three different upstream elements P1, P2 and P3 (for their location, see d) and the DGKA DMR region. (f) Influence of sequence orientation (arrow to the left/right: sense/antisense orientation) and DNA CpG methylation (grey/black bars: unmethylated/methylated insert) on the DGKA DMR gene enhancer activity in a luciferase reporter plasmid carrying a CpG-free EF1alpha minimal promoter (EF1a). Graphs (e,f) depict mean±s.e.m. of four independent replicates in HEK293T cells. Vector maps (e,f) indicate DMR inserts (blue), minimal promoter (red) and luciferase (grey), hatched boxes indicate lack of vector insert. (g) Chromatin conformation capture depicting the interaction of the DGKA DMR with the surrounding DGKA locus in two patient fibroblasts with low- or high-DMR methylation. Data show mean±s.e.m. of three independent experiments. Insert shows gene regions tested with DGKA transcripts depicted in blue and CpG islands in green. ****P<0.0001, Student’s t-test. DMR, differentially methylated region; UTR, untranslated region.
