T-47D and MCF-7 breast cancer cells were grown in SILAC-isotope containing media and treated with either vehicle control or R5020, a synthetic progestin for 3 h. PR (a) or ERα (b) RIME was conducted and the proteins that were quantitatively enriched in both cell lines are shown. Only proteins that were enriched with a FDR < 1% were included.c, Peptide coverage of the PR protein following ERα RIME in T-47D cells. The identified peptides are highlighted and one of the peptides covers the ‘Bus’ region representing the PR-B isoform.d, Comparison of binding at different time points and treatment of MCF-7 breast cancer cells with progesterone. ERα ChIP-seq at 3 h and 24 h results in comparable binding. Correlation between progesterone (PG) and R5020 (RO) at 3 and 24 h.e, MCF-7 cells were grown in oestrogen rich complete media and treated with progesterone or vehicle control for 3 h. ERα ChIP was conducted and peaks that occurred in at least two of three independent replicates were considered. Venn diagram showing the changes in ERα binding following progesterone treatment of MCF-7 cells.f, Uncropped western blots fromFig. 1c.

For each experiment, all binding sites identified as overlapping in at least two samples are merged and retained, and normalized read counts computed at each site for each sample.a, Clustering correlation heat maps, based on Pearson correlations read scores (with replicate numbers in the labels), show good reproducibility between replicates and similarity of natural and synthetic hormone treatments.b, PCA plots showing the two most significant principal components (with samples labelled with treatment type: ‘C’ for full-media control conditions, ‘P’ for progesterone, and ‘R’ for R5020). The data from the two cell lines is shown.

a,b, T47-D (a) or MCF-7 (b) cells were hormone-deprived and treated with vehicle control, oestrogen alone or progesterone alone. ERα ChIP-seq was conducted and we assessed the binding at the regions previously shown to be reprogrammed by oestrogen plus progesterone. The ERα reprogramming data under both oestrogen and progesterone conditions in the T-47D cells is fromFig. 2b. In the absence of oestrogen, progesterone does not induce ERα binding. In the absence of progesterone, oestrogen does not induce ERα binding to the locations shown to acquire reprogrammed ER binding events under dual hormone conditions.

a, Validation of dependence on PR for ERα binding and overlap between ERα binding and FOXA1 binding. T-47D cells were grown in full, oestrogen-rich media and transfected with siControl or siRNA to PR. ERα ChIP was conducted followed by qPCR of several novel ERα binding events only observed under progesterone treatment conditions. In the absence of PR, ERα is not able to associate with the progesterone-induced binding sites. The figure represents one biological replicate of three completed replicates and the error bars represent standard deviation of the technical ChIP-PCR replicates.b, Venn diagram showing the ERα binding events that were conserved in T-47D cells (that is, not altered by progesterone when compared to oestrogen alone) and the ERα binding events that were reprogrammed by progesterone treatment, when overlapped with FOXA1 ChIP-seq data from T-47D cells. The FOXA1 ChIP-seq data from T-47D cells was from ref.19.c, Differential gene changes in MCF-7 and T-47D cells following treatment with progesterone or R5020 for 3 h. Heat map showing gene changes relative to matched controls. Eight replicates were included.d, Table showing the differentially regulated genes in the two cell lines and in the two treatment conditions.e, Overlap between genes regulated by progesterone (in both cell lines) and gene regulated by the synthetic progestin R5020 (in both cell lines).

a, RNA-seq was conducted after progesterone or R5020 treatment for 3 h. GSEA analysis was conducted on progesterone/R5020 repressed genes with lost ERα binding events observed in T47-D cells. The progesterone-decreased ERα binding regions correlate with progesterone downregulated genes.b, Kaplan–Meier survival curve in 1,959 breast cancer patients based on a gene signature derived from the progesterone regulated genes and progesterone regulated ERα binding events.c, For a gene to be considered it was differentially regulated by progesterone/progestin (as measured by RNA-seq) and the gene had a differentially regulated ERα binding event within 10 kb of the transcription start site. This resulted in 38 genes.d, Performance of progesterone induced gene signature at separating based on survival over 392 patients in top or bottom 10% of expression compared to null distribution ofP values computed using 1,000 randomly selected 38-gene signatures.e, Copy number alterations on chromosome 11 in T-47D and MCF-7 cells. Green is copy number neutral, blue is copy number loss and red is copy number gain. T-47D cells have an amplification of the chromosome 11 region encompassing thePGR gene and MCF-7 cells have a copy number loss of this genomic region.

