Inhibition of AR-Mediated Transcription by Binding of Oct1 to a Motif Enrichedin AR-Occupied Regions

BACKGROUND. The androgen receptor (AR) plays roles in prostate development and cancer (PCa). In response to androgens, the AR binds to androgen-response elements (AREs) to modulate gene transcription. The responses of such genes are dependent on the cellular milieu and on sequences around the AREs, which attract other transcription factors. Previously, bioinformatic analysis of 62 AR-occupied regions (ARORs) in PCa cells revealed enrichment for both AREs and a TTGGCAAATA-like motif. We undertook the present study to investigate the significance of the TTGGCAAATA-like motif. METHODS. Prostate cancer cell lines, LNCaP and C4-2B, were analyzed by transient transfections of wild-type and mutant reporter constructs, electro-mobility shift assays (EMSAs), and RT-qPCR analysis of endogenous genes. RESULTS. In two of six tested ARORs, point mutations in the TTGGCAAATA-like motif resulted in inhibition of DHT-mediated enhancer activity. EMSA revealed that Oct1 bound the motif, and that the mutations that abolished DHT responsiveness in the transfection assays augmented Oct1 binding. These results suggest a role for Oct1 as a context-dependent negative coregulator of AR. Consistent with this, siRNA kmockdown of Oct1 increased the DHT-mediated enhancer activity of transfected reporters as well as an endogenous AR target gene, transglutaminase 2. CONCLUSIONS. Oct1 negatively regulates DHT-mediated enhancer activity in a subset of ARORs. The enrichment of ARORs for the Oct-binding, TTGGCAAATA-like motif may reflect a mechanism that utilizes Oct1 to keep AR activity in check at some ARORs, while augmenting AR activity in other ARORs. Therefore, Oct1 may have regulatory functions in prostate development and cancer progression. Prostate 69: 392-400, 2009. (C) 2008 Wiley-Liss, Inc.