Cell type-dependent transcriptional activity of TRβ1. Cells (4 × 10⁵ to 8 × 10⁵/60-mm dish) were plated 24 h before transfection in DMEM containing 10% fetal bovine serum. The cells were transfected with the appropriate plasmids by use of Lipofectamine (pTK28m, 0.2 μg; pCH110 and pCDM-TRβ1, 0.2 μg). After 24 h, all media were replaced with fresh DMEM containing 10% T3-depleted serum in the presence or absence of T3 (100 nM), and the dishes were incubated for an additional 24 h. The cells were harvested and lysed, and CAT activity was determined. The data shown are the results of three independent experiments, each with duplicates (mean ± standard deviation; n = 3). (A) CV1 cells and GC cells. (B) HeLa, A431, and MCF-7 cells. (C) RKO cells.

Expression of Ear-2 in tissues and cultured cells, as determined by Northern blot analysis. (A and B) An RNA human tissue blot was purchased from Clontech and probed with human Ear-2 cDNA (A) and TRβ1 cDNA (B) labeled with [³²P]dCTP. (C) Ten micrograms of total RNAs prepared from SK-Hep-1, RKO, GC, and CV1 cells was loaded onto a 1.2% agarose gel, and the blot was prepared as described in Materials and Methods. The blot was hybridized with ³²P-labeled Ear-2 cDNA.

The C-terminal region of TRβ1 is critical for the interaction of TRβ1 with Ear-2. (A) Molecular size of Ear-2. Ear-2 (lane 2) and TRβ1 (lane 1) were prepared by in vitro transcription-translation in the presence of [³⁵S]methionine, and 5 μl of the programmed lysates was analyzed by SDS-PAGE (10% gel). The dried gel was autoradiographed. (B) Binding of TRβ1 to intact Ear-2. The binding of ³⁵S-labeled Ear-2 to GST-TRβ1 was carried out as described in Materials and Methods. E. coli-expressed GST (2 μg) or GST-TRβ1 (2 μg) noncovalently bound to GSH-Sepharose beads was incubated with increasing concentrations of in vitro-translated ³⁵S-labeled Ear-2 (5, 10, and 20 μl [lanes 1 and 4, 2 and 5, and 3 and 6, respectively]). The bound proteins were analyzed by SDS-PAGE. Lane 7 shows the input from 10 μl of ³⁵S-labeled Ear-2. (C) T3-independent binding of Ear-2 to TRβ1. GSH-Sepharose-bound GST–Ear-2 (10 μg) or GST (10 μg) was incubated with ³⁵S-labeled TRβ1 in the presence or absence of T3 (100 nM) as described in Materials and Methods. After washing was done, bound ³⁵S-labeled TRβ1 was detected as described above. Lanes 1 and 3, without T3; lanes 2 and 4, with T3; lanes 1 and 2 were from the binding of ³⁵S-labeled TRβ1 to GST alone; lanes 3 and 4 were from the binding of ³⁵S-labeled TRβ1 to GST–Ear-2. (D) Mapping of the region of TRβ1 binding to Ear-2. ³⁵S-labeled wild-type TRβ1 (lanes 1 to 3), domains C, D, and E (lanes 4 to 6), domains D and E (lanes 7 to 9), domain E (lanes 10 to 12), and domain E with a deletion of amino acids 420 to 461 (EΔ420-461) were prepared from the corresponding T7 expression plasmids, pCJ3, pJL08, pJL05, pCJ4, and pCJ7, respectively (11). The ³⁵S-labeled proteins were incubated with GST alone (lanes 2, 5, 8, 11, and 14) or GST–Ear-2 (3, 6, 9, 12, and 15) basically as described in panel A. One-tenth the inputs are shown as markers in lanes 1, 4, 7, 10, and 13.

