Figure 6.

SET9 is recruited to the AR-regulated PSA promoter to methylate histone H3K4 and facilitate AR recruitment. (A) LNCaP cells grown in steroid-depleted media for 72-h were treated with 10 nM DHT over a time-course of 0–120 min and then subject to ChIP analysis using an anti-SET9 antibody, or non-specific isotype control, followed by quantitative PCR analysis using primers specific to the proximal (ARE I) and distal (ARE III) regions of the PSA promoter. (B) Representative SET9 knockdown and AR levels from the stable doxycycline-inducible SET9 knockdown LNCaP cell line (LNCaP-SET9_K/D) compared to the equivalent non-silencing (N/S) cell line (LNCaP-N/S_K/D). (C) ChIP analysis of histone H3K4 mono-methylation (H3K4me1) at ARE III in LNCaP-SET9_K/D cells treated with and without doxycycline for 48 h and 10 nM DHT for 0 or 120 min. (D) Representative western analysis of global histone H3K4me1 and H3K9me2 modifications in LNCaP cells transiently transfected with 25 and 50 nM N/S or SET9 siRNAs. (E) An anti-AR antibody was used in ChIP analysis as in (B). Data represents three independent repeats ± standard error (A and E).