Fig. 2

Discovery of an undescribed early enhancer of Nr5a1 gene. a, b and c Nr5a1-accessible chromatin regions harbor differential epigenetic marks of active enhancers depending on gonadotrope stage of differentiation. Epigenetic modifications of histone H3 associated with ATAC-seq open regions were investigated using chromatin immunoprecipitation assays in αT1–1, αT3–1 and LβT2 cell lines. Analyzed marks were lysine 4 tri- and monomethylation H3K4me3 (a) and H4K4me1 (b), specific of promoters and enhancers, respectively, and lysine 27 acetylation H3K27ac (c), enriched on active cis-regulatory sequences. Quantitative PCR was performed using sets of primers targeting ATAC-seq open regions. Raw qPCR data were normalized to input. The final results were expressed as fold over the control region. ANOVA followed by Dunnett’s multiple comparison tests was performed independently for each cell line and each histone modification. Results are the mean ± SEM of six independent experiments. Significant difference with the control region: “a” p < 0.05; “b” p < 0.01. d Nr5a1-accessible chromatin regions display differential cis-regulatory activity depending on gonadotrope differentiation stage. αT1–1, αT3–1 and LβT2 cells were transiently transfected with the potential cis-regulatory regions cloned in a pGL3b luciferase reporter system containing a minimal prolactin promoter (Pluc–Prl). Relative luciferase activity was measured as indicated in “Materials and Methods.” ANOVA followed by Dunnett’s multiple comparison tests was performed independently for each cell line. Results are normalized to control Pluc–Prl plasmid and are the mean ± SEM of six independent experiments. Significant difference with the control construct: “a” p < 0.05; “b” p < 0.01