Fig. 6
The ESR1-YAP1 fusion protein recruits the 26S proteasome for activated transcription of an ERE-driven luciferase reporter. a MS data depicted as a heatmap (displayed as in Fig. 1c) for ESR1-YAP1-dependent coactivators recruited to EREs from HNE. Recombinant purified WT or ESR1-YAP1 proteins were added to duplicate ERE DNA pulldown reactions. Fold change cutoff was ≥1.5 for enrichment over unliganded WT ERα. *ESR1 normalized ESR1 (see Supplementary Table 2). **YAP1 YAP1 corrected for ESR1. b Immunoblotting validation of the 26S proteasome being recruited to ESR1-YAP1. Independent ERE DNA pulldown samples were used to detect proteins recruited to ESR1-YAP1 compared to WT with/without E2 (i) or compared to WT or Y537S ERα (ii). Levels of ERα bound to the EREs were determined with an ERα antibody recognizing an N-terminal (N-term) epitope. TBP served as a loading control. 3% HNE, 3% of the starting HeLa S3 NE employed in the ERE DNA pulldown. c A 26S proteasome inhibitor, MG132, reduces ESR1-YAP1 transcriptional activity on an ERE-driven luciferase reporter. HeLa cells grown in phenol red-free, charcoal-stripped media were co-transfected with a vector expressing YFP-tagged ESR1-YAP1 protein and pERE-E1b-luc. Cells were then treated with vehicle (0.1% DMSO) or 1 μM MG132 for overnight. Luciferase activity (RLU relative light units) was assayed from whole-cell lysates. Data are represented as mean ± SEM (n = 3); ***p < 0.001
