TCF-4N, an alternatively spliced isoform of TCF-4, lacks DNA binding domain but retains β-catenin-interacting domain. (A) The amino acid sequence of TCF-4N (GenBank accession number AF363725), a novel TCF-4 isoform independently cloned from developing mouse pituitary and 3T3-L1 preadipocytes, is schematically diagramed relative to full-length TCF-4E. The β-catenin interaction domain is depicted as a black box, and the DNA binding domain is depicted as a gray box. (B) Engineered forms of TCF-4E and TCF-4N designed to lack the N-terminal β-catenin interaction domain (ΔNTCF-4E and ΔNTCF-4N) are diagramed. (C) 293T cells in 10-cm plates were transfected with 10 μg each of the indicated expression vectors by calcium phosphate coprecipitation. Cells were lysed after 48 h, and Myc-tagged β-catenin was immunoprecipitated (IP) with an anti-Myc (αmyc) antibody. Immunoprecipitated β-catenin complexes were separated by SDS-PAGE, followed by immunoblot analysis for TCF-4 and β-catenin. These results are representative of at least three independent experiments.

TCF-4N inhibits activation by β-catenin of a TCF-responsive reporter gene. 293T cells were transfected with pTOPFLASH (25 ng), which is a TCF-responsive reporter construct (solid bars), along with expression vectors for β-catenin (10 ng), TCF-4N (25, 50, or 100 ng) and ΔNTCF-4E (100 ng) as indicated. pFOPFLASH (25 ng), a reporter containing mutated TCF consensus binding sites, was transfected as a control (open bars). Samples were normalized to β-galactosidase activity to correct for variations in transfection efficiency. Luciferase activity is reported as activation (mean ± standard deviation) relative to pTOPFLASH alone. These results are representative of at least three independent experiments.

TCF-4N and β-catenin synergize to activate transcription from non-TCF-responsive promoters. (A) 293T cells were transfected with 5 ng of a cyclin D1 promoter-luciferase construct (cyclinD1-luc; black bars), a cyclin D1-luciferase construct with mutations in the consensus TCF binding site, mtTCF(1) (open bars), or with mutations in the one consensus site and four cryptic TCF binding sites, mtTCF(0-4) (grey bars). Reporter genes were cotransfected with expression vectors for β-catenin (250 ng), TCF-4N (125, 250, and 500 ng), and ΔNTCF-4E (500 ng) as indicated. Samples were normalized to β-galactosidase activity to correct for variations in transfection efficiency. Luciferase activity is reported as activation (mean ± standard deviation) relative to the reporter gene alone. (B) 293T cells were transfected with a Gal4-responsive reporter gene (250 ng), which contains multimerized Gal4 binding sites upstream of a minimal promoter and the luciferase gene. Cells were cotransfected with expression constructs for the Gal4 DNA binding domain fused to β-catenin (Gal4-βCat; 250 ng) and either 125, 250, or 500 ng of TCF-4N (solid bars), TCF-4E (open bars), or ΔNTCF-4N (gray bars) as indicated. Luciferase activity is reported as activation (mean ± standard deviation) relative to the reporter gene with Gal4-βCat. These results are representative of at least three independent experiments.

TCF-4N and β-catenin synergize to coactivate SF-1. (A) 293T cells were transfected with an inhibin-luciferase reporter gene (300 ng). Expression constructs for SF-1 (75 ng), β-catenin (300 ng), and TCF-4N (125, 250, and 500 ng) were cotransfected as indicated. Samples were normalized to β-galactosidase activity to correct for variations in transfection efficiency. Luciferase activity is reported as activation (mean ± standard deviation) relative to the reporter gene alone. (B) 293T cells were cotransfected with a LexA-responsive reporter gene (100 ng) and a LexASF-1 expression construct (10 ng). Expression constructs for β-catenin (500 ng) and TCF-4N (500 ng) were cotransfected as indicated. Luciferase activity is reported as activation (mean ± standard deviation) relative to the reporter gene alone. (C) SF-1 was immunoprecipitated from nuclear lysates of Y1 adrenocortical carcinoma cells. Immunoprecipitated SF-1 complexes were separated by SDS-PAGE, followed by immunoblot analysis for β-catenin. These results are representative of at least three independent experiments.

