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Volume 57, Issue 6, Pages 1047-1058 (March 2015)
Structure of a Biologically Active Estrogen Receptor-Coactivator Complex on DNA Ping Yi, Zhao Wang, Qin Feng, Grigore D. Pintilie, Charles E. Foulds, Rainer B. Lanz, Steven J. Ludtke, Michael F. Schmid, Wah Chiu, Bert W. O’Malley Molecular Cell Volume 57, Issue 6, Pages (March 2015) DOI: /j.molcel Copyright © 2015 Elsevier Inc. Terms and Conditions
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Molecular Cell 2015 57, 1047-1058DOI: (10.1016/j.molcel.2015.01.025)
Copyright © 2015 Elsevier Inc. Terms and Conditions
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Figure 1 ERα, SRC-3, and p300 Form an Active Transcription Complex
(A) ChIP-seq analysis on the genome-wide binding sites for ERα (Odom lab dataset [Schmidt et al., 2010]), SRC-3, and p300 in MCF-7 cells. (B) SRC-3 and p300 coactivate ERα-mediated in vitro transcription synergistically in the presence of E2. Data are represented as mean ± SEM. Details of the in vitro transcription reaction are described in the Experimental Procedures. See also Figure S1. Molecular Cell , DOI: ( /j.molcel ) Copyright © 2015 Elsevier Inc. Terms and Conditions
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Figure 2 Cryo-EM Structure of ERE DNA and Estrogen-Bound ERα/SRC-3/p300 Complex (A) Coomassie staining of the purified ERα, SRC-3, and p300 proteins. (B) Cryo-EM images of ERE-DNA/ERα/SRC-3/p300 complexes boxed out by white boxes and DNA indicated by white arrows. (C) Cryo-EM density map of the complex derived from EMAN2. Density map shows at a threshold of 0.5. (D) The 3D statistical variance map of the ERα-coactivator complex structure. Shown in red is the variance higher than 0.45 standard variation overlapped with the density of the complex. Higher variance was observed in SRC-3a, p300 densities, and the interacting area between them. See also Figure S2. Molecular Cell , DOI: ( /j.molcel ) Copyright © 2015 Elsevier Inc. Terms and Conditions
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Figure 3 Antibody Labeling of the p300
(A and B) Binding of a p300-specific monoclonal antibody (Ab1) to the ERE-DNA/ERα/SRC-3/p300 complex (A) or to the biochemically isolated p300 (B). (C) Binding of another p300-specific monoclonal antibody (Ab2) to the biochemically isolated p300. The p300 density is annotated in dark or light blue, while the antibodies are annotated in yellow or brown. Densities were Weiner filtered according to the FSC plot (Figure S3I). See also Figure S3 and Movies S1, S2, and S3. Molecular Cell , DOI: ( /j.molcel ) Copyright © 2015 Elsevier Inc. Terms and Conditions
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Figure 4 Identification of SRC-3 and ERα in the Complex
(A) Segmentation of the complex (red, orange, and green) after computationally removing the p300 density. These components were assigned to SRC-3a, SRC-3b, and ERα as described in the text. The 2-fold symmetry axes of the ERα (green arrow) and a fitted LBD crystal structure (purple arrow) are also shown. White solid axis shows another 2-fold axis found by symmetry search. The AF-1 domain of ER in the left panel is hidden in order to make the LBD crystal structure visible. (B) Localization of ERα AF-1 domain in the complex using AF-1-specific monoclonal antibody (yellow). Densities were Weiner filtered according to the FSC plot (Figure S3I). See also Figure S4 and Movies S4 and S5. Molecular Cell , DOI: ( /j.molcel ) Copyright © 2015 Elsevier Inc. Terms and Conditions
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Figure 5 Structure of the ERE-DNA/ERα/SRC-3/p300 Complex and Functional Analysis of the Complex (A) An annotated cryo-EM density map and a cartoon illustration of the ERα-coactivator complex. The white arrow indicates the C-terminal SRCID of p300 that mainly interacts with SRC-3a. (B) Multiple domains of p300 can interact with SRC-3. Top: schematic representation of the seven-domain structure of p300. The yellow and brown bars represent the Ab1 recognition region (774–1045) and Ab2 recognition region (1921–2023), respectively. Bottom: interaction between SRC-3 and different FLAG-tagged p300 fragments as assayed by co-IP using FLAG antibody. (C) CID deletion mutant of SRC-3 loses the ability to recruit p300 to DNA-bound ERα in vitro. Purified ERα, SRC-3, and p300 were incubated in the presence of 1 μM E2 with immobilized biotinylated EREs. The DNA-bound ERα, SRC-3, and p300 and those remaining unbound (supernatant) were visualized by western blot analysis. (D) CID deletion mutant loses the ability to coactivate ERα-mediated transcription in vitro. Purified SRC-3 WT or ΔCID mutant were added to ERα and HeLa nuclear extract in an in vitro transcription assay (as detailed in the Experimental Procedures). Data are represented as mean ± SEM. (E) ERα and SRC-3 significantly increased p300 HAT activity toward histone H3 substrate. The left panel shows the 3H autoradiography of acetylated H3 and H4 proteins in the absence or presence of p300, SRC-3, and ERα. The right panel is the quantitation graph of the H3 and H4 acetylation levels. Data are represented as mean ± SEM. See also Figure S5. Molecular Cell , DOI: ( /j.molcel ) Copyright © 2015 Elsevier Inc. Terms and Conditions
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