Volume 10, Issue 5, Pages 1019-1032 (November 2002) A Dynamic Structural Model for Estrogen Receptor-α Activation by Ligands, Emphasizing the Role of Interactions between Distant A and E Domains  Raphaël Métivier, Alexander Stark, Gilles Flouriot, Michael R Hübner, Heike Brand, Graziella Penot, Dominique Manu, Stefanie Denger, George Reid, Martin Koš, Robert B Russell, Olivier Kah, Farzad Pakdel, Frank Gannon  Molecular Cell  Volume 10, Issue 5, Pages 1019-1032 (November 2002) DOI: 10.1016/S1097-2765(02)00746-3

Figure 1 hERα A Domain Silences Ligand-Independent Transactivation and Transrepression (A) Scheme of hERα, its functions (AFs, transcription activation functions), and the epitopes targeted by the antibodies used. We examined the transactivation ability of the hERα devoid of its A domain (hERαΔ1-37) as compared to the wild-type protein in ERα-negative cell lines. (B) Activation of a Luciferase reporter gene controlled by an ERE and the thymidine kinase gene promoter (ERE-TK-Luc) in HepG2 AF-1-permissive cells. In yeast, we followed the activity of a LacZ gene controlled by 3 EREs and the cytochrome C1 gene minimal promoter (3ERE-Cyc1-LacZ). After estrogen (E2) or ethanol (vehicle, EtOH) treatment, reporter activities were normalized by internal controls. Values are the reporter activity induction by E2, with the wild-type hERα activity set as 100%. (C) Wild-type and mutants hERα were expressed in HeLa AF-2-permissive cells in the presence or absence of transfected NCoR. Results are the mean ± SEM of the ERE-TK-Luc fold modulation (positive values, activation; negative, repression). (D) Physical interaction between truncated hERα and NCoR in the presence of EtOH, 1 μM E2, 50 μM 4-hydroxy-tamoxifen (OHT), or ICI164,384 (ICI). (E) Quantification (mean ± SD of normalized values against inputs) by real-time PCR of ChIP assays following endogenous NCoR recruitment to transfected ERE-TK-Luc reporter in HeLa cells after 3 hr treatment with ligands. Position of the specific band (Sp) amplified is shown. Molecular Cell 2002 10, 1019-1032DOI: (10.1016/S1097-2765(02)00746-3)

Figure 2 Endogenous hERα 46 kDa Isoform Devoid of A Domain Has Ligand-Independent Transrepression Ability (A) ChIP analysis of pS2, CyclinD1, and ERE-TK-Luc promoters occupancy by hERα and NCoR in MCF-7 cells. Two antibodies directed against hERα N- (H184) or C-terminal (HC20) regions were used. Specific (Sp) and unspecific (Unsp) regions of the promoters were amplified. Real-time PCRs are shown for the pS2 promoter. (B) Western blots show equivalent expression of endogenous hERα, NCoR, and control GAPDH during the 3 hr treatment with ligands. MDA-MB231 are ERα-negative cells. IVT states for in vitro-translated hERα 66 and hERα 46. (C) After ChIPping chromatin from MCF-7 cells with anti-hERα or anti-NCoR antibodies, a Re-ChIP step was performed. pS2 promoter occupancy was next followed by quantitative PCR. (D) After 3 hr treatment with ligands, reverse transcription/real-time PCR was performed using MCF-7 cells RNA. Values obtained for pS2 mRNA were normalized against GAPDH control. Asterisks indicate a significant difference between control and hormonal treatments (P < 0.01 by Student's test). Molecular Cell 2002 10, 1019-1032DOI: (10.1016/S1097-2765(02)00746-3)

Figure 3 Insights into the Mechanisms Involved in the Functions of the A Domain (A) ERα-positive MCF-7 or 2T3 cells were treated for 3 hr with E2 or EtOH. Proteins were subjected to immunoprecipitations (IPs) against hsp70 and hsp90. A rat preimmune serum was used as control. Retained (IP) or not (flow-through, FT) proteins were blotted with ERα antibodies (two different gels to see the hERα 46) and anti-hsp antibodies to control the IP efficiency. (B) Similar IPs performed using HepG2 or HeLa cells transfected with hERα or hERαΔ1-37. (C) Pictures of HepG2 or HeLa living cells treated for 3 hr with ligand demonstrate a similar localization of both hERα and hERαΔ1-37 tagged to the GFP. Endogenous hERα of MCF-7 cells was stained with H184 antibody. ERα-negative MDA-MB231 cells were used as a control. Nuclei are visualized by Hoechst staining. (D) ChIP assays in transfected HepG2 and HeLa cells show hERα and hERαΔ1-37 bind DNA in a similar manner. A Western blot controls hERα expression following ligand treatments. (E) Interaction of in vitro-labeled NCoR with N- and C-terminal domains of hERα. Controls are 200 ng of GST. (F) Isolated or combined A and B domains of the hERα were fused to the Gal4DBD and expressed in yeast cells. Activity (Miller Units) of a Gal4 responsive LacZ gene (3UASG-LacZ) is expressed as the mean ± SD. Molecular Cell 2002 10, 1019-1032DOI: (10.1016/S1097-2765(02)00746-3)

