Volume 13, Issue 5, Pages 725-738 (March 2004) Selective Recognition of Distinct Classes of Coactivators by a Ligand-Inducible Activation Domain  Mari Luz Acevedo, Kathleen C Lee, Joshua D Stender, Benita S Katzenellenbogen, W.Lee Kraus  Molecular Cell  Volume 13, Issue 5, Pages 725-738 (March 2004) DOI: 10.1016/S1097-2765(04)00121-2

Figure 1 Both Med220(RID) and SRC2(RID) Are Potent Inhibitors of ERα-Dependent Transcription (A) Schematics of Med220, SRC2, and the RID polypeptides. Med220(RID) has two NR boxes (1 and 2) and SRC2(RID) has three (1, 2, and 3). (B) ERα interacts with Med220(RID) and SRC2(RID) in a standard “GST pulldown” assay. Bound ERα was analyzed by Western blotting. (C) Med220(RID) and SRC2(RID) are potent inhibitors of ERα-dependent transcription. The plasmid pERE (bottom) was assembled into chromatin in the presence of ERα and E2 as indicated. The templates were subjected to in vitro transcription in the presence of increasing molar amounts, relative to ERα, of GST, GST-Med220(RID), or GST-SRC2(RID). (D) Quantification of multiple experiments like those shown in (C) (mean ± SEM for three or more determinations). (E) NR box mutant Med220(RID) and SRC2(RID) do not inhibit ERα-dependent transcription in vitro. W, wild-type RID; M, mutant RID. (F) Med220(RID) and SRC2(RID) do not inhibit Gal4-VP16-dependent transcription in vitro with a template containing five Gal4 sites and the AdE4 promoter (pGIE0). Molecular Cell 2004 13, 725-738DOI: (10.1016/S1097-2765(04)00121-2)

Figure 2 Med220(RID) and SRC2(RID) Have Distinct Effects on RNA pol II-Dependent and Chromatin-Dependent Functional Endpoints (A) SRC2(RID), but not Med220(RID), efficiently inhibits targeted nucleosomal histone acetylation by p300. pERE was assembled into chromatin by salt gradient dialysis and used in histone acetylation reactions containing p300, GST-SRC2(RID/PID), ERα, E2, and 3H-acetyl CoA. Wild-type (W) and NR box mutant (M) GST-fused RIDs were added as indicated. (B) Med220(RID), but not SRC2(RID), inhibits ERα-dependent transcription with naked DNA templates. In vitro transcription reactions were performed with chromatin (left) or naked DNA (right) templates. GST-fused RIDs were added as indicated. (C) Med220(RID), but not SRC2(RID), inhibits ERα-dependent transcription with naked DNA (middle) and TSA-hyperacetylated chromatin (right) templates. Quantification and summary of multiple in vitro transcription experiments like those shown in (B) (mean + SEM for three or more determinations). Molecular Cell 2004 13, 725-738DOI: (10.1016/S1097-2765(04)00121-2)

Figure 3 Med220(RID) and SRC2(RID) Interact in Distinct Ways with DNA-Bound ERα, yet Compete with Each Other for Binding to the Receptor (A) Distinct effects of Med220(RID) and SRC2(RID) on ERE-ERα complex formation in EMSAs using a single ERE (EREs; top) or tandem EREs (EREt; bottom). GST-Med220(RID) and GST-SRC2(RID) were added as indicated. The composition of the shifted complexes are indicated. (B) NR box mutant RIDs do not affect EREs-ERα complexes in EMSAs. Wild-type (W) and NR box mutant (M) GST-fused RIDs were added as indicated. (C) Med220(RID) and SRC2(RID) compete for binding to EREs-bound ERα. A fixed amount of GST-Med220(RID) was competed with increasing amounts of GST-SRC2(RID) (left) or vice versa (right). Molecular Cell 2004 13, 725-738DOI: (10.1016/S1097-2765(04)00121-2)

Figure 4 Med220(RID) and SRC2(RID) Selectively Inhibit the Formation of Coactivator Complexes with DNA-Bound ERα (A) Experimental set up for the immobilized template binding assays. (B) Mediator and SRCs interact with DNA-bound ERα. Western blotting was performed with a panel of Mediator subunit antibodies, a panspecific SRC antibody that recognizes SRC1, SRC2, and SRC3, and an antibody to ERα. The Mediator subunits are designated using the unified nomenclature of Rachez and Freedman (2001). (C) ERα-dependent binding of Mediator subunits to a naked DNA template in vitro requires a promoter. Binding experiments like those shown in (B) were performed using templates containing (+) or lacking (–) the AdE4 promoter. (D) GST-Med220(RID) and GST-SRC2(RID) interact with DNA-bound ERα in a ligand-dependent manner. Western blotting was performed with antibodies to ERα and GST. (E) GST-Med220(RID) and GST-SRC2(RID) selectively inhibit the interaction of Med220 and SRCs with DNA-bound ERα. Low (100 nM) and high (500 nM) amounts of the RIDs were added as indicated. (F) Quantification of multiple experiments like those shown in (E) (mean plus the SEM for three or more determinations). Results from experiments using a low amount (100 nM) of the GST-fused RIDs are shown (i.e., lanes 2, 3, 6, and 7 from [E]). Molecular Cell 2004 13, 725-738DOI: (10.1016/S1097-2765(04)00121-2)

