Negative regulation of the Wnt-beta-catenin pathway by the transcriptional repressor HBP1

Abstract

In certain cancers, constitutive Wnt signaling results from mutation in one or more pathway components. The result is the accumulation and nuclear localization of beta-catenin, which interacts with the lymphoid enhancer factor-1 (LEF)/T-cell factor (TCF) family of HMG-box transcription factors, which activate important growth regulatory genes, including cyclin D1 and c-myc. As exemplified by APC and axin, the negative regulation of beta-catenin is important for tumor suppression. Another potential mode of negative regulation is transcriptional repression of cyclin D1 and other Wnt target genes. In mammals, the transcriptional repressors in the Wnt pathway are not well defined. We have previously identified HBP1 as an HMG-box repressor and a cell cycle inhibitor. Here, we show that HBP1 is a repressor of the cyclin D1 gene and inhibits the Wnt signaling pathway. The inhibition of Wnt signaling and growth requires a common domain of HBP1. The apparent mechanism is an inhibition of TCF/LEF DNA binding through a physical interaction with HBP1. These data suggest that the suppression of Wnt signaling by HBP1 may be a mechanism to prevent inappropriate proliferation.

Fig. 1. HBP1 inhibits Wnt–β-catenin-activated gene expression. (A) Schematic diagram of Wnt signaling (adapted from Bienz and Clevers, 2000; Polakis, 2000). The asterisks mark Wnt pathway components with known mutation in cancer. (B) HBP1 inhibits Wnt1-activated gene expression. HEK293T cells were transfected with 1 µg of TOPFLASH, 2 µg of RSV–β-Gal, 5 µg of Wnt1, and 6 or 12 µg of pEFBOS-HBP1. One microgram of FOPFLASH with and without 5 µg of Wnt1 was also transfected as a negative control. TOPFLASH contains three LEF/TCF sites followed by the minimal TATA and luciferase. FOPFLASH contains three mutated LEF/TCF sites followed by the minimal TATA and luciferase. Cells were harvested 48 h after transfection, and luciferase and β-Gal activity were measured. The results are normalized for transfection efficiency and are expressed as a relative ratio of luciferase to β-Gal activities (denoted as relative activity). (C) HBP1 inhibits LiCl-activated gene expression. HEK293T cells were transfected with 1 µg of TOPFLASH, 2 µg of RSV–β-Gal, and 3, 6 or 12 µg of pEFBOS-HBP1 (indicated by ramp). Cells were treated with 15 mM LiCl for 12 h. One microgram of FOPFLASH was also transfected as a negative control. Cells were then harvested at 48 h after transfection. The results are normalized for transfection efficiency and are expressed as a ratio of luciferase to β-Gal activities. (D) HBP1 inhibits β-catenin activation through LEF/TCF DNA binding sites. HEK293T cells were transfected with 2 µg of RSV–β-Gal, 1 µg of TOPFLASH or 1 µg of FOPFLASH as indicated, 2 µg of β-catenin and 7.5 µg of pEFBOS HBP1 as indicated. Cells were harvested 48 h after transfection, and luciferase and β-Gal activity were measured. The results are normalized for transfection efficiency and are expressed as a relative ratio of luciferase to β-Gal activities (denoted as relative activity).

Fig. 1. HBP1 inhibits Wnt–β-catenin-activated gene expression. (A) Schematic diagram of Wnt signaling (adapted from Bienz and Clevers, 2000; Polakis, 2000). The asterisks mark Wnt pathway components with known mutation in cancer. (B) HBP1 inhibits Wnt1-activated gene expression. HEK293T cells were transfected with 1 µg of TOPFLASH, 2 µg of RSV–β-Gal, 5 µg of Wnt1, and 6 or 12 µg of pEFBOS-HBP1. One microgram of FOPFLASH with and without 5 µg of Wnt1 was also transfected as a negative control. TOPFLASH contains three LEF/TCF sites followed by the minimal TATA and luciferase. FOPFLASH contains three mutated LEF/TCF sites followed by the minimal TATA and luciferase. Cells were harvested 48 h after transfection, and luciferase and β-Gal activity were measured. The results are normalized for transfection efficiency and are expressed as a relative ratio of luciferase to β-Gal activities (denoted as relative activity). (C) HBP1 inhibits LiCl-activated gene expression. HEK293T cells were transfected with 1 µg of TOPFLASH, 2 µg of RSV–β-Gal, and 3, 6 or 12 µg of pEFBOS-HBP1 (indicated by ramp). Cells were treated with 15 mM LiCl for 12 h. One microgram of FOPFLASH was also transfected as a negative control. Cells were then harvested at 48 h after transfection. The results are normalized for transfection efficiency and are expressed as a ratio of luciferase to β-Gal activities. (D) HBP1 inhibits β-catenin activation through LEF/TCF DNA binding sites. HEK293T cells were transfected with 2 µg of RSV–β-Gal, 1 µg of TOPFLASH or 1 µg of FOPFLASH as indicated, 2 µg of β-catenin and 7.5 µg of pEFBOS HBP1 as indicated. Cells were harvested 48 h after transfection, and luciferase and β-Gal activity were measured. The results are normalized for transfection efficiency and are expressed as a relative ratio of luciferase to β-Gal activities (denoted as relative activity).

