Fig. 5
EHMT2/G9a is a negative regulator of ESR1 expression in endometrial cancer. a Schematic workflow used to perform DNA-oligo protein pull-downs. Biotin-conjugated wild-type or mutated DNA-oligos are immobilized on streptavidin magnetic beads and mixed with Ishikawa nuclear lysates. Captured proteins are then dimethyl labeled and analyzed by mass spectrometry. b A DNA oligo with the sequence of the ±25 bp surrounding the ERα binding site in the ESR1 Enhancer 1, in wild-type or chr6:152,002,679–TCT-to-T form, was used to perform DNA-oligo protein pull-downs in Ishikawa cells as described in a. The scatter plot shows the log2 ratios of all identified and quantified proteins in both experiments plotted against each other. Proteins significantly enriched at the wild-type sequence are highlighted in red, and proteins significantly enriched at the mutant sequence are highlighted in blue. c Gene expression correlation heatmap for all corresponding 24 differentially bound proteins identified in b in 589 endometrial cancer patients (TCGA). Dashed lines indicate the separation between positive and negative correlation scores. Genes are ranked by correlation with ESR1 gene expression. d Progression-free Kaplan-Meier curve of endometrial cancer patients (TCGA data) divided into two groups using the median of EHMT2/G9a expression (FPKM) as cutoff. e EHMT2/G9a and IgG (negative control) ChIP followed by western blot in Ishikawa endometrial cancer cells. Antibodies against EHMT2/G9a and ERα have been used. For EHMT2/G9a, two different exposure images were used for input and IP, as indicated by the vertical dashed bar. f EHMT2/G9a ChIP in Ishikawa cells stimulated for 6 h with 10 nM β-estradiol. Bar plot shows percentage of enrichment over the input (% Input) at the ESR1 Enhancer 1, ESR1 promoter, and CDK12 promoter (negative control) analyzed by quantitative PCR (qPCR). Mean of 2 independent experiments is shown. g Ishikawa endometrial cancer cells were stimulated for 72 h with 10 nM β-estradiol in combination or not with 100 nM ICI-182,780 (fulvestrant, negative control). In all conditions, cells where incubated either with a non-targeting (NT) siRNA or with an siRNA against EHMT2/G9a. Then, whole-cell extracts were analyzed by immunoblotting with antibodies against ERα, EHMT2/G9a, and GAPDH (loading control). h Differential ERα peak centered EHMT2/G9a average ChIP-seq density profiles in Ishikawa cells upon 6 h treatment with 10 nM β-estradiol (purple) or DMSO (blue, control). Ribbon indicates SEM. Paired Wilcoxon test was performed on the average score of the highlighted area for each individual region analyzed; P values are indicated. i Schematic representation of the ESR1 gene regulation working model. Upon tumorigenesis, ERα is re-located to an endometrial cancer specific ESR1 enhancer (Fig. 1f–g). ERα interacts with EHMT2/G9a (e) and binds both enhancer and promoter regions of ESR1, in a hormone-dependent fashion (f). In a subset of metastatic endometrial cancers, a somatic mutation is acquired at the tumor-specific ESR1 enhancer (Fig. 4a). In vitro analyses probing this mutation revealed a loss of EHMT2/G9a DNA binding capacity (b), and EHMT2/G9a knockdown in endometrial cancer cell lines leads to an increase of ESR1 expression levels (g)