Fig. 5

SRCs and KMT2C/2D promote cell growth and transcription of ERE-containing target genes in conditionally expressed (Dox-inducible) WT and mutant ERα MCF-7 cell lines. Prior to all assays, cell lines were grown in charcoal-stripped, phenol red-free media with/without Dox for at least 2 days. a Confirmation of FLAG-tagged WT, Y537S, and D538G ERα Dox-inducible expression in MCF-7 cells transduced with specific lentiviruses. Immunoblotting was performed on whole-cell extracts with FLAG, ERα (HC-20 antibody), or β-actin (loading control) antibodies. *, Nonspecific band. →, Position of endogenous ERα. b KMT2D and SRC-3 co-immunoprecipitate with mutant ERα. Whole-cell extracts from Dox-inducible MCF-7 cells were subject to immunoprecipitation of FLAG, followed by immunoblotting. For analysis, intensities were normalized for different precipitated ERα levels by Image J. c Dox-inducible expression of Y537S and D538G ERα in MCF-7 cells results in activation of two canonical ERα target genes independent of E2. Cell lines were treated with Dox and the WT ERα line was treated (±) 10 nM E2 overnight, followed by RNA isolation. Relative levels of GREB1 or TFF1 mRNAs were determined by RT-qPCR with ESR1 mRNA as the normalizer. Data are represented as mean ± SEM (n = 3); **p < 0.01; ***p < 0.001. d, e Cell viability (measured by Cell Titer Glo as RLU) of Dox-inducible WT, Y537S, and D538G ERα expressing MCF-7 lines (induced as above). *p < 0.05; **p < 0.01; ***p < 0.001. d Cell viability is reduced in all three cell lines upon treatment with SRC inhibitor SI-1 (1 μM) after 3 days' exposure. DMSO served as the vehicle control. Data are represented as mean ± SEM (n = 3). e Cell viability of Dox-inducible WT and Y537S ERα-expressing MCF-7 lines, but not the D538G-expressing ERα line, is reduced upon knockdown of KMT2C and KMT2D after 3 days exposure to 100 nM total siRNA (50 nM each). Data are represented as mean ± SEM (n = 3). f Knockdown of KMT2C and KMT2D reduces Y537S ERα-enhanced expression of GREB1 and TFF1. Cells were transfected with siRNAs (100 nM total, 50 nM each). Relative levels of GREB1 or TFF1 mRNAs were determined by RT-qPCR using ACTB mRNA as the normalizer. Data are represented as mean ± SEM (n = 3); *p < 0.05; **p < 0.01; ***, p < 0.001. g Dox-induced FLAG-tagged WT, Y537S, and D538G ERα proteins occupy EREs of GREB1 and TFF1 genes in MCF-7 cells, implying direct transcriptional regulation. In contrast, ERα proteins minimally occupy a negative control region from intron 4 of the CCND1 gene, which lacks endogenous ERα binding [36]. Where indicated, WT ERα cells were treated with 100 nM E2 for 45 min. ChIP assays employed an antibody against FLAG to IP the FLAG-tagged ERα proteins and associated DNA. Isolated DNA was assayed by ChIP-qPCR. Representative data were plotted relative to percentage of starting input chromatin and are represented as mean of triplicate qPCR reactions ± SEM. Supplementary Figure 7d shows a repeated ChIP assay. h KMT2D occupies EREs of GREB1 and TFF1 genes in a Dox-dependent manner correlating with increased Y537S ERα occupancy. ChIP-qPCR was performed using an antibody to KMT2D. Representative data were plotted as above and the CCND1 gene intron 4 served as a negative control region. Supplementary Figure 7e shows a repeated ChIP assay