Fig. 1. HBP1 inhibits Wnt–β-catenin-activated gene expression. (A) Schematic diagram of Wnt signaling (adapted from Bienz and Clevers, 2000; Polakis, 2000). The asterisks mark Wnt pathway components with known mutation in cancer. (B) HBP1 inhibits Wnt1-activated gene expression. HEK293T cells were transfected with 1 µg of TOPFLASH, 2 µg of RSV–β-Gal, 5 µg of Wnt1, and 6 or 12 µg of pEFBOS-HBP1. One microgram of FOPFLASH with and without 5 µg of Wnt1 was also transfected as a negative control. TOPFLASH contains three LEF/TCF sites followed by the minimal TATA and luciferase. FOPFLASH contains three mutated LEF/TCF sites followed by the minimal TATA and luciferase. Cells were harvested 48 h after transfection, and luciferase and β-Gal activity were measured. The results are normalized for transfection efficiency and are expressed as a relative ratio of luciferase to β-Gal activities (denoted as relative activity). (C) HBP1 inhibits LiCl-activated gene expression. HEK293T cells were transfected with 1 µg of TOPFLASH, 2 µg of RSV–β-Gal, and 3, 6 or 12 µg of pEFBOS-HBP1 (indicated by ramp). Cells were treated with 15 mM LiCl for 12 h. One microgram of FOPFLASH was also transfected as a negative control. Cells were then harvested at 48 h after transfection. The results are normalized for transfection efficiency and are expressed as a ratio of luciferase to β-Gal activities. (D) HBP1 inhibits β-catenin activation through LEF/TCF DNA binding sites. HEK293T cells were transfected with 2 µg of RSV–β-Gal, 1 µg of TOPFLASH or 1 µg of FOPFLASH as indicated, 2 µg of β-catenin and 7.5 µg of pEFBOS HBP1 as indicated. Cells were harvested 48 h after transfection, and luciferase and β-Gal activity were measured. The results are normalized for transfection efficiency and are expressed as a relative ratio of luciferase to β-Gal activities (denoted as relative activity).

Fig. 1. HBP1 inhibits Wnt–β-catenin-activated gene expression. (A) Schematic diagram of Wnt signaling (adapted from Bienz and Clevers, 2000; Polakis, 2000). The asterisks mark Wnt pathway components with known mutation in cancer. (B) HBP1 inhibits Wnt1-activated gene expression. HEK293T cells were transfected with 1 µg of TOPFLASH, 2 µg of RSV–β-Gal, 5 µg of Wnt1, and 6 or 12 µg of pEFBOS-HBP1. One microgram of FOPFLASH with and without 5 µg of Wnt1 was also transfected as a negative control. TOPFLASH contains three LEF/TCF sites followed by the minimal TATA and luciferase. FOPFLASH contains three mutated LEF/TCF sites followed by the minimal TATA and luciferase. Cells were harvested 48 h after transfection, and luciferase and β-Gal activity were measured. The results are normalized for transfection efficiency and are expressed as a relative ratio of luciferase to β-Gal activities (denoted as relative activity). (C) HBP1 inhibits LiCl-activated gene expression. HEK293T cells were transfected with 1 µg of TOPFLASH, 2 µg of RSV–β-Gal, and 3, 6 or 12 µg of pEFBOS-HBP1 (indicated by ramp). Cells were treated with 15 mM LiCl for 12 h. One microgram of FOPFLASH was also transfected as a negative control. Cells were then harvested at 48 h after transfection. The results are normalized for transfection efficiency and are expressed as a ratio of luciferase to β-Gal activities. (D) HBP1 inhibits β-catenin activation through LEF/TCF DNA binding sites. HEK293T cells were transfected with 2 µg of RSV–β-Gal, 1 µg of TOPFLASH or 1 µg of FOPFLASH as indicated, 2 µg of β-catenin and 7.5 µg of pEFBOS HBP1 as indicated. Cells were harvested 48 h after transfection, and luciferase and β-Gal activity were measured. The results are normalized for transfection efficiency and are expressed as a relative ratio of luciferase to β-Gal activities (denoted as relative activity).

