The transactivation and the phosphorylation of AR are disrupted by the XPD/R683W mutation. (A) GM03348D (XPD/WT), TTD12PV (XPD/R112H), XPJCLO (XPD/R683W) and TTD8PV (XPD/R722W) fibroblasts were transiently co-transfected with 100 ng of pGL3.PSA-Luc, pCMV.β-gal and either pSG5.AR (AR) or pSG5.RARα (RARα) or the corresponding empty vector. The cells were then treated with a specific ligand for AR (DHT, 10⁻⁷ M) or RARα (t-RA, 10⁻⁸ M). Luciferase activity was measured 24 h later and normalized to β-galactosidase activity. The results are the mean of three different experiments. (B) In vivo phosphorylation of AR was investigated in HeLa (WT), HD2 (XPD) and XPD cells overexpressing XPD/WT upon AR immunoprecipitation, autoradiography ([³²P]-AR) and western blotting (AR). Quantitative analysis of AR phosphorylation in WT and XPD cells represents the ratio autoradiography/western blot signals ([³²P]-AR/AR). (C) AR interacts with TFIIH. (Left panel) Endogenous AR was immunoprecipitated (IP:AR, lane 5) from LNCaP cells and after washes (150 mM NaCl), co-precipitated TFIIH was detected using an anti-XPB antibody. The control IP (IP neg, lane 4) was performed with a non-specific antibody. The input (lane 1) represents 10% of the total volume of LNCaP extract used for the immunoprecipitation. His-AR (lane 2) and purified XPB (lane 3) were used as size markers for the immunodetection of the endogeneous AR and XPB in LNCaP cells. (Right panel) Highly purified-recombinant AR was incubated with Sf9 cell extracts overexpressing TFIIH complex containing a FLAG-tagged p34, in the presence or absence of DHT (10⁻⁶ M). Immunoprecipitation was performed using an anti-FLAG antibody (IP:IIH) and after extensive washes (300 mM NaCl) and SDS–PAGE, bound proteins were detected with an anti-AR, XPD, p62, p44 and cdk7 antibodies. (D) Equal amounts of highly purified-recombinant AR was incubated with Sf9 cell extracts overexpressing separately the subunit XPB, XPD, p44 or cdk7 of TFIIH in the presence or absence of DHT (10⁻⁶ M). After AR immunoprecipitation, co-precipitated TFIIH subunits were detected by western blot. The control IP (lane 1) was performed without AR. (E) AR, A/B.AR, ARΔA/B, PPARα or A/B.AR S515A were incubated with purified-recombinant TFIIH in the presence or absence of DHT (10⁻⁶ M) as indicated. After SDS–PAGE, each protein was resolved by Coomassie blue staining (Stain.) and radioactive labelling was analysed by autoradiography (Auto.). The complete Coomassie blue stainings and western blots of the different purified-recombinant proteins are shown in Supplementary Figure S1A. (F) WT (HeLa) and XPD (HD2) cells were transiently co-transfected with 100 ng of pGL3.PSA-Luc, pCMV.β-gal and pSG5 AR/WT, /S515A, or /S515E before DHT (10⁻⁷ M) treatment. The results were obtained as described in A.

AR phosphorylation status selectively promotes the recruitment of ubiquitin–proteasome components at the PSA promoter. WT and XPD cells were transiently transfected to overexpress either AR/WT, AR/WT together with XPD/WT or AR/S515E (as indicated at the top of each panel). (A–D) Expression of the PSA gene: RT–qPCR analysis was performed at indicated times after DHT (10⁻⁷ M) treatment. The values were normalized relative to the GAPDH mRNA expression. The results of three independent experiments are presented as n-fold induction relative to non-treated cells. (E–H) After DHT treatment, the recruitment of RNA pol II (yellow curve), TFIIH (via its XPB and cdk7 subunits, blue and green curve, respectively) and AR (red curve) were analysed by ChIP assays at the PSA proximal promoter. The results are presented as percentage of DNA immunoprecipitated relative to the input (% input). (I–L) Recruitment of the MDM2 (green curve) and CHIP (yellow curve) E3 ligases on the PSA promoter. The recruitment of the ubiquitinated AR-containing fraction (brown curve) was also analysed by ChIP/re-ChIP assays using first an anti-ubiquitin antibody and second with an anti-AR antibody. (M–P) Recruitment of SUG1 (red curve), β5 (light blue) and S1 (dark blue) subunits of the proteasome at the PSA promoter was analysed by ChIP assays. Note that the recruitment of S1 has not been analysed in XPD cells transiently co-transfected with plasmids encoding AR/WT and XPD/WT (O).

