Fig 2: NCOR2 loss results in a hypermethylation phenotype in LNCaP and C4–2 cells(A) Volcano plots representing DMPs following NCOR2 knockdown in LNCaP (left) and C4–2 (right) cells, hypomethylated, blue; hypermethylated, red.(B) Venn diagram of DMPs identified in each cell.(C) Binning analysis depicting the median methylation calculated for genomic regions relative to TSS (left) and CpG island (right) loci. For TSS, each bin represents 100 bp, with the TSS centered at bin 50. For CpG islands, shore (orange) and shelf (blue) bins represent 200 bp, while islands (green) are variable depending on genomic length but centered on bin 30.(D) Relative proportions of all CpGs (top) or DMPs (middle, bottom) that annotate to ChromHMM regions.(E) Peak centered densities of non-DMP CpG sites (black) or hypermethylated DMPs (red) centered at ChromHMM promoter or enhancer regions.(F) Comparison of NCOR2 knockdown gene expression changes annotated with DMR promoters and/or DMR enhancers relative to genes not annotated. Distributions are compared by Kolmogorov-Smirnov (KS) test. Mean value (black dot) and IQR (horizontal lines) are shown.(G) Representative genomic view of an NCOR2-dependent gene locus with annotated enhancer hypermethylation (GPR108). Tracks (from top), RefSeq gene (exons [red] and introns [black]); methylation detected in shCTL (black) or shNCOR2 C4–2 cells (green, blue); ChromHMM regions (color code is the same as shown in [D]); DMR regions (green); CpG island regions (purple).

Fig 3: Data integration identifies effects on SEs(A and B) Upset plot showing the intersection between DEGs upon NCOR2 knockdown, expressed genes associated with differentially methylated enhancer regions upon NCOR2 knockdown (DMR.enh), and expressed genes associated with NCOR2 binding found under basal (EtOH) conditions in both LNCaP (A) and C4–2 (B) cells. Significant overlaps are denoted by a red asterisk (*p < 0.05; **p < 0.01; ***p < 0.001).(C) Overlaps of NCOR2 binding by ChIP-seq with identified SEs from LNCaP and C4–2 cells. Peak overlaps are shown for both basal (EtOH) and androgen-supplemented (DHT) conditions, and significance was determined by hypergeometric distribution.(D) Functional enrichment analysis of genes annotated to NCOR2/SE shared regions from (C). The top 15 significant (p adjusted < 0.05) gene sets that related to the functional categories (cell differentiation, chromatin organization, proliferation, and metastasis) are displayed as counts within their respective functional category.(E) Heatmap showing the expression levels of DEGs from CWR22 shNCOR2 recurrent tumors compared with control recurrent tumors (1,189) and the scaled expression (Z score) of those genes in mCRPC patient samples. The column annotations indicate pathology classification (Pathology), NEPC score, and AR signaling score (AR score). The row annotations indicate FC direction of CWR22 recurrent tumor DEGs (shNCOR2 compared with shCTL) and if that gene is regulated by an SE (identified in LNCaP and C4–2 cells).

