SBF-1 may occupy a novel binding site AR-DBD for blocking the interaction between the transcription factor and its target gene
Above results suggest that SBF-1 may have a novel binding site in AR. To find the site, we constructed an ARE-1 sequence, which known to be a consensus recognition site for the AR (Denayer, Helsen, Thorrez, Haelens & Claessens, 2010). This constructed sequence was incubated with each purified ARWT or AR∆DBD (Androgen receptor lacking DBD). As the result, there is a shift band in case of ARWT but not in AR∆DBD with the Kd values of 370 nM in ARWT and N/A in AR∆DBD, respectively (Fig.4A), suggesting that the AR-DBD is a potential target site of SBF-1. We further analyzed the binding affinity between SBF-1 and AR∆DBD. By adding 16 different doses of SBF-1 to PC3 cells transfected with the previously constructed and GFP-tagged AR∆DBD, a very low binding affinity of SBF-1 with AR∆DBD was calculated as 698 µM (Fig. 4B). Furthermore, we expressed and purified AR∆DBDand used ITC technique to determine the thermodynamic parameters of the interaction between SBF-1 and AR∆DBD, the result shows that SBF-1 totally failed to show any binding signal with the purified AR∆DBD (Fig. 4C). Structural analysis revealed the existence of SBF-1 and AR-DBD binding (Fig. S1A). We further analyzed the gene expression enrichment related to the AR through ChIP-qPCR assay, and used AR ChiP grade antibody to determine the IGF-1 enrichment status as shown previously in Fig. 2A and B. We confirmed that IGF-1 was reduced due to the treatment of SBF-1. In Fig. 4E, we found that DHT induced a significant increase in the AR target gene IGF-1 enrichment while SBF-1 completely inhibited this enrichment. SBF-1 also inhibited the PCNA gene expression. Computer docking and DNA pull down assays proved this result (Fig. S1 and S2). These results suggest that SBF-1 may directly block AR from binding to target genes by occupying its DBD.