Fig. 1

Proteomics of co-regulators recruited to the Y537S ERα LBD point mutant. a Schematic diagram of WT, Y537S, and D538G ERα point mutants (mutations indicated by arrows), and the ESR1-YAP1 fusion protein. Numbers refer to amino-acid residues in ERα and YAP1. AF1 activation function 1, DBD DNA-binding domain. Hinge, region between DBD and ligand-binding domain (LBD); AF2 Activation function 2, WW WW domain, TAD transcription activation domain. In ESR1-YAP1, blue represents ERα residues (1–365); yellow indicates YAP1 residues (230–504). b Mutant ERα proteins display E2-independent transcriptional activity. Vectors expressing YFP or YFP-tagged WT, Y537S, D538G, or ESR1-YAP1 proteins were co-expressed with an ERE-dependent luciferase reporter (pERE-E1b-luc) in HeLa cells grown in charcoal-stripped fetal bovine serum (FBS). Cells were then treated with/without 10 nM E2 for overnight. Luciferase activity (RLU relative light units) was assayed from whole-cell extracts of the cells transfected in triplicate. Data are represented as mean ± SEM (n = 3); ***p < 0.001. c MS data depicted as a heatmap for WT or Y537S ERα-dependent coactivators recruited to EREs. (Top) Schematic of ERE DNA pulldown assay using HeLa S3 NE as the source of co-regulators (modified from ref. [21]) and purified ERα proteins. (Bottom) MS data were analyzed from duplicate reactions using a label-free method and depicted as in ref. [21]. Peptides number of peptides detected; amount (vsESR1) amount normalized by sum of area under the curve for six N-terminal ESR1 peptides (see Supplementary Table 2). Fold change represents the ratio of amount detected normalized to unliganded WT ERα. Fold change cutoff used was ≥1.5. SRC-1 to -3 (gene symbols: NCOA1, NCOA2, NCOA3), p300 (gene symbol: EP300), CBP (gene symbol: CREBBP). d Immunoblotting validation of KMT2D and SRC-3 enrichment with purified Y537S ERα bound to EREs using independent DNA pulldown samples in the absence of E2. ERα protein binding was assayed by an N-terminal (N-term) antibody. TBP serves as a loading control. 2% input represents 2% of the starting HNE employed in the ERE DNA pulldown. e Y537S ERα and the KMT2D complex interact directly in an enhanced manner relative to unliganded WT ERα. ERE DNA pulldown assays were performed with purified ERα proteins and a purified KMT2D “fusion” complex [22], and then analyzed by immunoblotting. (i) Representative immunoblot probing for select KMT2D complex members and ERα. (ii) Quantification of KMT2D signal across three independent pulldown assays. Binding was quantified using Image J and normalized to ERα signal