Fig. 2. HBP1 inhibits expression of cyclin D1. (A) Lef-1 and HBP1 have opposite effects on the cyclin D1 promoter. HEK293T cells were transfected with 1 µg of –963 cyclin D1 promoter, 2 µg of RSV–β-Gal, CMV–Lef-1 (1, 2 or 4 µg), pEFBOS-HBP1 (5, 7.5 or 10 µg). Cells were harvested 48 h after transfection, and luciferase and β-Gal activity were measured. The results are normalized for transfection efficiency and are expressed as a ratio of luciferase to β-Gal activities. (B) HBP1 inhibits β-catenin activation of the cyclin D1 promoter. HEK293T cells were transfected with 1 µg of –963 cyclin D1, 2 µg of RSV–β-Gal, indicated amounts of β-catenin, and 7.5 µg of pEFBOS-HBP1. Cells were harvested 48 h after transfection, and luciferase and β-Gal activity were measured. The results are normalized for transfection efficiency and are expressed as a ratio of luciferase to β-Gal activities. (C) HBP1 inhibits endogenous expression of two β-catenin/LEF/TCF targets, cyclin D1 and c-myc. Ten micrograms of pEFBOS-HBP1 were transiently transfected into HCT116 cells along with 2 µg of pMACS K^k. Transfected cells were selected and RNA isolated. As a control, RNA from cells transfected only with pMACS K^k was also isolated. RT–PCR was performed using 2 µg of RNA from each cell population. Results of the RT–PCR using primers to cyclin D1, c-myc and 18S RNA were run on an agarose gel. RT–PCR cycle number for each primer-pair was optimized in order to calibrate into the linear range (see Materials and methods).

Fig. 2. HBP1 inhibits expression of cyclin D1. (A) Lef-1 and HBP1 have opposite effects on the cyclin D1 promoter. HEK293T cells were transfected with 1 µg of –963 cyclin D1 promoter, 2 µg of RSV–β-Gal, CMV–Lef-1 (1, 2 or 4 µg), pEFBOS-HBP1 (5, 7.5 or 10 µg). Cells were harvested 48 h after transfection, and luciferase and β-Gal activity were measured. The results are normalized for transfection efficiency and are expressed as a ratio of luciferase to β-Gal activities. (B) HBP1 inhibits β-catenin activation of the cyclin D1 promoter. HEK293T cells were transfected with 1 µg of –963 cyclin D1, 2 µg of RSV–β-Gal, indicated amounts of β-catenin, and 7.5 µg of pEFBOS-HBP1. Cells were harvested 48 h after transfection, and luciferase and β-Gal activity were measured. The results are normalized for transfection efficiency and are expressed as a ratio of luciferase to β-Gal activities. (C) HBP1 inhibits endogenous expression of two β-catenin/LEF/TCF targets, cyclin D1 and c-myc. Ten micrograms of pEFBOS-HBP1 were transiently transfected into HCT116 cells along with 2 µg of pMACS K^k. Transfected cells were selected and RNA isolated. As a control, RNA from cells transfected only with pMACS K^k was also isolated. RT–PCR was performed using 2 µg of RNA from each cell population. Results of the RT–PCR using primers to cyclin D1, c-myc and 18S RNA were run on an agarose gel. RT–PCR cycle number for each primer-pair was optimized in order to calibrate into the linear range (see Materials and methods).