Figure 4 Molecular and Structural Determinants for the Interaction between the hERα A and E Domains (A) Alscript (Barton, 1993) figure showing the alignment of A domain sequences from several ERα. Color coding is: gray for amino acids outside of defined domains, yellow for conserved hydrophobic, red for conserved polar, and blue for conserved small residues. Abbreviations are defined in the experimental procedures section. Jpred2 was used to predict the presence of secondary structure. (B) Comparison between the NR interacting domain sequences (ID) determined in CoAs and CoRs and the putative helix of the A domain in tetrapod ERα. (C) Interaction of 200 ng of each fusion proteins or GST and 1 μl of [35S-Met]-labeled hERα D-F domains were tested in the presence of EtOH, 1 μM E2, 50 μM OHT, or ICI. (D) Two-hybrid experiments performed with mutated peptides fused to the ADGal4 in yeast cells containing wild-type or mutated A domains fused to the Gal4DBD. β-galactosidase activity (Miller Units) is expressed as the mean ± SD of values from four independent experiments. (E) Alscript comparison of the structure of the region including the KCK motif in hERα and hERβ determined in the presence of antagonist and agonist. PDB accession is indicated. (F) 200 ng of GST fusion proteins or GST was incubated with 1 μl of [35S-Met]-labeled ERs as in (C). Molecular Cell 2002 10, 1019-1032DOI: (10.1016/S1097-2765(02)00746-3)

Figure 5 Structural Evidence for a Competition between H12, A Domain, and NCoR for a Same Cleft (A and B) GRASP (Nicholls et al., 1991) and Molscript (Kraulis, 1991) pictures illustrating the hERα LBD overall surface, with the two possible hydrophobic clefts in which the A domain could lodge. Hydrophobic residues are in yellow, identified determinants in red for negatively charged and blue for the positively charged residues. The A domain backbone is modeled as a green helix. Mutated residues are shown. (C) Potential competition between the interaction of the A domain and hERα dimerization. (D) 200 ng of GST fusion proteins or GST was incubated with 1 μl of [35S-Met]-labeled point mutated hERα D-F and D-FΔH12 domains, in the presence of EtOH or 50 μM OHT. (E) Binding of labeled hERα, hERαΔ1-37, or NCoR to 100 ng of GST/NCoR or GST/hERα C-F was competed by increasing amounts (50, 100, 250, and 500 ng) of wild-type or mutated A domain. (F) Quantification of competition assays performed with A domain mutants. Amounts required to reduce the interactions by 50% are indicated. Control comes from a GST purification cut by thrombin. (G and H) Increasing amounts of NCoR, wild-type or mutated A domain do not affect dimerization of hERα. Molecular Cell 2002 10, 1019-1032DOI: (10.1016/S1097-2765(02)00746-3)

Figure 6 In Vivo Evidence for the A Domain-Mediated Repression of hERα Ligand-Independent Activities (A) Control of the expression of in vitro-labeled A domains fused to the SV40 NLS. (B) MCF-7 cells were transfected with increasing amounts (25, 50, 100, 200, or 400 ng) of competitors shown, and treated by EtOH or 10 nM E2 for 3 hr. pS2 mRNA levels were quantified as in Figure 2. (C and D) HeLa and HepG2 cells were transfected with the ERE-TK-Luc reporter, pCH110 control for transfection, 25 ng of plasmids expressing hERα or hERαΔ1-37, and increasing amounts of plasmids expressing SV40 NLS fused A domains. Results are the mean ± SEM of the normalized reporter fold repression or activation. Molecular Cell 2002 10, 1019-1032DOI: (10.1016/S1097-2765(02)00746-3)

Figure 7 Functional Validation of the Model (A) Ligand-independent activity of the point mutants in AF-1-permissive cells (HepG2), with the 100% set to hERαΔ1-37. Expression of the diverse proteins in HepG2 cells is shown. (B) Transrepressive ability of the mutant proteins in the presence of EtOH or OHT in AF-2- (HeLa) permissive cells coexpressing NCoR. The 100% was fixed to the hERαΔ1-37 activity in the presence of EtOH and to the wild-type protein activity in the presence of OHT. Results are the mean ± SEM of normalized activities. (C) Estrogen-induced activity of the point mutants. The 100% is the wild-type protein activity. (D) EMSA control the association of in vitro-translated point mutants to a 32P-labeled ERE. The position of specific complexes is indicated. The asterisk points an unspecific band. H222 antibody supershifted the specific bands. (E) Association of key point mutants to hsp in HepG2 cells, as in Figure 3. Molecular Cell 2002 10, 1019-1032DOI: (10.1016/S1097-2765(02)00746-3)

Figure 8 Model for the Structural Modulation of hERα Activity by Its A Domain (A) GRASP illustration of the general equilibrium that follows the hERα C-terminal region depending upon ligand and cofactors binding. For full description, see the Discussion. Helices figuring the A domain, H12, CoAs, and CoRs are, respectively, in green, blue, red, and pink. E380 is depicted in red and the K529,K531 residues are in blue. (B) STAMP structural superimposition of our model to the PPARα/SMRT (silencing mediator for retinoid and thyroid hormone receptors) structure (1kkq). Molecular Cell 2002 10, 1019-1032DOI: (10.1016/S1097-2765(02)00746-3)