TRβ1 interacts with Ear-2 in cells. (A) Preparation of anti-Ear-2 antibody PC-9. PC-9 was prepared by injection of mice with expression plasmid pCDM-Ear-2 as described in Materials and Methods. Antisera (1 μl; lane 2) and preimmune sera (1 μl; lane 3) were screened by immunoprecipitation with in vitro-translated ³⁵S-labeled Ear-2 (3 μl). The immunoprecipitates were analyzed by SDS–10% PAGE. The dried gel was autoradiographed. Lane 1 shows ³⁵S-labeled Ear-2 as a marker (control [C]). (B) Coimmunoprecipitation of TRβ1 and Ear-2. CV1 cells (4 × 10⁵/60-mm dish) were plated 1 day before transfection. The cells were transfected with 2 μg of pCLC51 and/or 2 μg of pCDM-Ear2. After being incubated overnight, the cells were metabolically labeled with [³⁵S]Met-Cys (40 μCi) for 3 h at 37°C. Cellular extracts (500 μg) were immunoprecipitated with anti-TRβ1 antibodies J51 and J52 (2.5 μg each) (17) (lane 2) or PC-9 (5 μl of sera; lanes 3 and 6) or with antibody MOPC (5 μg; lanes 4 and 7). The immunoprecipitates were analyzed by SDS–10% PAGE. Lanes 1 and 5 show in vitro-translated ³⁵S-labeled TRβ1 and Ear-2, respectively, as markers (control [C]). The dried gel was autoradiographed.

The binding of TRβ1 or Ear-2 to TRE is modulated by TRβ1 or Ear-2. ³⁵S-labeled Lys-TRE, TRβ1, and Ear-2 receptors were prepared as described in Materials and Methods. (A) A constant amount of ³²P-labeled Lys-TRE was incubated with a constant amount of TRβ1 (2 μl of programmed lysates) in the presence of increasing concentrations of the Ear-2 protein. Identical amounts of Ear-2 were used in lane pairs 3 and 4, 5 and 6, 7 and 8, 9 and 10, and 11 and 12. The ratios of Ear-2 to TRβ1 are indicated. After electrophoresis, the gel was dried and autoradiographed. (B) Interaction of Lys-bound Ear-2 and TRβ1 with antibodies. A constant amount of ³²P-labeled Lys-TRE was incubated with 2 μl of in vitro-translated TRβ1 (lanes 2 and 3), Ear-2 (1 μl; lanes 4 and 5), or 2 μl of TRβ1 and 1 μl of Ear-2 (lanes 6 to 9). Lys-bound TRβ1 was supershifted by MAb C4 (1 μg; lanes 3 and 7), and the binding of Ear-2 to Lys-TRE was blocked by PC-9 (1 μl; lanes 5 and 8). In lane 9, a control antibody (MOPC) (1 μg) was used. (C) The binding of TRβ1-RXRβ heterodimers to Lys-TRE is inhibited by the binding of Ear-2. A constant amount (10⁵ cpm) of ³²P-labeled Lys-TRE was incubated with 2 μl of in vitro-translated TRβ1 and/or 1 μl of Ear-2 in the absence or presence of 1 μg of MAb C4, MOPC, and/or RXRβ, as indicated. After gel electrophoresis, the dried gel was autoradiographed.

The differential transcriptional repression by Ear-2 is cell type and T3 dependent. (A) RKO cells were transfected with the CAT reporter plasmid (pTK28m; 0.2 μg) and the TRβ1 expression plasmid (pCDM-TRβ1; 0.2 μg) in the absence (lanes 1 to 4) or the presence of increasing concentrations of pCDM-Ear-2 (lanes 5 to 10, 11 to 16, and 17 to 22). The ratios of Ear-2 to TRβ1 plasmids used in the transfection are shown (pCDM-TRβ1, 0.2 μg; pCDM-Ear-2, 0.025, 0.05, 0.1, 0.2, 0.4, and 0.8 μg for the groups in lanes 11 to 16 and 17 to 22, respectively). Lanes 5 to 10 were transfected with the same increasing concentrations of pCDM-Ear-2 but without pCDM-TRβ1. CAT activity was determined as described in Materials and Methods. (B) T3-dependent transcriptional repression by Ear-2 in RKO cells. The CAT activity in panel A was quantified and plotted. (C) CV1 cells were transfected with the CAT reporter plasmid (pTK28m; 0.2 μg) and the TRβ1 expression plasmid (pCDM-TRβ1; 0.2 μg) in the absence (lanes 1 to 4) or the presence of increasing concentrations of pCDM-Ear-2 (0.2 and 0.4 μg for the groups in lanes 5 and 6 and lanes 7 and 8, respectively). CAT activity was determined as described in Materials and Methods. Data are averages of three independent experiments, each with duplicates (mean ± standard deviation; n = 3).