Fig. 2. HBP1 inhibits expression of cyclin D1. (A) Lef-1 and HBP1 have opposite effects on the cyclin D1 promoter. HEK293T cells were transfected with 1 µg of –963 cyclin D1 promoter, 2 µg of RSV–β-Gal, CMV–Lef-1 (1, 2 or 4 µg), pEFBOS-HBP1 (5, 7.5 or 10 µg). Cells were harvested 48 h after transfection, and luciferase and β-Gal activity were measured. The results are normalized for transfection efficiency and are expressed as a ratio of luciferase to β-Gal activities. (B) HBP1 inhibits β-catenin activation of the cyclin D1 promoter. HEK293T cells were transfected with 1 µg of –963 cyclin D1, 2 µg of RSV–β-Gal, indicated amounts of β-catenin, and 7.5 µg of pEFBOS-HBP1. Cells were harvested 48 h after transfection, and luciferase and β-Gal activity were measured. The results are normalized for transfection efficiency and are expressed as a ratio of luciferase to β-Gal activities. (C) HBP1 inhibits endogenous expression of two β-catenin/LEF/TCF targets, cyclin D1 and c-myc. Ten micrograms of pEFBOS-HBP1 were transiently transfected into HCT116 cells along with 2 µg of pMACS K^k. Transfected cells were selected and RNA isolated. As a control, RNA from cells transfected only with pMACS K^k was also isolated. RT–PCR was performed using 2 µg of RNA from each cell population. Results of the RT–PCR using primers to cyclin D1, c-myc and 18S RNA were run on an agarose gel. RT–PCR cycle number for each primer-pair was optimized in order to calibrate into the linear range (see Materials and methods).

Fig. 3. HBP1 repression of Wnt–β-catenin-activated gene expression requires the repression domain but not DNA binding. (A) HBP1 domain requirement for repression of LEF/TCF-dependent gene expression. HEK293T cells were transfected with 1 µg of TOPFLASH, 2 µg of RSV–β-Gal, 2 µg of β-catenin, 10 µg of pEFBOS-HBP1 or the indicated HBP1 mutant. Cells were harvested 48 h after transfection and results were scored as a relative ratio of luciferase to β-Gal activities (denoted as relative activity). (B) HBP1 domain requirement for repression of Wnt1 gene activation. HEK293T cells were transfected with 1 µg of TOPFLASH, 2 µg of RSV–β-Gal, 5 µg of Wnt1, and 6 µg of pEFBOS-HBP1 or the indicated HBP1 mutant. Cells were harvested 48 h after transfection. The results are normalized for transfection efficiency and are expressed as a relative ratio of luciferase to β-Gal activities. (C) WT HBP1 and pmHMG/HBP1 both suppress β-catenin-induced gene expression. WT HBP1 and pmHMG were transfected in equal amounts to titrate the repression response to β-catenin activation of TOPFLASH. HEK293T cells were transfected with 1 µg of TOPFLASH, 2 µg of RSV–β-Gal, 2 µg of β-catenin, and 3, 6 or 12 µg of pEFBOS-HBP1 or pmHMG (indicated by the ramp). Cells were harvested 48 h after transfection. Luciferase and β-Gal activity were measured. The results were normalized for transfection efficiency and are expressed as a ratio of luciferase to β-Gal activities. Anti-FLU western blotting for HBP protein level is shown for transfected samples below. (D) HBP1 does not bind the TOPFLASH LEF/TCF DNA binding site. GST–HBP1(HMG), GST–HBP1(pmHMG) and GST–TCF4(HMG) proteins were used to score the ability of GST–HBP1(HMG) to bind the LEF/TCF site present in TOPFLASH and the cyclin D1 promoter. Two hundred nanograms of each protein were used in each gel-shift reaction with radioactively labeled TOPFLASH probe or the high affinity (HA) HBP1 site probe as a control for HBP1 DNA binding. A 100-fold molar excess of wild-type and mutant competitors was used in all cases. GST–TCF4(HMG) was used as a positive control for binding TOPFLASH. (E) Repression of the n-myc promoter by HBP1 requires both DNA binding and a repression domain. C33A cells were trans fected with 3 µg of PUC–n-myc–CAT, 2 µg of RSV–β-Gal and 10 µg of pEFBOS-HBP1 or the indicated HBP1 mutant. Cells were harvested 48 h after transfection and results were scored as CAT activity normalized with RSV–β-Gal activity. (F) Repression of the cyclin D1 promoter by HBP1 requires a repression domain, but not an intact DNA binding domain. The –963 cyclin D1–luciferase construct was analyzed as in Figure 2B, except that β-catenin was omitted. Both wild-type and the indicated HBP1 mutants were used.

