Fig. 4

ERα controls the cis-regulatory activity of the α enhancer. a A 65-bp core sequence of the α enhancer is conserved across mammalian genomes. The α enhancer genomic sequences of 31 mammalian species were retrieved on Ensembl Web site and aligned using Clustal Omega software. A 65-bp core sequence displays clear conservation among the different species. In this core sequence, a perfect match with the canonical ERE-binding site was observed (red bar and ERE-binding motif). b The ERE-binding site in the 65-bp core sequence is essential for α enhancer cis-regulatory activity in immature αT3–1 cells. αT3–1 cells were transiently transfected in complete steroid-containing culture medium with Pluc constructs containing either a minimal prolactin promoter (Pluc–Prl) used as control, a full-length α enhancer (Pluc–α enh), a truncated α enhancer (Pluc–α enh Δ65, harboring a deletion of the 65-bp core sequence), a reduced α enhancer (Pluc–α enh +65, containing only the 65-bp core sequence) or a mutated α enhancer (Pluc–α enh MutERE with the following mutation in the ERE-binding site: GATCAATGTGATC). Prl minimal promoter is represented in dark followed by luciferase coding sequence shown in green, α enhancer is represented in blue, containing the 65-bp core sequence in orange, and the ERE-binding site in yellow. ERE mutation is indicated with asterisks. Relative luciferase activity was measured as indicated in “Materials and Methods.” Results were normalized to Pluc–Prl used as control and are the mean ± SEM of six independent experiments. Comparisons with control were performed with ANOVA followed by Dunnett’s multiple comparison tests. Significant difference with Pluc–Prl control: “c” p < 0.001. c Erα but not Erβ is expressed in immature αT3–1 cells. Total RNA from αT1–1, αT3–1 and LβT2 cells was extracted and reverse transcribed. The mRNA levels of Erα and Erβ were quantified as indicated in “Materials and Methods.” Results are normalized to Gapdh and are the mean ± SEM of four independent experiments. ANOVA followed by Bonferroni’s post hoc comparisons test was performed to compare Erα expression in the different cell lines. Significant difference: “a” p < 0.05 and “b” p < 0.01. Nd: not detected. d Endogenous ERα binds to the α enhancer chromatin in immature αT3–1 cells. ERα binding on α enhancer chromatin was investigated using ChIP assays in the αT3–1 cell line using the anti-estrogen receptor alpha ChIP-grade antibody (abcam ab32063). Quantitative PCR was performed using primers targeting the α enhancer sequence (α enh). Raw qPCR data were normalized to input. The final results were expressed as fold over the control region (Ctr region). Results are the mean ± SEM of four independent experiments. Significant difference with the control region using Student’s t-test: “c” p < 0.001. e The cis-regulatory activity of α enhancer is strictly dependent on Erα expression level. αT3–1 cells were transiently co-transfected with control (Pluc–Prl) or full-length α enhancer (Pluc–α enh) constructs and with scramble or Erα SiRNA. Relative luciferase activity was measured as indicated in “Materials and Methods.” Results were normalized to control Pluc–Prl plasmid and are the mean ± SEM of six independent experiments. Significant difference with the scramble SiRNA using Student’s t test “c” p < 0.001. f ERα agonist and antagonist modulate α enhancer cis-regulatory activity. αT3–1 cells were transiently transfected with control (Pluc–Prl) or full-length α enhancer (Pluc–α enh) constructs. Transfected cells were treated with either vehicle, E2 or ICI 182,780 at the indicated concentrations. Relative luciferase activity was measured as indicated in “Materials and Methods.” Results were normalized for control Pluc–Prl and are the mean ± SEM of six independent experiments. ANOVA followed by Dunnett’s multiple comparison tests was performed to compare drugs at different concentrations against vehicle. Significant difference with the vehicle: “c” p < 0.001