Figure 3: The DGKA DMR is regulating DGKA activation in fibroblasts.

(a) Upper panel: map of the interrogated region at the DGKA gene (transcripts shown in blue), CpG islands (green) and location of PCR amplicons used in ChIP analysis (red). lower panel: putative transcription factor-binding sites for EGR1 and FOS identified by in silico prediction at the DGKA DMR. (b) ChIP analysis showing EGR1 and FOS signals at the DGKA locus in patient fibroblasts with differential DGKA DMR methylation (high methylation, n=4; low methylation, n=4). Dots show mean values from four independent immunoprecipitations per fibroblast sample, bars depict mean±s.e.m. in each group. Rabbit IgG served as control. (c,d) DGKA mRNA transcription 48 h after siRNA-mediated knockdown of EGR1 in irradiated (2 Gy) patient-derived fibroblasts (n=4 high-methylation and n=4 low-methylation fibroblasts) (c) and global DGK activity (d). siCon, non-targeting siRNA control, siEGR1, siRNA directed against EGR1. Dots show mean values from duplicate experiments for each fibroblast (triplicates for d), bars depict mean±s.e.m. for each group. (e) Luciferase reporter assay in HEK293T cells on overexpression of EGR1 showing signal from a reporter containing the DGKA DMR and an adjacent minimal promoter (EF1a). Grey/black bars: unmethylated/methylated insert after in vitro methylation of the reporter plasmid. (f) Site-directed mutagenesis of the two predicted EGR1-binding sites at the DGKA DMR and influence on enhancer activity in a luciferase reporter construct. Relative luciferase activity in a reporter vector carrying the DGKA DMR (blue), a minimal promoter (EF1a, red) and luciferase (grey) are shown. P values refer to comparison with WT-EF1a reporter. Data from e and f show mean±s.e.m. of at least four individual measurements. EF1a, CpG-free EF1alpha minimal promoter; EV, empty vector; WT, wild type; hatched boxes indicate lack of vector insert *P<0.05, **P<0.01, ***P<0.001, Student’s t-test.