Fig 4: NCOR2 loss accelerates the molecular features of ADT resistance during PC progression in vivo(A) Gene set variation analysis of established pathways associated with ADT-R in PC.(B) Gene expression profiles for SYP, CHGB, NTN1, and AR in CWR22 groups.(C) IHC showing representative examples of SYP expression in shCTL-RT and shNCOR2-RT samples (left); scale bars, 20 μm. Quantification of IHC in RT is shown on the right. Data are summarized as boxplots representing lower, middle, and upper quartiles. Whiskers indicate maximum and minimum values, excluding outliers defined as 1.5× IQR (A–C).(D) Heatmap of scaled expression depicting the 41 most significant DEGs in NCOR2-low relative to NCOR2-high tumors within the SU2C cohort that were also identified in RT-shNCOR2 (left). Column annotations show grouped neuroendocrine scores (NE.quartiles) for each tumor. Kaplan-Meier curves of overall survival proportions between major tumor clusters are identified.(E) Volcano plots depicting NCOR2-dependent DMPs (red = upregulated, blue = downregulated) determined at different stages of disease.(F) Proportion of DMPs in each comparison; hypermethylated, red; hypomethylated, blue.(G) Total number of NCOR2 and stage-specific DMPs in PRE-ADT and RT.(H) Representative genomic view of the HOXA3 locus showing the average methylation levels within each CWR22 group; CpG island regions, purple; arrows, example DMPs; mean value, black dot; IQR, horizontal lines.(I) Binning analysis depicting the median methylation calculated for genomic regions relative to TSS (top) and CpG island (bottom) loci. For TSS, each bin represents 100 bp, with the TSS centered at bin 50. For CpG islands, shore (orange) and shelf (blue) bins represent 200 bp, while islands (green) are variable depending on genomic length but centered on bin 30.

Fig 5: VGLL3 represses ESR1 transcription by binding TEAD and recruiting NCOR2.a List of transcriptional regulators identified by VGLL3-TurboID mass spectrometry. b VGLL3 interacts with TEAD. MCF-7 cells were transiently transfected with plasmids expressing the indicated proteins. Protein–protein interaction was examined by IP-western blot using the indicated antibodies. c TEAD is required for VGLL3-induced ERa downregulation. Wild-type and TEAD1-4 qKO MCF-7 cells expressing a control vector or Flag-VGLL3 cDNA were subjected to immunoblot analysis. d YAP/TAZ are dispensable for VGLL3-induced ERa downregulation. Wild-type or YAP/TAZ dKO MCF-7 cells transduced with control vector or Flag-VGLL3 cDNA were subjected to immunoblot analysis. e Heatmap and line graph of the ChIP-seq profiles for doxycycline (dox) inducible HA-tagged VGLL3 (iHA-VGLL3), TEAD2 (iHA-TEAD2), and control vector (iHA-Ctrl) in LATS1/2 deficient cells or iHA-VGLL3 in LATS1/2 & TEAD1-4 6KO cells at the summits of iHA-VGLL3 peaks. f Scatterplots depicting the correlation between iHA-VGLL3 and iHA-TEAD2 ChIP-seq signals in LATS1/2 deficient cells. g Enrichment of the TEAD motif in VGLL3 ChIP-seq. MEME-AME Motif enrichment analysis for VGLL3 binding peaks against JASPAR CORE database (1404 profiles) and ordered by log (p-value). h The TEAD motif (JASPAR Matrix ID: MA1121.1) was enriched at VGLL3 ChIP-seq peaks. i De novo motif analysis for the VGLL3 binding peaks by MEME-Suit. j Venn diagram showing the overlap of VGLL3 (purple), TEAD2 (yellow) or YAP (blue) peaks in LATS1/2 dKO MCF-7 cells. k NCOR2 is required for VGLL3 to repress ESR1. MCF-7 cells with CRISPR-cas9 sgRNA targeting individual putative VGLL3 binding partners were transfected with Flag-VGLL3 or control vector. Immunoblot analysis was performed with the indicated antibodies. l NCOR2 is required for ESR1 repression by LATS1/2 deletion. MCF-7 cells were infected with CRISPR sgRNA targeting individual VGLL3 binding partners or in combination with sgRNA targeting LATS1/2. m VGLL3 interacts with NCOR2. MCF-7 cells expressing Flag-VGLL3, or control vector, were immunoprecipitated with Flag antibody. Western blotting for co-precipitated endogenous NCOR2 was determined. n VGLL3 mediates TEAD-VGLL3-NCOR2 complex formation. MCF7 transfected with indicated proteins and immunoprecipitated with anti-Myc-tag or IgG control antibodies were subjected to immunoblot with indicated antibodies. Asterisk indicates non-specific band. Source data are provided in the Source Data file.