Fig. 7

TLX recruits HDACs and LSD1 as co-repressors to suppress the AR gene expression. a ChIP assay of AR gene promoter performed in LNCaP cells infected with pWPI-TLX. Top: schematic diagram shows the locations of the identified TLX-binding sites P1 and P2 located at the 5′-UTR and proximal region of AR gene promoter, and also the locations of PCR primers used for ChIP assay. Sonicated DNA fragments extracted from LNCaP-TLX infectants were immunoprecipitated with various antibodies (against TLX, HDACs, and LSD1) and IgG as antibody specificity control. PCR of AR 5′-UTR and promoter region was performed using primers flanking the TLX-binding motifs at P1 and P2 sites, and also the 8-kb upstream distal region as negative control. DNA fragments flanking the P1 and P2 sites could be intensely PCR-amplified from extracted DNA immunoprecipitated by TLX, HDAC1, HDAC3, and LSD1, but barely or negatively by HDAC5 and IgG. b GST pull-down assay performed in LNCaP-TLX infectants. Cellular extracts from LNCaP-TLX infectants were incubated with GST-TLX or GST-TLXΔLBD fusion proteins, and the pull-down proteins were probed by antibodies against HDAC1, HDAC3, HDAC5, and LSD1. c Immunoprecipitation (IP) assay performed in FLAG-TLX-infected LNCaP cells. Cell lysates were immunoprecipitated with a FLAG-antibody or IgG as IP control and immunoblot-analyzed with HDACs and LSD1 antibodies. d Luciferase reporter assay of AR1.8-Luc performed in TLX-transfected HEK293 cells treated or untreated with pan-inhibitors of HDACs, NaBut (0.5–2.0 mM), TSA (0.25–1.0 µg/ml), and VPA (2–8 mM). *P < 0.05 versus empty vector; ^#P < 0.05 versus untreated TLX-transfected cells. Data are presented as mean ± SD of triplicate assays. e Luciferase reporter assay of AR1.8-Luc in TLX-transfected HEK293 cells treated with LSD1 inhibitor pargyline (3–6 mM). *P < 0.05 versus empty vector; ^#P < 0.05 versus untreated TLX-transfected cells. Data are presented as mean ± SD of triplicate assays. f qRT-PCR analysis of AR expression in LNCaP-TLX infectants treated with pargyline (6 mM). *P < 0.05 versus untreated cells. Data are presented as mean ± SD of triplicate assays. g Luciferase reporter assay of ARE-Luc in LNCaP-TLX infectants treated or untreated with pargyline and DHT. *P < 0.05 versus DHT-stimulated and pargyline-untreated cells. h Schematic diagram depicts the hypothesized mechanism of TLX-mediated repression of AR transcriptional activity via its recruitment of HDACs and LSD1 as co-repressors and its significance in induction of androgen insensitivity in CRPC progression