Ear-2 suppresses basal and T3-induced responses of the human S14 promoter. The data are from a representative experiment that was repeated three times. Each bar is the mean + standard deviation of three plates with the indicated treatment. Bars 1, 3, 5, and 7 were hepatocytes maintained at 5.5 mM glucose and no T3; bars 2, 4, 6, and 8 were cells maintained at 27.5 mM glucose and 500 nM T3. As indicated below the bars, TRβ1 and Ear-2 were cotransfected as indicated in Materials and Methods. At each condition, the addition of T3 led to a significant (P, <0.05) increase in luciferase activity. At each condition, the cotransfection of Ear-2 led to a significant (P, <0.05) decrease in luciferase activity.

Lack of binding of the Ear-2 mutant to TRE and its partial repression of TRβ1-mediated transcriptional activity. (A) The Ear-2 mutant has the same molecular size as wild-type Ear-2. ³⁵S-labeled TRβ1 (lanes 1 and 2), Ear-2 (lanes 3 and 4), and mutant Ear-2 (lanes 5 and 6) proteins were prepared by in vitro transcription-translation in the presence of [³⁵S]Met-Cys with a TNT kit. One (lanes 1, 3, and 5) or 2 (lanes 2, 4, and 6) μl from each programmed lysate was analyzed by SDS–10% PAGE. The dried gel was exposed overnight. (B) The Ear-2 mutant does not bind to TRE either as a homodimer or as a heterodimer with RXR. Two microliters of TRβ1 (lanes 2 and 3), 1 μl of Ear-2 (lanes 4 and 5), and 1 μl of Ear-2 mutant (lanes 6 and 7) prepared by in vitro transcription-translation were incubated with ³²P-labeled Lys-TRE in the absence (lanes 2, 4, and 6) or the presence (lanes 3, 5, and 7) of 1 μg of RXRβ. (C) Relative protein expression of Ear-2 and the Ear-2 mutant. Cellular lysates (50 μg) of transfected CV1 cells (1 and 2 μg of pCDM-Ear-2 in lanes 2 and 3, respectively, or 1 and 2 μg of the pCDM-Ear-2 mutant in lanes 4 and 5, respectively) were analyzed by Western blot analysis with an anti–Ear-2 antibody (1:2,000 dilution of PC-9) as described in Materials and Methods. The arrow indicates Ear-2. (D) Partial repression of the T3-dependent transcriptional activity of TRβ1 by mutant Ear-2. CV1 cells were transfected with the CAT reporter plasmid (pTK28m; 0.2 μg) and the TRβ1 expression plasmid (pCDM-TRβ1; 0.2 μg) in the absence (lanes 1 to 4) or the presence of increasing concentrations of pCDM-Ear-2 (1 and 2 μg for lanes 5 and 6, respectively) or the pCDM-Ear-2 mutant (1 and 2 μg for lanes 7 and 8, respectively). The data were from three independent experiments, each with duplicates (mean ± standard deviation; n = 3). The ratios of Ear-2 and Ear-2 mutant proteins were determined from the results of Western blot analysis in panel C (see above).

Reversal of the Ear-2 repression of the T3-dependent transcriptional activity of TRβ1 by SRC-1 in CV1 cells. CV1 cells were cotransfected with the CAT reporter plasmid (pTK28m; 0.2 μg), the TRβ1 expression plasmid (pCDM-TRβ1; 0.2 μg), the β-galactosidase expression vector (pCH110; 0.2 μg), and the SRC-1 expression vector (pCR3.1 hSRC-1A; 1 μg) in the absence (lanes 1 to 4, 9 to 12, and 17 to 20) or the presence of increasing concentrations of pCDM-Ear-2 (lanes 6 to 9, 0.025 μg; lanes 14 to 17, 0.05 μg; lanes 22 to 25, 0.1 μg) as described in Materials and Methods. The total plasmids transfected were normalized to 3 μg by using the vector control plasmid, pCR3.1. The final ratios of Ear-2 to TRβ1 are indicated. The data were from three independent experiments, each with duplicates (mean ± standard deviation; n = 3).

