Figure 1: G9a is required for the E2-induced expression of endogenous ERα target genes in breast cancer cells.

(a) Western blot analysis of G9a protein levels and H3K9me2 levels in control (shCtrl) and G9a knockdown (shG9a) MCF-7 (left) and T-47D (right) cells. Tubulin and total H3 were used as loading controls. (b,c) G9a is required for E2-induced activation of GREB1 and PR. qPCR analysis of gene expression in control (shCtrl) and G9a knockdown (shG9a) MCF-7 (b) and T-47D (c) cells treated with 10 nM of E2 for 3 or 6 h. Gene expression was normalized to GAPDH and is shown as fold relative to the expression of each gene in the control cells without E2 treatment that was arbitrarily set as ‘1’. Error bars indicate the mean ± s.e.m. of three experiments. Significant fold changes are indicated as follows: *P<0.05; ^**P<0.01 (Student’s t-test). (d) Gene expression heatmap of the E2-activated genes in control and G9a knockdown MCF-7 cells ±E2. Heatmap values represent the log2 fold change of read counts relative to the counts in the control cells without E2 induction (lane 1). E2-activated genes are divided into three groups: group A: G9a-dependent genes (downregulated in G9a knockdown cells); group B: G9a-repressed genes (further upregulated in G9a knockdown cells); and group C: G9a-independent genes (no change in G9a knockdown cells) from top to bottom. (e) Venn diagram showing the numbers of E2-activated genes assigned to each of the three groups as defined in d in MCF-7 cells. P<1e-143 (Fisher’s exact test).