a, MCF-7 cells were transfected with control vector, PR-A or PR-B expressing vectors. Western blotting confirmed the expression of the appropriate PR isoform.b, Growth was assessed following oestrogen plus progesterone treatment. The graph represents the average of three independent biological replicates and the error bars represent standard deviation.c, Assessment of MCF-7 xenograft tumour growth by physical measurement of tumour volume. Ten tumours for each condition (two in each of five mice per condition) were included. The data were analysed using at-test and the error bars represent ±s.e.m.d, T-47D xenografts were established in NSG mice. Ten tumours for each condition (two in each of five mice per condition) were included. All were grown in the presence of oestrogen (E2) pellets and subsequently supplemented with vehicle, progesterone, tamoxifen or tamoxifen plus progesterone. Normalized tumour growth is shown. The data were analysed using at-test and the error bars represent ±s.e.m.e, Final T-47D xenograft tumour volumes are shown.f, Final T-47D xenograft tumour volumes plotted graphically.

a, Histological analysis of MCF-7 xenograft tumours in untreated, oestrogen or oestrogen plus progesterone conditions. Tumours were taken from 25 day treated conditions. The human xenograft cells expressed GFP, permitting discrimination between human tumour cells and mouse host cells. MCF-7 xenograft experiment in ovariectomized mice.b, In order to map ERα binding events by ChIP-seq in MCF-7 xenograft tumours, we repeated the experiment in ovariectomized mice to eliminate any issues related to the endogenous mouse progesterone. Ten tumours for each condition (two in each of five mice per condition) were included. Growth of xenograft tumours under different hormonal conditions, Control, oestrogen alone (E2) and oestrogen plus progesterone (E2 + Prog). The data were analysed using at-test and the error bars represent ±s.e.m.c, ChIP-seq for ERα and PR were conducted in six matched tumours from each hormonal condition. Also included were two tumours from no hormone conditions. Correlation heat map of all samples.

Representative images of primary breast cancer explant tissue sections treated with vehicle, oestrogen (E2), the progestin R5020 or oestrogen plus progestin (E2 + R5020).a,b, These sections were probed with anti-Ki67 (brown) to label proliferating cells (a) or haematoxylin and eosin (b). Each image is of a single tissue segment from a selection of 3–4 sections per sample treatment. Scale bars, 100 μm.c, Confocal microscopy images (representative fields from each of the triplicate fragments) of a representative primary breast cancer explant tissue treated with vehicle, oestrogen (E2), the progestin R5020 (Progestin) or oestrogen plus progestin (E2 + R5020) and probed with anti-ERα (green), anti-PR (red) and anti-Ki67 to assess proliferating cells (blue).

a, Kaplan–Meier analysis of breast cancer specific survival within the METABRIC cohort. Only within luminal A tumours (based on PAM50 gene expression signature), tumours were stratified based on copy number loss ofPGR or not. In total 19% of luminal A tumours contain a copy number loss of thePGR genomic locus and these patients have a poorer clinical outcome.b, All ERα⁺ cases were stratified based onPGR copy number status, showing tumours with heterozygous and homozygous deletions separately.c, Chromosome 11 in tumours with neutral or gainedPGR versus those with copy number loss of thePGR gene (defined by line).d, Chromosome 11 copy number status between ERα positive and negative tumours.e, Visual representation of all ERα⁺ tumours with a copy number alteration at thePGR genomic locus, showing the copy number changes relative to thePGR gene (highlighted below) and the surrounding∼2.2 Mb of genomic sequence.

a, TCGA ERα⁺ breast cancers were assessed for copy number changes inPGR. The number of tumours in each category, based on copy number changes. Only ERα⁺ breast cancers were included.b, Correlation between PR mRNA levels and copy number status in all luminal breast cancers within the TCGA cohort. The heterozygous and homozygous deletions are combined.c, Frequency of copy number alterations across entire genome in TCGA breast cancer cohort, stratified based on subtype using PAM50 signature. Chromosome 11, which encompassesPGR gene is highlighted and the frequency of copy number loss of thePGR genomic region is provided.d, Copy number changes on chromosome 11 within the METABRIC cohort, based on subtype stratification (PAM50 signature).

This Supplementary table contains all RIME proteomic SILAC data from Figures 1a, Figure 1b and Extended data figure 1. (XLSX 2776 kb)

Peak numbers following ERα, PR and p300 ChIP-seq in T-47D and MCF-7 cell lines. The number of peaks for the different conditions are shown, these include estrogen, estrogen plus progesterone and estrogen plus R5020 treatment. Also included are the common peaks observed under estrogen plus progesterone and estrogen plus R5020 conditions. (XLSX 39 kb)

Enriched pathways based on the ERα binding events induced by progesterone and R5020. The enriched pathways that occur in both T-47D and MCF-7 cells are shown. The values represent the Odds ratio. (XLSX 47 kb)