Fig. 6.

TRAF4 overexpression alters AR genomic binding profile and increases its interaction with FOXA1. (A) Venn diagram and distribution of AR genomic binding peaks in TRAF4-overexpressing cells or control LNCaP cells. Cells were cultured in an androgen-deprived medium for 2 d followed by ChIP using an AR antibody. ChIP seq analysis was then performed. (B) Top gained AR-binding peaks in TRAF4-overexpressing cells are enriched with forkhead domain family and homeodomain family-binding motifs. Left panel, the heatmap of AR-binding intensity in TRAF4-overexpressing and control cells. Right panel, top enriched forkhead domain family and homeodomain family transcription factor-binding motifs. (C) AR gained more binding at the FOXA1-binding sites in TRAF4-overexpressing cells compared to vector control cells (blue peak vs. green peak). The red peak represents shared AR-binding sites between TRAF4- and vector-expressing cells. (D) TRAF4 overexpression enhances the association between AR and FOXA1. LNCaP cells were infected with adenovirus expressing TRAF4 or GFP control. A co-IP experiment was then carried out using an AR antibody followed by a western blot analysis using a FOXA1 antibody to detect AR-associated FOXA1. (E) AR ubiquitination mutation abolished the TRAF4-promoted AR-FOXA1 interaction. Shown is a co-IP experiment in flag-AR WT or ubiquitination mutant (K913/911R)-expressing cells. Cells were treated with siAR to reduce the levels of endogenous AR.