Repression of the T3-independent and T3-dependent transcriptional activities of TRβ1 by wild-type Ear-2 and mutant Ear-2 and its reversal by SRC-1 in RKO cells. (A) Repression by wild-type Ear-2 and mutant Ear-2. RKO cells (8 × 10⁵ cells/60 mm-dish) were transfected with the CAT reporter plasmid (pTK28m; 0.5 μg), the β-galactosidase expression vector (pCH110; 0.2 μg), and pCDM-TRβ1 (0.2 μg) in the absence of Ear-2 (lanes 1 to 4), the presence of Ear-2 (pCDM-Ear-2, 0.4 μg for lanes 5 and 6), or the presence of mutant Ear-2 (pCDM-Ear-2 mutant, 0.4 μg for lanes 7 and 8). (B) Relative expression of Ear-2 and Ear-2 mutant proteins. Cellular lysates of transfected RKO cells (50 μg) were analyzed by Western blot analysis with PC-9 (1:2,000 dilution) as described in Materials and Methods. Lanes 2 and 3 and lanes 4 and 5 are duplicates. (C) Reversal of the repression effect of Ear-2 by SRC-1. RKO cells were transfected as described in Materials and Methods, except in the absence of SRC-1 (bars 1 to 4, 7, and 8) or the presence of SRC-1 (bars 5, 6, 9, and 10; pCR3.1 hSRC-1A; 1 μg). The total DNA concentration in all dishes was kept at 3 μg by supplementation with the vector control plasmid, pCR3.1. CAT activity in panels A and C was determined as described in Materials and Methods. The data in panels A and C were from three independent experiments, each with duplicates (mean ± standard deviation; n = 3).

Ear-2 binds to SRC-1, GR, and ER. E. coli-expressed GST (4 μg; lanes 2, 5, and 8) or GST–Ear-2 (4 μg; lanes 3, 6, and 9) noncovalently bound to GSH-Sepharose beads was incubated with 10 μl of in vitro-translated ³⁵S-labeled SRC-1 (lanes 2 and 3), GR (lanes 5 and 6), or ER (lanes 8 and 9) for 2 h at 4°C. After washing was done, the bound proteins were analyzed by SDS–10% PAGE. The gel was dried and autoradiographed. Lanes 1, 4, and 7 show 10% inputs of ³⁵S-labeled SRC-1, GR, and ER, respectively.

Ear-2 represses the hormone-dependent transcriptional activity of GR and ER. (A) CV1 cells (4 × 10⁵ to 8 × 10⁵/60-mm dish) were cotransfected with the CAT reporter plasmid (pMSG-CAT; 0.2 μg), pCMV-GR (0.2 μg), and pCH110 (0.2 μg) in the absence (bars 1 to 4) or the presence (bars 5 and 6) of pCDM-Ear-2. The total plasmids transfected were normalized to 3 μg. Twenty-four hours before cells were harvested, Dex (100 nM) was added to the dishes (bars 2, 4, and 6). The success of transfection was monitored by β-galactosidase activity. The CAT activity was normalized by using equal amounts of lysate proteins. The final GR/Ear-2 ratio is shown. The data were from three independent experiments, each with duplicates (mean ± standard deviation; n = 3). (B) CV1 cells were cotransfected with the luciferase reporter plasmid (pTK-ERE-Luc; 0.2 μg), the ER expression plasmid (HEGO; 0.2 μg), and pCH110 (0.2 μg) in the absence (bars 1 to 4) or the presence (bars 5 and 6) of pCMV-Ear-2. The total plasmids transfected were normalized to 3 μg. E2 (100 nM) was added to the dishes (bars 2, 4, and 6) 24 h before cells were harvested. The luciferase activity was normalized by using equal amounts of lysate proteins. The final ER/Ear-2 ratio is shown. The data were from three independent experiments, each with duplicates (mean ± standard deviation; n = 3).