Fig. 4

Genome-wide transcriptomic analysis reveals regulation of ligand-independent estrogen receptor (ER) targets, and of novel target genes by ERα mutants in T47D and MC7 cells. a T47D and MCF7 cell lines were hormone-deprived for 3 days, treated with vehicle (veh) or 1 nM of estradiol (E2) for 24 h, RNA was isolated and RNA sequencing analysis was performed. The heat map shows normalized log2 fold change (FC) of genes differentially regulated in mutants vs wild-type (WT) in the absence of ligand (FC >2, p value <0.005). The genes are sorted based on E2 regulation in WT (red arrow ligand-independent E2 activated genes, blue arrow ligand-independent E2 downregulated genes, green circle ligand-independent non-E2 regulated genes, i.e. “novel target genes”). b Hormone-deprived T47D and MCF7 cells were treated with veh, 1 nM E2, 1 μM of fulvestrant (Ful) or 1 nM E2 plus 1 μM of Ful for 24 h. RNA was isolated, and GREB1 or insulin-like growth factor-binding protein 4 (IGFBP4) expression was assessed by quantitative RT-qPCR (one-way analysis of variance (Anova) for comparison of basal level, Student’s t test for comparison of Ful response in the presence of E2, *p < 0.05, **p < 0.01). The experiment was repeated twice with three biological replicates each time. c Cells were hormone-deprived, treated with 1 nM of E2 for 45 minutes, and chromatin-immunoprecipitation (ChIP) assays were performed on the ER binding sites on GREB1 and IGFBP4 promoters. The data are presented as fold enrichment compared to IgG control (one-way Anova, **p < 0.01). The experiment was repeated twice with three biological replicates each time and the figure shows a representative experiment. d The chi-square test was used to assess the statistical significance of overlaps in venn diagrams. Left panel overlap of E2-regulated genes in WT cells between the cell lines (chi-square test, **p < 0.01). Right panel overlap of ligand-independent target genes between different mutations within each cell line and between the two cell lines (chi-square test, **p < 0.01)