PMC

Construct maps. Schematic representations of the Gal4-β-catenin (A) and corepressors SMRT/NCoR (B) used in this study. Gal4-DBD fusion proteins of wild-type and deletion mutants of β-catenin were used to map the domain required for association with SMRT and NCoR, and full-length NCoR, s-SMRT, and VP16 fusion proteins VP16/NCoR.ID and VP16/SMRT.ID were used in mammalian two-hybrid assays.

Mapping the interaction between SMRT/NCoR and TCF4. A, direct interaction between SMRT/NCoR and TCF4. GST-SMRT-(982–1496) and GST-NCoR-(1944–2453) were expressed in bacteria and purified, and the pulldown assays were carried out with [³⁵S]methionine-labeled and in vitro translated TCF4 as described under “Experimental Procedures.” B, schematic representation of the TCF4 protein and deletion mutants used to map the domains required for association with corepressors. C, high mobility group (HMG) of TCF4 is required for interaction with NCoR. Wild-type and deletion mutants of TCF4 were in vitro translated and labeled with [³⁵S]methionine and incubated with GST-NCoR-(1944–2453). GST was pulled down, and the radioactively labeled TCF4 was detected in the precipitates by SDS-PAGE followed by autoradiography.

Mapping the interaction between SMRT and NCoR and β-catenin by mammalian two-hybrid assays. A, CV-1 cells were cotransfected with Gal4/β-catenin vectors (10 ng/well), pFR-Luc (100 ng/well), Renilla (10 ng/well), together with 100 ng/well of SMRT, NCoR, or empty vector pCMX. Reporter activity was determined after 48 h of transfection. B, CV-1 cells were transiently transfected as described in A except that VP16/SMRT.ID, VP16/NCoR.ID, or empty vector VP16 was used. C, Western blot analysis of the expression of Gal4/β-catenin fusion proteins. D, cells were transiently transfected with CCND1 promoter reporter CCND1-Luc (100 ng/well), 0–100 ng/well of VP16/SMRT.ID, VP16/NCoR.ID, or VP16, together with Renilla (10 ng/well). Reporter activity was monitored after 48 h of transfection. E, Western blot analysis of the expression of VP16-corepressor fusion proteins. A, C, and D, all data represent mean ± S.D. of three independent experiments, and p values are shown for comparisons versus empty vector controls. RLU, relative luciferase units.

Model for negative regulation of Wnt signaling pathway by corepressors SMRT and NCoR. In cells with a low level of corepressors SMRT and NCoR or aberrant cytoplasmic localization (left), Wnt signals enable the accumulation of β-catenin in the nucleus, where it binds to TCF4, displaces the corepressor complexes containing HDACs, and enhances transcriptional activation of Wnt-responsive target genes by recruiting additional coactivators, such as the histone acetylase CBP/p300. In cells with high level of SMRT and NCoR (right), SMRT and NCoR can form complexes with both β-catenin and TCF4. In response to Wnt signals, TCF4 and β-catenin can still be recruited to the promoter region of Wnt signaling target genes. However, SMRT and NCoR are also indirectly recruited by both β-catenin and TCF4 to the same binding sites. Because SMRT and NCoR can associate with HDACs and form a corepressor complex with additional proteins, the transcriptional activity of Wnt signaling target genes is inhibited.

Mapping the interaction between SMRT and NCoR and β-catenin by GST-pulldown assays. A, VP16/SMRT.ID and VP16/NCoR.ID were translated and labeled with [³⁵S]methionine in vitro and incubated with GST-tagged full-length β-catenin (GST-β-cat) or with GST alone and subject to SDS-PAGE. B, GST or GST-NCoR-(1944–2453) was incubated with cell lysates from 293T or LNCaP cells. GST was pulled down by extensive washing, and β-catenin was detected in the precipitates by SDS-PAGE and Western blotting with β-catenin-specific antibody. The 1st two lanes represent the input from 293T and LNCaP cells. C, schematic representation of the GST fusion proteins of wild-type and deletion mutant β-catenin used in GST-pulldown as described in D; the right panel indicates the ability of these constructs to bind corepressors SMRT and NCoR. D, armadillo repeat domain is required for the association of β-catenin with corepressors SMRT and NCoR. [³⁵S]Methionine-labeled VP16/SMRT.ID and VP16/NCoR.ID were incubated with GST alone or GST-β-cat constructs. GST was pulled down, and radioactively labeled SMRT and NCoR fusions were detected in the precipitates by SDS-PAGE followed by autoradiography.

SMRT and NCoR inhibit the expression of endogenous CCND1. A, SMRT and NCoR were expressed in SW480 cells. Cells were plated onto 6-well plates and transfected with increasing amounts of SMRT or NCoR (0, 500, 1000, and 2000 ng/well). Western blot analysis was performed with anti-FLAG M2 and anti-SMRT antibodies to detect NCoR and SMRT, respectively. B, CCND1 mRNA expression is down-regulated by overexpression of SMRT or NCoR. SW480 cells were plated onto 6-well plates and transfected with increasing amounts of SMRT or NCoR. After 48 h of incubation, total RNA was isolated, and the expression level of CCND1 and β-actin mRNAs was determined by semiquantitative RT-PCR as described under “Experimental Procedures.” PCR products were detected by ethidium bromide. Similar results were obtained from two additional experiments. C, CCND1 protein levels were decreased in SW480 cells overexpressing SMRT or NCoR. SW480 cells were transfected with increasing amounts of SMRT or NCoR as described in A and B. After 48 h of incubation, cell lysates were prepared, and the expression level of CCND1, β-catenin, TCF4, andβ-actin was determined by SDS-PAGE followed by Western blotting as described under “Experimental Procedures.” Similar results were obtained from one additional experiment.