Fig. 3. HBP1 repression of Wnt–β-catenin-activated gene expression requires the repression domain but not DNA binding. (A) HBP1 domain requirement for repression of LEF/TCF-dependent gene expression. HEK293T cells were transfected with 1 µg of TOPFLASH, 2 µg of RSV–β-Gal, 2 µg of β-catenin, 10 µg of pEFBOS-HBP1 or the indicated HBP1 mutant. Cells were harvested 48 h after transfection and results were scored as a relative ratio of luciferase to β-Gal activities (denoted as relative activity). (B) HBP1 domain requirement for repression of Wnt1 gene activation. HEK293T cells were transfected with 1 µg of TOPFLASH, 2 µg of RSV–β-Gal, 5 µg of Wnt1, and 6 µg of pEFBOS-HBP1 or the indicated HBP1 mutant. Cells were harvested 48 h after transfection. The results are normalized for transfection efficiency and are expressed as a relative ratio of luciferase to β-Gal activities. (C) WT HBP1 and pmHMG/HBP1 both suppress β-catenin-induced gene expression. WT HBP1 and pmHMG were transfected in equal amounts to titrate the repression response to β-catenin activation of TOPFLASH. HEK293T cells were transfected with 1 µg of TOPFLASH, 2 µg of RSV–β-Gal, 2 µg of β-catenin, and 3, 6 or 12 µg of pEFBOS-HBP1 or pmHMG (indicated by the ramp). Cells were harvested 48 h after transfection. Luciferase and β-Gal activity were measured. The results were normalized for transfection efficiency and are expressed as a ratio of luciferase to β-Gal activities. Anti-FLU western blotting for HBP protein level is shown for transfected samples below. (D) HBP1 does not bind the TOPFLASH LEF/TCF DNA binding site. GST–HBP1(HMG), GST–HBP1(pmHMG) and GST–TCF4(HMG) proteins were used to score the ability of GST–HBP1(HMG) to bind the LEF/TCF site present in TOPFLASH and the cyclin D1 promoter. Two hundred nanograms of each protein were used in each gel-shift reaction with radioactively labeled TOPFLASH probe or the high affinity (HA) HBP1 site probe as a control for HBP1 DNA binding. A 100-fold molar excess of wild-type and mutant competitors was used in all cases. GST–TCF4(HMG) was used as a positive control for binding TOPFLASH. (E) Repression of the n-myc promoter by HBP1 requires both DNA binding and a repression domain. C33A cells were trans fected with 3 µg of PUC–n-myc–CAT, 2 µg of RSV–β-Gal and 10 µg of pEFBOS-HBP1 or the indicated HBP1 mutant. Cells were harvested 48 h after transfection and results were scored as CAT activity normalized with RSV–β-Gal activity. (F) Repression of the cyclin D1 promoter by HBP1 requires a repression domain, but not an intact DNA binding domain. The –963 cyclin D1–luciferase construct was analyzed as in Figure 2B, except that β-catenin was omitted. Both wild-type and the indicated HBP1 mutants were used.