Figure 5 Selective Inhibitory Effects of Med220(RID) and SRC2(RID) with ERα(L540Q) (A) Med220(RID), but not SRC2(RID), interacts with EREs-bound ERα(L540Q) in EMSAs. GST-Med220(RID) and GST-SRC2(RID) were added as indicated. (B) The transcriptional activity of ERα(L540Q) in vitro with chromatin templates is stimulated by exogenously added p300. Purified recombinant p300 was added as indicated. (C) Distinct inhibitory effects of Med220(RID) and SRC2(RID) with wild-type (left) and L540Q (right) ERα. All transcription reactions contained exogenously-added p300 to activate ERα(L540Q). GST-fused RIDs were added as indicated. Each bar represents the mean + SEM for three or more determinations. (D) The transcriptional activity of ERα(L540Q) in vitro with chromatin templates is stimulated by TSA with the AdE4 promoter, but not the pS2 promoter. Each bar represents the mean + SEM for three determinations. (E) E2-dependent transcription of the endogenous pS2 gene in 231/ERα(L540Q) cells is not observed in the presence of TSA. (F) Control experiments for (E) showing that ERα and p300 levels do not change and that histone H4 is hyperacetylated by the TSA treatment. Molecular Cell 2004 13, 725-738DOI: (10.1016/S1097-2765(04)00121-2)

Figure 6 Selective Recruitment of Coactivators by ERα(L540Q) to an Estrogen-Regulated Gene In Vivo (A) E2 stimulates transcription of the endogenous pS2 gene in 231/ERα cells, but not in 231/ERα(L540Q) cells. mRNA levels after treatment with E2 were determined by Q-PCR. Each bar represents the mean + SEM for three determinations. (B) The 231/ERα, 231/ERα(L540Q), and 231/ERα(82G) cell lines express similar amounts of ERα, Med220, SRCs, and p300 as assessed by Western blotting. (C) Schematic representation of the human pS2 promoter and the PCR product from the ChIP assays. (D) Selective recruitment of coactivators by ERα(L540Q) to the pS2 promoter. 231/ERα, 231/ERα(L540Q), and 231/ERα(82G) cells were used in ChIP assays with antibodies for ERα, Med220, p300, and SRCs (panspecific) (right). Serial dilutions of the input material for each condition are shown (left). Treatment with E2 was for 45 min. (E) The presence of RNA pol II and acetylated H4 at the pS2 promoter is not increased after E2 treatment in 231/ERα(L540Q) cells. 231/ERα and 231/ERα(L540Q) cells were used in ChIP assays with antibodies for RNA pol II and acetylated H4 as described for (D). (F) Time course of ERα and Med220 recruitment to the pS2 promoter after E2-treatment in 231/ERα and 231/ERα(L540Q) cells. Molecular Cell 2004 13, 725-738DOI: (10.1016/S1097-2765(04)00121-2)

Figure 7 Global Analysis of E2-Dependent Gene Expression in 231/ERα and 231/ERα(L540Q) Cells (A) E2 represses transcription of the endogenous c-Myc gene in both 231/ERα and 231/ERα(L540Q) cells. mRNA levels after treatment with E2 were determined by Q-PCR. Each bar represents the mean + SEM for three determinations. (B) ChIP analysis of factor recruitment to the c-Myc promoter in 231/ERα and 231/ERα(L540Q) cells using the factor antibodies described in Figures 6D and 6E. The schematic diagram (top right) shows the human c-Myc promoter and the PCR product from the ChIP assays. (C) Venn diagram summary of the microarray expression analyses with the 231/ERα and 231/ERα(L540Q) cells. Genes were scored as E2-stimulated if they showed ≥ 2-fold enhanced expression after 1 hr and/or 2 hr of E2 treatment. (D) Confirmation of E2-responsiveness for the five genes listed in (C) by Q-PCR (mean + SEM for three determinations). Molecular Cell 2004 13, 725-738DOI: (10.1016/S1097-2765(04)00121-2)