Fig. 3

The α enhancer regulates Nr5a1 expression specifically in immature gonadotropes. a The α enhancer exhibits different DNA CpG methylation status according to gonadotrope differentiation stage. Genomic DNA of αT1–1, αT3–1 and LβT2 cells was extracted and bisulfited. The FL enhancer and α enhancer-bisulfited sequences were amplified and cloned. A minimum of five clones per cell line was sequenced. Top: Schematic representation of enhancer sequences with location of CpGs (open-circle lollipops). Numbering is relative to Nr5a1 1A promoter TSS. The state of CpG methylation for each cell line, methylated (black circles) or unmethylated (open circles), is indicated below. b The regulation of Nr5a1 expression is dependent on the α enhancer sequence in immature αT3–1 cells. Deletion of genomic sequence of the FL and α enhancers was carried out in αT3–1 cells using CRISPR/Cas9 and two independent specific guide RNA (gRNA) couples flanking each enhancer sequence: α gRNA1–gRNA3 or α gRNA2–gRNA4 for the α enhancer and FL gRNA1–gRNA3 or FL gRNA2–gRNA4 for the FL enhancer as described in Additional file 7. Untargeting control gRNA was used as control. For each gRNA couples, three independent homozygous clones were tested for Nr5a1 expression by RT-qPCR. Nr5a1 expression level was normalized to Gapdh. Data are the normalized mean ± SEM of six independent experiments. WT and Δ FL enh αT3–1 or Δ α enh αT3–1 clones were compared with ANOVA followed by Dunnett’s multiple comparison tests. Significant difference with WT: “c” p < 0.001. c The α enhancer is a functional enhancer of Nr5a1 in immature αT3–1 cells. The α enhancer was decommissioned in αT3–1 cells using CRISPR/dCas9 fused with the lysine-specific histone demethylase LSD1 coding sequence (dCas9–LSD1). The dCas9–LSD1 was targeted to the α enhancer genomic sequence using α gRNA1–gRNA3 or α gRNA2–gRNA4 gRNA couples. Untargeting control gRNA (Ctr gRNA) was used as control. The 25% highly transfected cells were retrieved using cytometry cell sorting and tested for Nr5a1 expression by RT-qPCR. Nr5a1 expression level was normalized to Gapdh. Data are the normalized mean ± SEM of three independent experiments and are compared to control untargeting gRNA using Student’s t test “b” p < 0.01. d The α enhancer interacts with Nr5a1 pituitary promoter in progenitor and immature cells. Top: Quantitative chromatin conformation capture (c) assay was carried out in αT1–1 and αT3–1 cells. Chimeric DNA fragments were detected using a fixed forward primer targeting the α enhancer and several forward primers targeting regions upstream, inside or downstream from the 1G pituitary promoter sequence as shown in the schematic diagram of Nr5a1 structure. Primers positions are indicated with red arrows for Ce (external to locus control region), FL (fetal Leydig enhancer), 1G (1G promoter), α (α enhancer) and Ci (internal to locus control region). Exons are indicated as dark bars, regulatory regions as green bars and α enhancer as a purple bar. Numbering is relative to 1A promoter TSS. Bottom: Histograms showing qPCR measurements of chimeric fragments in 3C library. Raw qPCR data were normalized to input and to Ci/α chimeric DNA used as a control of non-specific ligation events. RP23 225F7 bacterial artificial chromosome (BAC) was used to create template enabling quantitative measurement of chimeric regions in the 3C library. Data are the mean ± SEM of four independent experiments and were analyzed with ANOVA followed by Dunnett’s multiple comparison tests. Significant difference with Ci/α: “b” p < 0.01; “c” p < 0.001, nd: not detected