Fig. 3. HBP1 repression of Wnt–β-catenin-activated gene expression requires the repression domain but not DNA binding. (A) HBP1 domain requirement for repression of LEF/TCF-dependent gene expression. HEK293T cells were transfected with 1 µg of TOPFLASH, 2 µg of RSV–β-Gal, 2 µg of β-catenin, 10 µg of pEFBOS-HBP1 or the indicated HBP1 mutant. Cells were harvested 48 h after transfection and results were scored as a relative ratio of luciferase to β-Gal activities (denoted as relative activity). (B) HBP1 domain requirement for repression of Wnt1 gene activation. HEK293T cells were transfected with 1 µg of TOPFLASH, 2 µg of RSV–β-Gal, 5 µg of Wnt1, and 6 µg of pEFBOS-HBP1 or the indicated HBP1 mutant. Cells were harvested 48 h after transfection. The results are normalized for transfection efficiency and are expressed as a relative ratio of luciferase to β-Gal activities. (C) WT HBP1 and pmHMG/HBP1 both suppress β-catenin-induced gene expression. WT HBP1 and pmHMG were transfected in equal amounts to titrate the repression response to β-catenin activation of TOPFLASH. HEK293T cells were transfected with 1 µg of TOPFLASH, 2 µg of RSV–β-Gal, 2 µg of β-catenin, and 3, 6 or 12 µg of pEFBOS-HBP1 or pmHMG (indicated by the ramp). Cells were harvested 48 h after transfection. Luciferase and β-Gal activity were measured. The results were normalized for transfection efficiency and are expressed as a ratio of luciferase to β-Gal activities. Anti-FLU western blotting for HBP protein level is shown for transfected samples below. (D) HBP1 does not bind the TOPFLASH LEF/TCF DNA binding site. GST–HBP1(HMG), GST–HBP1(pmHMG) and GST–TCF4(HMG) proteins were used to score the ability of GST–HBP1(HMG) to bind the LEF/TCF site present in TOPFLASH and the cyclin D1 promoter. Two hundred nanograms of each protein were used in each gel-shift reaction with radioactively labeled TOPFLASH probe or the high affinity (HA) HBP1 site probe as a control for HBP1 DNA binding. A 100-fold molar excess of wild-type and mutant competitors was used in all cases. GST–TCF4(HMG) was used as a positive control for binding TOPFLASH. (E) Repression of the n-myc promoter by HBP1 requires both DNA binding and a repression domain. C33A cells were trans fected with 3 µg of PUC–n-myc–CAT, 2 µg of RSV–β-Gal and 10 µg of pEFBOS-HBP1 or the indicated HBP1 mutant. Cells were harvested 48 h after transfection and results were scored as CAT activity normalized with RSV–β-Gal activity. (F) Repression of the cyclin D1 promoter by HBP1 requires a repression domain, but not an intact DNA binding domain. The –963 cyclin D1–luciferase construct was analyzed as in Figure 2B, except that β-catenin was omitted. Both wild-type and the indicated HBP1 mutants were used.

Fig. 3. HBP1 repression of Wnt–β-catenin-activated gene expression requires the repression domain but not DNA binding. (A) HBP1 domain requirement for repression of LEF/TCF-dependent gene expression. HEK293T cells were transfected with 1 µg of TOPFLASH, 2 µg of RSV–β-Gal, 2 µg of β-catenin, 10 µg of pEFBOS-HBP1 or the indicated HBP1 mutant. Cells were harvested 48 h after transfection and results were scored as a relative ratio of luciferase to β-Gal activities (denoted as relative activity). (B) HBP1 domain requirement for repression of Wnt1 gene activation. HEK293T cells were transfected with 1 µg of TOPFLASH, 2 µg of RSV–β-Gal, 5 µg of Wnt1, and 6 µg of pEFBOS-HBP1 or the indicated HBP1 mutant. Cells were harvested 48 h after transfection. The results are normalized for transfection efficiency and are expressed as a relative ratio of luciferase to β-Gal activities. (C) WT HBP1 and pmHMG/HBP1 both suppress β-catenin-induced gene expression. WT HBP1 and pmHMG were transfected in equal amounts to titrate the repression response to β-catenin activation of TOPFLASH. HEK293T cells were transfected with 1 µg of TOPFLASH, 2 µg of RSV–β-Gal, 2 µg of β-catenin, and 3, 6 or 12 µg of pEFBOS-HBP1 or pmHMG (indicated by the ramp). Cells were harvested 48 h after transfection. Luciferase and β-Gal activity were measured. The results were normalized for transfection efficiency and are expressed as a ratio of luciferase to β-Gal activities. Anti-FLU western blotting for HBP protein level is shown for transfected samples below. (D) HBP1 does not bind the TOPFLASH LEF/TCF DNA binding site. GST–HBP1(HMG), GST–HBP1(pmHMG) and GST–TCF4(HMG) proteins were used to score the ability of GST–HBP1(HMG) to bind the LEF/TCF site present in TOPFLASH and the cyclin D1 promoter. Two hundred nanograms of each protein were used in each gel-shift reaction with radioactively labeled TOPFLASH probe or the high affinity (HA) HBP1 site probe as a control for HBP1 DNA binding. A 100-fold molar excess of wild-type and mutant competitors was used in all cases. GST–TCF4(HMG) was used as a positive control for binding TOPFLASH. (E) Repression of the n-myc promoter by HBP1 requires both DNA binding and a repression domain. C33A cells were trans fected with 3 µg of PUC–n-myc–CAT, 2 µg of RSV–β-Gal and 10 µg of pEFBOS-HBP1 or the indicated HBP1 mutant. Cells were harvested 48 h after transfection and results were scored as CAT activity normalized with RSV–β-Gal activity. (F) Repression of the cyclin D1 promoter by HBP1 requires a repression domain, but not an intact DNA binding domain. The –963 cyclin D1–luciferase construct was analyzed as in Figure 2B, except that β-catenin was omitted. Both wild-type and the indicated HBP1 mutants were used.