SMRT and NCoR constitutively occupy the Wnt/β-catenin target gene promoters. A, LiCl induced β-catenin (β-cat) stabilization and nuclear accumulation in 293T cells. Nuclear extracts were prepared from 293T cells after treatment with 20 mm LiCl for the indicated times, and Western blot analysis of β-catenin is shown. As a loading control, Western blot analysis of histone H1 was done. B–E, SMRT and NCoR constitutively occupied on Wnt/β-catenin target gene promoters. 293T cells were treated with 20 mm LiCl for 0–4 h. Chromatin was isolated and sonicated as described, and ChIP assays were performed. Specific primer sets covering TBE sites of CCND1 (B), c-MYC (C), DKK1 (D), and AXIN2 (E) were used for PCR. PCR products were resolved by agarose gel ethidium bromide staining (left panel), and the percentage of the signal corresponding to each PCR product was determined by densitometric scanning and is indicated on the right panel. Each data point is the average of duplicates from a representative experiment. Experiments were repeated twice.

SMRT and NCoR repress gene transcriptional activity of TCF4. A, triplicate wells of CV-1 cells were transiently transfected with the OT-Luc (100 ng/well), TCF4 (10 ng/well), β-catenin (10 ng/well), increasing amounts of either SMRT or NCoR (0, 50, 100, and 200 ng/well), the molar equivalent of empty vector (pCMX), and 10 ng/well null Renilla for normalization. B, cells were transfected with increasing amounts of SMRT/NCoR as described in A, and Western blot analysis of the expression of NCoR and SMRT was performed with appropriate antibodies as described under “Experimental Procedures.” C, CV-1 cells were transiently transfected as described above except that a constant amount of SMRT (25 ng/well) and increasing amounts of β-catenin (0, 10, 50, and 100 ng/well) were used. D, CV-1 cells were transiently transfected with CCND1 promoter reporter (CCND1-Luc, 100 ng/well), increasing amounts of SMRT or NCoR (0, 25, 50, and 100 ng/well), plus the molar equivalent of pCMX. After transfection, cells were cultured in fresh medium for 48 h, harvested, analyzed, and plotted as described under “Experimental Procedures.” Data represent mean values of triplicate experiments, and standard deviations are shown by error bars. A, C, and D, p values are shown for comparison versus empty vector controls. RLU, relative luciferase units.

Recruitment of SMRT and NCoR to the endogenous TCF4-binding sites in promoters of target genes. A, ChIP analysis was performed with chromatin isolated from SW480 cells, and PCRs were performed using specific primers covering the TBE sites of the CCND1 and AXIN2 promoters or primers amplifying the promoter region of GAPDH, which does not contain any known TBE. PCR products were resolved by agarose gel and stained with ethidium bromide (left panel), and the percentage of the signal corresponding to each PCR product was determined by densitometric scanning as indicated on the right panel that is representative of three different experiments. B and C, ChIP Re-ChIP assays. Chromatin prepared from SW480 cells was first immunoprecipitated (1st IP) with TCF4 antibody (B) or β-catenin antibody (C). After extensive washing, immunoprecipitates were eluted by incubation with 10 mm dithiothreitol at 37 °C for 30 min, diluted 20 times with RIPA buffer, followed by a second immunoprecipitation (ChIP Re-IP) with the indicated antibodies as described under “Experimental Procedures.” Specific primer sets covering the TBE sites of the CCND1, AXIN2, and DKK1 promoters were used for PCR.

Knockdown of SMRT or NCoR enhances the transcriptional activity of β-catenin/TCF4 and promotes cell proliferation. A, knockdown of endogenous SMRT and NCoR. 293T cells were transfected with 150 ng/well of empty vector pSilencer or vectors encoding specific siRNA against SMRT or NCoR. 48 h after transfection, total RNA was isolated, and RT-PCR was performed to determine the expression of NCoR and SMRT mRNA. B and C, luciferase reporter assays. Triplicate 293T cells were cotransfected with either OT-Luc (B) or CCND1-Luc (C), together with 150 ng/well of either SMRT siRNA, NCoR siRNA, or control vector pSilencer and Renilla (10 ng/well). Sixteen hours after transfection, cells were treated with or without 20 mm LiCl for 24 h, and cell lysates were prepared, and reporter activity was determined as described under “Experimental Procedures.” Data represent mean ± S.D. of three independent experiments, and p values are shown for comparisons versus empty vector controls. D and E, cell proliferation assays. SW480 cells were plated onto 48-well plates and transfected with indicated constructs (150 ng/will). Sixteen hours after transfection, fresh complete medium containing 10% fetal bovine serum was added, and cells were cultured for 5 days. Crystal violet staining of cell cultures (D) and corresponding measurements of OD570 (E) are shown. Data in D and E represent one of three independent experiments. RLU, relative luciferase units.