TCF-4N and β-catenin synergize to coactivate C/EBPα. (A) 293T cells were transfected with a leptin promoter reporter gene (250 ng; solid bars) or mtLeptin-luc (open bars), containing a mutated C/EBPα binding site. Expression constructs for β-catenin (250 ng), C/EBPα (50 ng), and TCF-4N (125, 250, and 500 ng) were cotransfected as indicated. Samples were normalized to β-galactosidase activity to correct for variations in transfection efficiency. Luciferase activity is reported as activation (mean ± standard deviation) relative to the reporter gene alone. (B) 293T cells were cotransfected with a Gal4-responsive reporter gene (250 ng) and an expression construct for the Gal4 DNA binding domain fused to the transactivation and basic region of C/EBPα (5 ng). Expression constructs for β-catenin (250 ng) and TCF-4N (500 ng) were cotransfected as indicated. Luciferase activity is reported as activation (mean ± standard deviation) relative to the reporter gene alone. These results are representative of at least three independent experiments. (C) Nuclear extracts (20 μg) prepared 1 to 4 days after induction of 3T3-L1 cell differentiation were analyzed by immunoblot for expression of β-catenin and C/EBPα. RNA prepared 1 to 4 days after induction of 3T3-L1 cell differentiation was analyzed for expression of TCF-4N by RNase protection assay with a riboprobe specific for the unique 3′ untranslated region.

β-Catenin and TCF-4N interact with C/EBPα and p300 to activate the leptin promoter. (A) The amino acid sequence of mouse C/EBPα is schematically diagrammed, with the four conserved regions within the transactivation domain indicated. The CR2-C/EBPα and CR1/3/4-C/EBPα deletion constructs are shown schematically relative to full-length p42C/EBPα. (B) 293T cells were transfected with Leptin-luc (250 ng) and 50 ng of expression vectors for full-length C/EBPα (solid bars), CR2-C/EBPα (open bars), or CR1/3/4-C/EBPα (gray bars). Expression constructs for β-catenin (250 ng) and TCF-4N (500 ng) were cotransfected as indicated. Samples were normalized to β-galactosidase activity to correct for variations in transfection efficiency. Luciferase activity is reported as fold activation (mean ± standard deviation) relative to the reporter gene alone. (C) 293T cells in 10-cm plates were transfected with 5 μg each of the indicated expression vectors by calcium phosphate coprecipitation. Cells were lysed after 48 h, and β-catenin was immunoprecipitated with anti-Flag antibody. Immunoprecipitated β-catenin complexes were separated by SDS-PAGE, followed by immunoblot analysis for C/EBPα and β-catenin. (D) 293T cells were transfected with Leptin-luc (250 ng) and expression constructs for C/EBPα (50 ng), β-catenin (250 ng), and TCF-4N (500 ng) as indicated. Cells were cotransfected with 250 ng of either empty vector (solid bars) or human p300 (open bars). Luciferase activity is reported as activation (mean ± standard deviation) relative to the reporter gene alone. Results are representative of at least three independent experiments.

TCF-4N partially rescues the block of differentiation caused by ectopic expression of β-catenin. (A) 3T3-L1 preadipocytes were infected with a control retrovirus (Hygro) or a retrovirus containing the coding region for TCF-4N. Control and TCF-4N-expressing cells were reinfected with a control retrovirus (Neo) or a retrovirus containing the coding region for S33Y β-catenin. Two days after confluence, cells were treated with inducers of adipogenesis. Two weeks later, cells were stained with Oil Red-O to visualize the degree of lipid accumulation. The amount of Oil Red-O was quantified after extraction and is displayed as fold absorbance relative to the empty vector control. Cells were lysed, and expression of C/EBPα was analyzed by immunoblot (inset). (B) Control (Neo) and S33Y β-catenin-expressing cells were reinfected with a retrovirus alone (Hygro) or a retrovirus containing the coding region for TCF-4N and analyzed as in A. (C) Model for the regulation of β-catenin by TCF-4N. Expression of TCF-4N inhibits interactions between β-catenin and TCF/LEF transcription factors. Complexes containing β-catenin, TCF-4N, and p300 coactivate multiple transcription factors, including the adipogenic transcription factor C/EBPα.