Estrogen response element-dependent regulation of transcriptional activation of estrogen receptors α and β by coactivators and corepressors
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Figure 1
ERE sequence impacts ERα and ERβ transcriptional activity. CHO-K1 cells were co-transfected with (A) ERα or (B) ERβ plus the indicated pGL3-ERE-luciferase reporter and pRL-CMV as described in Materials and methods. Twenty-four hours after transfection, the cells were treated with EtOH, 10 nM E2, 100 nM 4-OHT, or 10 nM E2 plus 100 nM 4-OHT for 30 h. Cell extracts were prepared and assayed as described in Materials and methods. Data are displayed as luciferase activity divided by the RL-luc activity in each well and normalized by EtOH control (which is set to 1). Within each experiment, each treatment was performed in triplicate. The data shown are the means±s.e.m. from at least 11 separate experiments. *P<0.05, E2 values that are statistically different from the EtOH control value.
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Figure 2
Coregulators influence ligand-independent ER activity. In (A and B) CHO-K1 cells were transfected with ERα, EREc38-luciferase reporter, and the amounts of the indicated coactivators. In (C) CHO-K1 cells were transfected with ERα, pGL3-pro-luciferase reporter (no EREs), and the amounts of the indicated coactivators. In (D–H) CHO-K1 cells were transfected with the indicated ERE-luciferase reporter, ERα or ERβ, and 250 ng of the indicated coregulators. All cells were co-transfected with RL-luc reporter control. Cells were treated with EtOH for 30 h and processed as described in Fig. 1 and in Materials and methods. Values are the fold-induction over EtOH activity in the absence of added coregulator and are the means±s.e.m. of three to eight different experiments. *P<0.05, values that are statistically different from the control value.
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Figure 3
Transcriptional response of ERα and ERβ to coregulators on EREc38. CHO-K1 cells were transfected with ERα or ERβ and EREc38-luciferase reporter, pRL-CMV, and 250 ng of the indicated coregulator as described in Materials and methods. Cells were treated with EtOH, 10 nM E2, 100 nM 4-OHT, or both E2 and 4-OHT, as indicated, for 30 h and processed as described in Fig. 1 and in Materials and methods. The maximal activity with 10 nM E2 was set at 100 (absolute fold values are shown in Fig. 1). Values are the means±S.E.M. of three to eight different experiments. (A, B, D, and E) *P<0.05, values that are statistically different from the E2-induced value for that receptor subtype in the absence of coregulator. (C) Δ P<0.05, values that are significantly different (Student’s t-test) between the 4-OHT activity without and with SMRT or NCoR.
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Figure 4
Transcriptional response of ERα and ERβ to coregulators on EREc13. CHO-K1 cells were transfected with ERα or ERβ, EREc13-luciferase, pRL-CMV, and 250 ng of the indicated coregulators as described in Materials and methods and Figs 1 and 3. Values are the means±s.e.m. of three to nine different experiments. *P<0.05, values that are statistically different from the E2-induced value for that receptor subtype in the absence of coregulator.
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Figure 5
Natural variations on ERE palindrome sequence impact the effect of coactivators and corepressors on E2-induced ERα and ERβ transcription. CHO-K1 cells were transfected with ERα or ERβ and (A) pS2, (B) PR1148, or (C) Fos-1211 ERE-luciferase reporters plus pRL-CMV and 250 ng of each of the indicated coregulators as described in Materials and methods. (D) The impact of the addition of either NCoR or SMRT on E2-induced ERα or ERβ activity from the indicated ERE-luciferase reporter. Cells were treated with 10 nM E2, 100 nM 4-OHT, or both (as indicated) for 30 h and processed as described in Fig. 1 and in Materials and methods. The maximal activity with 10 nM E2 was set at 100% and the actual fold-induction values are shown in Fig. 1A and 1B for E2-ERα and ERβ respectively. Values are the means±s.e.m. of three to nine different experiments. *P<0.05, values that are statistically different from the E2-induced value for that receptor subtype in the absence of coregulator.
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Figure 6
Coactivator expression in transiently transfected CHO-K1 cells. CHO-K1 cells were transfected with the indicated amount of the expression vector, ER subtype, and ERE. Western blots were performed on cell extracts using antibodies against (A) SRC-1 and β-actin, (B) GRIP1 and GAPDH, and (C) ACTR and β-actin. These blots are representative of experiments that were repeated twice.
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Figure 7
Synergism between SRA and p160 coregulators depends on ER subtype and ERE sequence. CHO-K1 cells were transfected with ERα or ERβ and the indicated ERE-luciferase reporters plus pRL-CMV and 250 ng SRA plus each of the indicated p160 coactivators as described in Materials and methods and Fig. 1. The maximal activity with 10 nM E2 was set at 100. Values are the means±s.e.m. of three different experiments. *P<0.05, values that are statistically different from both the E2-induced value for that receptor subtype/ERE combination with either SRA or the indicated coactivator alone. The inset shows the relative activity of each coactivator alone with E2-ERα or E2-ERβ on the indicate ERE. These data are taken from Figs 3, 4, and 5.
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Figure 8
The RID of ACTR, but not GRIP1, interacts with ERα and ERβ bound to the pS2 ERE in vitro. (A) Human ERα was incubated with [32P]pS2-ERE as described in Materials and methods. GST-ACTR or GST-GRIP1 (0.5, 1, or 2 μg) were added to ERα and [32P]pS2-ERE in lanes 2–4 and 6–7 respectively, as indicated. GST-ACTR and GST-GRIP alone did not bind pS2-ERE (lanes 5 and 9). Lane 10 included 1 μl ERα-specific antibody G20. (B) Rat ERβ was incubated with [32P]pS2-ERE as described in Materials and methods. GST-ACTR or GST-GRIP1 (0.5, 1, or 2 μg) were added to ERβ and [32P]pS2-ERE in lanes 2–4 and 6–7 respectively, as indicated. GST-ACTR and GST-GRIP alone did not bind pS2-ERE (lanes 5 and 9). Lane 10 included 1 μl ERβ-specific antibody Y-19. NS indicates non-specific binding of baculovirus proteins to the ERE; SS indicates the super-shifted complex formed between the ER antibody and the ER-pS2 complex. This autoradiograph is representative of two independent EMSA experiments for both ERα and ERβ showing similar results. (C and D) EMSA data were quantitated as described in Materials and methods. Values are the % of (C) ERα or (D) ERβ binding in the absence of added coregulator or antibody. %BD=ER-ERE bound; %SS=ER-ERE supershifted with the added GST fusion protein or antibody. Values are the average of two separate experiments±s.d.
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Figure 9
Effect of GST-coregulators on ER-ERE binding in vitro. EMSA data were quantitated as described in Materials and methods. Values are the % of (A and C) ERα or (B and D) ERβ binding to (A and B) EREc38 or (C and D) EREc13 in the absence of added coregulator or antibody. %BD=ER-ERE bound; %SS=ER-ERE supershifted with the added GST fusion protein or antibody. Values are the average of two to three separate experiments±s.e.m.
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Figure 10
Model for ERE sequence-, ER subtype-, coregulator- and ligand state-dependent transcription by ER. Coactivators and corepressors regulate ERα and ERβ transcriptional activity depending on different conformations of the receptors in their unliganded versus E2-occupied as they bind to EREs from different genes. Please see Discussion for further details.
- © 2004 Society for Endocrinology
