PMC

Two-step ChIP test of co-occupancy of coactivators. Cells were treated with DHT (1 h) (A) or with CPA (1 h) after IL-1 for 1 h (B). Aliquots were first immunoprecipitated with IgG against Tip60 (first IP). The bound materials and supernatant were collected and reimmunoprecipitated with IgGs against Tip60, Brg1, GRIP1, p300, PBP, pCAF, pCIP, SRC1, or CARM1.

CBP/p300 and CARM1 are required for DHT but not SARM-dependent activation. (A) ChIP analysis of CBP/p300, CARM1, and other histone-modifying factors on PSA promoter after treatment with either DHT or IL-1β and SARM. (B) KLK2 promoter, another AR-responsive promoter, was examined after challenging with SARM and IL-1β, revealing that CBP/p300 were not recruited on KLK2 promoter. (C) Injection of anti-CBP IgG blocked the DHT-dependent activation of a reporter containing ARE. (D) Plasmid rescue experiments were performed as indicated after microinjection of anti-CBP IgG. (E) The function of CARM1 was assessed in cells injected with the ARE reporter. Rescue experiments with CARM1 expression plasmids confirmed a requirement for the functional methyltransferase activity.

Evaluation of the recruitment of coactivators to AR-dependent promoter in response to 5α-dihydrotestosterone (DHT) and SARMs. (A) Pretreatment of cells with IL-1β abolished CPA or bicalutamide-mediated repression of a reporter containing androgen receptor response element (ARE). (B) ChIP assay to monitor the occupancy of prostate-specific antigen (PSA) promoter by Tip60, pCAF, SRC1, p/CIP, GRIP1, PBP, BRG1, acetylated histones H3 and H4, and RNA polymerase II (POL II) at the indicated times (min) after treatment with either DHT or IL-1β and SARM. α, Antibody. Soluble chromatin was prepared from LNCaP prostate cancer cells treated with either DHT for 1 h or IL-1β and CPA for 1 h.

Differential mode of p160 factors for the DHT- versus SARM-dependent activation. (A) Injection of anti-SRC-1 IgG blocked both the DHT-dependent and bicalutamide/IL-1β-dependent activation of a reporter containing ARE. (B and C) After microinjection of anti-SRC-1 IgG, each LXXLL mutant of SRC-1 plasmid rescued the activation in the presence of DHT (B) or bicalutamide and Il-1β (C). Effects of mutating each LXXLL motif in SRC1 on DHT-dependent and bicalutamide/IL-1β-dependent activation are shown. (D) LNCaP cells stably infected with the AR virus or the control vector were generated and were starved for 5 days and then challenged with bicalutamide (10 μM), R1881 (100 pM), or DHT (10 nM). After 1 h of treatment, ChIP assay was performed with various IgGs. (E) Schematic representation of the action of coactivator use in SARMs-dependent gene activation.