Silencing either cdk7 or MDM2 promotes CHIP recruitment at the PSA promoter. WT cells stably expressing AR/WT were transfected with either a pool of non-targeting si-RNAs (used as control, si-ctl, panels A, E, I, M, Q), si-cdk7 (panels B, F, J, N, R), si-MDM2 (panels C, G, K, O) or si-CHIP (panels D, H, L, P). (A–D) PSA gene expression was analysed by RT–qPCR at indicated times after DHT (10⁻⁷ M) treatment in cells transfected with si-ctl (A), si-cdk7 (B), si-MDM2 (C) or si-CHIP (D). The values were normalized relative to the GAPDH mRNA expression. The results of three independent experiments are presented as n-fold induction relative to non-treated cells. (E–H) The recruitment of RNA pol II (yellow bars) and AR (orange bars) at the PSA promoter was analysed by ChIP assays. The results are presented as percentage of DNA immunoprecipitated relative to the input (% input). (I–L) Recruitment of the E3 ligases MDM2 (green bars) and CHIP (yellow bars) at the PSA promoter was next investigated. (M–P) ChIP assays analysed the recruitment of the proteasomal subunits β5 (green bars), S1 (blue bars) and SUG1 (red bars). (Q–R) [³⁵S] Pulse chase of AR protein in cells stably expressing AR/WT and transfected with either si-ctl (Q) or si-cdk7 (R). Experiments were performed as described in Figure 2. The values are the mean±s.e.m. of three independent experiments.

Pulse chase of AR protein in WT and XPD cells. (A–F) WT (A, C) and XPD cells (B, D) were transiently transfected in order to overexpress AR/WT (A, B, lower panels), AR/S515A (C) or AR/S515E (D). XPD cells were also co-transfected in order to simultaneously overexpress AR/WT and XPD/WT (F). Control experiments were performed with cells transfected with empty vectors (A, B, upper panels). Following a [³⁵S] pulse, cells were maintained in the presence of DHT (10⁻⁷ M) for the indicated time points (0, 0.5, 1, 2, 4 and 8 h). After immunoprecipitation, AR was resolved by SDS–PAGE and [³⁵S] labelling quantified with a phosphoimager. Western blots were performed in parallel to localize AR (see Supplementary Figure S2). Arrows indicate the position of the radiolabelled [³⁵S]-AR. Graphs depict AR protein levels normalized to that observed in absence of DHT treatment (arbitrary units, au). The values are the mean±s.e.m. of three independent experiments. (E) Summary of AR turnover following DHT treatment in WT cells overexpressing either AR/WT (black curve) or AR/S515A (blue curve), and in XPD cells overexpressing either AR/S515E (yellow curve) or AR/WT in the absence (red curve) or presence of XPD/WT (green curve). The [³⁵S]-AR levels are presented as percentages, 100% being the [³⁵S]-AR levels in absence of DHT treatment. Data are the mean±s.e.m. of three independent experiments.

AR interacts with MDM2 and CHIP and is ubiquitinated by both E3 ligases. (A) AR/WT, /S515A or /S515E, immunoprecipitated (IP:AR/) from DHT (10⁻⁷ M) treated HeLa cells, were incubated with either purified-recombinant MDM2 or CHIP E3 ligases, in the presence or absence of HSP90α, as indicated. After extensive washes (300 mM KCl), western blot analyses were performed with AR-, MDM2-, CHIP- and HSP90α-specific antibodies. The control IP (lane 1) was performed using a non-specific antibody. Inputs are shown in the Supplementary Figure S3. (B) Recombinant purified AR was incubated with the ubiquitin-activating enzyme E1 (UBE1) and E2 (UbcH5a) in the presence of either MDM2 (1 μM, lanes 3 and 4) or CHIP (1 μM, lanes 7 and 8) E3 ligase as indicated on top of the panel. His-Ub_WT (wild type) or His-Ub_K0 (in which all seven lysine residues critical for polyubiquitination are replaced with alanine) have been added to the reaction, when indicated. Western blot analyses were then performed with Ubiquitin and AR-specific antibodies.

TFIIH-mediated phosphorylation of AR regulates its turnover by triggering its degradation by the ubiquitin/proteasome pathway. Upon the DHT ligand induction, the transactivation complex is formed once AR homodimer has targeted its responsive element (ARE) at the PSA promoter; co-factors are assembled at the promoter together with RNA pol II and the general transcription factors (GTFs) including TFIIH. In WT cells, the AR/S515 phosphorylation by TFIIH (via its cdk7 subunit) promotes the recruitment of both MDM2 E3 ligase that helps for AR polyubiquitination and the proteasome. In XPD cells (bearing the XPD/R683W mutation), AR/S515 phosphorylation is strongly inhibited, preventing the recruitment of MDM2. The E3 ligase CHIP is thus preferentially recruited, which allows with a lesser efficiency the AR polyubiquitin/proteasome process resulting in a much slower turnover.