Fig. 5: Nuclear PTGES3 facilitates AR-mediated transcription.

a, LNCaP cells containing ARE–luciferase reporter were transfected with pCDH-control vector (gray) or pCDH-PTGES3 (blue). Cells were treated with DMSO, 1 nM DHT or 1 nM DHT plus 5 μM apalutamide (APA). Luciferase activities over Renilla were normalized with control (n = 3 as biological replicates; mean ± s.d.). Statistical significance was determined using two-sided t-test (DMSO group *P = 0.0435; DHT group ***P = 0.0002; DHT + APA group **P = 0.0040). b, LNCaP cells were fixed sequentially by EGS and formaldehyde. Dual crosslinking ChIP experiments were performed using indicated antibodies. Precipitated DNA was used as a template to amplify the indicated genomic regions by real-time PCR (n = 3 as biological replicates; mean ± s.d.). Statistical significance was determined using two-sided t-test (**P < 0.01; ***P < 0.001; NS, not significant). c, Venn diagram showing overlaps of AR ChIP–seq peaks in LNCaP(sgCTRL) and LNCaP(sgPTGES3). d, Peak distribution of ChIP-AR peaks in LNCaP(sgCTRL) and LNCaP(sgPTGES3) across different genomic locations. e, ChIP-AR read-density heatmaps of LNCaP(sgCTRL) and LNCaP(sgPTGES3) centered at combined ChIP-AR peaks (n = 38,122). f, The five most enriched known HOMER motifs in the top 2,000 significant ChIP-AR peaks in LNCaP(sgCTRL) and LNCaP(sgPTGES3). g, An IGV screenshot of ChIP-AR enrichment in LNCaP(sgCTRL) and LNCaP(sgPTGES3) at enhancer and promoter sites of KLK3 and KLK2 genes. h, LNCaPi cells were infected with sgCTRL, sgPTGES3 or sgAR. Cells were collected for ATAC-seq (n = 2 as biological replicates). The heatmap shows the differentially accessible ATAC-seq peak regions in sgPTGES3 or sgAR as compared to that in sgCTRL. Each ATAC-seq peak region was scaled to the same size from the start to the end region. Flanking ±500 bp regions are also shown. The top panel shows the corresponding ATAC-seq signal intensity. i, The volcano plot shows the differentially accessible ATAC-seq peak regions upon PTGES3 knockdown as compared to controls. Each dot represents an individual ATAC-seq peak. Differentially accessible ATAC-seq peaks are highlighted in red color (P < 0.05 and log2 Foldchange >1 or <−1). The nearest gene to several representative differentially accessible ATAC-seq peaks have been highlighted. j, A graph depicting the overlap in genomic loci with differentially accessible ATAC-seq peaks upon PTGES3 knockdown that are or are not also bound by AR as measured by ChIP–seq in LNCaP cells⁵² (Stello2018). The figure shows the percentage of these differentially accessible ATAC-seq peaks that overlapped with the AR ChIP–seq dataset. k, Plots showing ATAC-seq peak coverage in the KLK3 gene body and flanking ±5 kb region. sgCTRL ATAC-seq peaks are colored in blue and sgPTGES3 ATAC-seq peaks in red. The upstream promoter region of KLK3 contains overlapping ATAC-seq peaks and AR Chip–seq peaks⁵². AR binding motif was identified in this region.