Extended Data Fig. 7: Cistromic and WNT-driven phenotypic characteristics of the class-2 FOXA1 mutants.

a, De novo motif analyses of the wild-type-specific, common and class-2-specific FOXA1-binding site subsets defined from either sequencing-read fold changes (left) or peak-calling scores (right) of ChIP–seq data. Wild-type and class-2 cistromes were generated from n = 3 and n = 2 independent biological replicates, respectively. Only the top 5,000 or 10,000 peaks from each subset were used as inputs for motif discovery (see Methods) (HOMER, hypergeometric test). b, c, Per cent of wild-type or class-2 binding sites with perfect match to the core FOXA1 motif (5′-T[G/A]TT[T/G]AC-3′) (b) and the consensus FOXA1 motifs identified from these sites (c). d, e, Per cent of binding sites in the three FOXA1-binding-site subsets containing known motifs of the labelled FOXA1 or AR cofactors (d), and enrichment of the cofactor motifs in the three binding site subsets relative to the background (e). f, Genomic distribution of wild-type-specific, common and class-2-specific binding sites in prostate cancer cells. g, Differential expression of genes in FOXA1 class-2 mutant CRISPR clones relative to FOXA1 wild-type clones (n = 2 biological replicates (limma two-sided test)). h, Distinct transcription factor motifs within the promoter (2-kb upstream) of differentially expressed genes. Transcription factors with the highest enrichment (fold change, per cent of upregulated genes with the motif and significance) are highlighted and labelled (two-tailed Fisher’s exact test). i, Immunoblots showing the expression of β-catenin and vimentin in a panel of wild-type and heterozygous or homozygous class-2 mutant 22RV1 CRISPR clones. j, Immunoblots showing the phosphorylation status of β-catenin and expression of direct WNT target genes in select class-2 mutant 22RV1 clones. Immunoblots in i and j are representative of two independent experiments; every individual clone serves as a biological replicate. For gel source data, see Supplementary Fig. 1. k, Representative images of Boyden chambers showing invaded cells stained with calcein AM dye. l, Quantified fluorescence signal from invaded cells (n = 2 biological replicates per group; two-way ANOVA and Tukey’s test). Mean ± s.e.m. is shown and dots are individual data points. m, Absolute counts of disseminated cell foci in individual zebrafish embryos as a measure of metastatic burden. n, Per cent metastasis at day 2 and day 3 in zebrafish embryos injected with either the normal HEK293 cells (negative controls) or 22RV1 prostate cancer cells virally overexpressing wild-type, class-1 or class-2 mutant FOXA1 variants (n > 20 for each group). o, Fluorescent signal from the invaded wild-type or class-2-mutant 22RV1 cells after androgen starvation (5% charcoal-stripped serum medium for 72 h) or treatment with the WNT inhibitor XAV939 (20 μM for 24 h; n = 2 biological replicates per group; two-way ANOVA and Tukey’s test). Mean ± s.e.m. and individual data points are shown.

Source Data