Figure 2. ChIP analysis of the endogenous PSA gene promoter region.

LNCaP cells were cultured in charcoal-stripped serum (S) for three days and certain cultures were treated for 4 hr with DHT (10 nM) (D) or bicalutamide (15 μM) (CDX), or vehicle control (S). ChIP assays were performed using primers sets which amplified the PSA promoter region, the enhancer region, or a distal region upstream of known PSA regulatory elements. Immunoprecipitations were carried out using antibodies directed against SIRT1, androgen receptor (AR), NCoR, polymerase II (Pol II) and an irrelevant protein (Rag1). The bound and input DNA were analyzed by ABI 7500 Real-Time PCR system (Applied Biosystems, Foster City, California) by the ΔΔ Ct method. The results are presented as the relative level of the protein associated with the PSA promoter or enhancer, normalized to irrelevant control antibody and input DNA. A) SIRT1 on the PSA promoter. B) SIRT1 on the PSA enhancer. C) AR on the PSA promoter. D) AR on the PSA enhancer. E) Pol II on the PSA promoter. F) Pol II on the PSA enhancer. G) NCoR on the PSA promoter. H) NCoR on the PSA enhancer. I) Immunoblot analysis of AR protein levels in DU145 cells transfected with wt-AR or empty vector. J) Bicalutamide-induced recruitment of SIRT1 to the endogenous PSA promoter requires the AR. DU145 cells were cultured in DMEM+10% charcoal-treated FBS. The cells were transfected with AR or mock-transfected, and treated with bicalutamide (CDX) or DHT (D). ChIP assays were performed using antibodies directed against SIRT1.