Coactivator AIB1 links estrogen receptor transcriptional activity and stability

Abstract

Agonist-mediated degradation of estrogen receptor alpha (ERalpha) has been associated with its transcriptional activity. However, the mechanism by which ERalpha is targeted for degradation and whether there is a direct functional link between ERalpha stability and ERalpha-mediated transactivation have not been elucidated. Here we provide evidence that the p160 coactivator, AIB1, uniquely mediates agonist-induced, but not antagonist-induced, ERalpha degradation. We show that AIB1 recruitment by ERalpha is not only necessary but also sufficient to promote degradation. Suppression of AIB1 levels leads to ERalpha stabilization in the presence of 17beta-estradiol and, despite increased ERalpha levels, reduced recruitment of ERalpha to endogenous target gene promoters. In addition, association of RNA polymerase II with ERalpha target promoters is lost when AIB1 is suppressed, leading to inhibition of target gene transcription. AIB1 thus plays a dual role in regulating ERalpha activity, one in recruiting transcription factors including other coactivators involved in gene activation and the other in regulating ERalpha protein degradation mediated by the ubiquitin-proteosome machinery.

Fig. 1.

Suppression of AIB1 by RNAi leads to ERα protein stabilization in the presence of E2. (A) Suppression of AIB1 inhibits E2-induced ERα degradation. MCF-7 cells were seeded in hormone-free condition before siRNA transfection. Forty-eight hours posttransfection, cells were treated with either vehicle (–) or 100 nM E2 (+) for 16 h. The ERα protein expression was analyzed by Western blotting (Left). The expression of calnexin protein was included as a loading control. (Right) Quantitative analyses of the ERα expression relative to the calnexin level in three independent experiments are represented. (B) siRNA specific to each of the p160 coactivators leads to efficient inhibition of the target protein. siRNA targeting the luciferase gene was used as a negative control. (C) The carboxyl-terminal region of AIB1 is necessary to mediate the ERα degradation by E2. (Top) Schematic diagram of AIB1, SRC-1, and the HA-tagged SRC-1/AIB1 is shown; the site in AIB1 targeted by siAIB1 is indicated by an asterisk. (Middle and Bottom) Western blots.

Fig. 2.

Suppression of AIB1 inhibits E2-induced ERα degradation but does not affect the ERα stability in the presence of OHT, fulvestrant, or GW5638. Solutions used in the experiments were vehicle control (C), 100 nM E2, 10 nM fulvestrant (F), 1 μM GW5638 (GW), and 1 μM OHT (T).

Fig. 3.

AIB1 binding to ERα is sufficient to induce its degradation. (A Upper) Schematic diagram showing the replacement of the nuclear receptor interacting sequences in AIB1 with CoRNR boxes in subAIB1. (A Lower) Interaction of ³⁵S-labeled in vitro translated AIB1 and subAIB1 with GST or GST-RXR ΔAF2 in the absence or presence of 9-cis-retinoic acid was assessed by GST pull-down assay. (B) Protein expression of ERα was examined by Western blot analysis in MCF-7 cells mock-transfected or transfected with vector alone or subAIB1 in the presence of vehicle (C), E2, or OHT (T).

Fig. 4.

AIB1 interacts with the ubiquitin ligase E6-AP both in vivo and in vitro. (Top) Diagram showing the structure of E6-AP. The HECT and catalytic domains are as indicated. (A) AIB1 interacts with E6-AP in vivo as determined by coimmunoprecipitation. (B) AIB1 interacts with E6-AP directly in vitro as assessed by GST pull-down assay. (C) E6-AP is not necessary for E2-induced ERα degradation.

Fig. 5.

AIB1 is required for optimal association of ERα with target gene promoter and ERα transactivation. (A) The association of ERα, AIB1, and Pol II with the pS2 promoter upon E2 stimulation was determined by ChIP analyses in MCF-7 cells. (B) The protein levels of ERα in cells after E2 treatment at different time points were determined by Western blotting. (C) Inhibition of the AIB1 expression abolishes the E2-dependent transactivation of the pS2 gene. The levels of hnpS2 were measured by quantitative RT-PCR. (D) Suppression of AIB1 has a more marked effect on the E2-inducible PR up-regulation than does SRC-1. The protein levels of PR were determined by Western blotting.