Fig. 3. HBP1 repression of Wnt–β-catenin-activated gene expression requires the repression domain but not DNA binding. (A) HBP1 domain requirement for repression of LEF/TCF-dependent gene expression. HEK293T cells were transfected with 1 µg of TOPFLASH, 2 µg of RSV–β-Gal, 2 µg of β-catenin, 10 µg of pEFBOS-HBP1 or the indicated HBP1 mutant. Cells were harvested 48 h after transfection and results were scored as a relative ratio of luciferase to β-Gal activities (denoted as relative activity). (B) HBP1 domain requirement for repression of Wnt1 gene activation. HEK293T cells were transfected with 1 µg of TOPFLASH, 2 µg of RSV–β-Gal, 5 µg of Wnt1, and 6 µg of pEFBOS-HBP1 or the indicated HBP1 mutant. Cells were harvested 48 h after transfection. The results are normalized for transfection efficiency and are expressed as a relative ratio of luciferase to β-Gal activities. (C) WT HBP1 and pmHMG/HBP1 both suppress β-catenin-induced gene expression. WT HBP1 and pmHMG were transfected in equal amounts to titrate the repression response to β-catenin activation of TOPFLASH. HEK293T cells were transfected with 1 µg of TOPFLASH, 2 µg of RSV–β-Gal, 2 µg of β-catenin, and 3, 6 or 12 µg of pEFBOS-HBP1 or pmHMG (indicated by the ramp). Cells were harvested 48 h after transfection. Luciferase and β-Gal activity were measured. The results were normalized for transfection efficiency and are expressed as a ratio of luciferase to β-Gal activities. Anti-FLU western blotting for HBP protein level is shown for transfected samples below. (D) HBP1 does not bind the TOPFLASH LEF/TCF DNA binding site. GST–HBP1(HMG), GST–HBP1(pmHMG) and GST–TCF4(HMG) proteins were used to score the ability of GST–HBP1(HMG) to bind the LEF/TCF site present in TOPFLASH and the cyclin D1 promoter. Two hundred nanograms of each protein were used in each gel-shift reaction with radioactively labeled TOPFLASH probe or the high affinity (HA) HBP1 site probe as a control for HBP1 DNA binding. A 100-fold molar excess of wild-type and mutant competitors was used in all cases. GST–TCF4(HMG) was used as a positive control for binding TOPFLASH. (E) Repression of the n-myc promoter by HBP1 requires both DNA binding and a repression domain. C33A cells were trans fected with 3 µg of PUC–n-myc–CAT, 2 µg of RSV–β-Gal and 10 µg of pEFBOS-HBP1 or the indicated HBP1 mutant. Cells were harvested 48 h after transfection and results were scored as CAT activity normalized with RSV–β-Gal activity. (F) Repression of the cyclin D1 promoter by HBP1 requires a repression domain, but not an intact DNA binding domain. The –963 cyclin D1–luciferase construct was analyzed as in Figure 2B, except that β-catenin was omitted. Both wild-type and the indicated HBP1 mutants were used.

Fig. 3. HBP1 repression of Wnt–β-catenin-activated gene expression requires the repression domain but not DNA binding. (A) HBP1 domain requirement for repression of LEF/TCF-dependent gene expression. HEK293T cells were transfected with 1 µg of TOPFLASH, 2 µg of RSV–β-Gal, 2 µg of β-catenin, 10 µg of pEFBOS-HBP1 or the indicated HBP1 mutant. Cells were harvested 48 h after transfection and results were scored as a relative ratio of luciferase to β-Gal activities (denoted as relative activity). (B) HBP1 domain requirement for repression of Wnt1 gene activation. HEK293T cells were transfected with 1 µg of TOPFLASH, 2 µg of RSV–β-Gal, 5 µg of Wnt1, and 6 µg of pEFBOS-HBP1 or the indicated HBP1 mutant. Cells were harvested 48 h after transfection. The results are normalized for transfection efficiency and are expressed as a relative ratio of luciferase to β-Gal activities. (C) WT HBP1 and pmHMG/HBP1 both suppress β-catenin-induced gene expression. WT HBP1 and pmHMG were transfected in equal amounts to titrate the repression response to β-catenin activation of TOPFLASH. HEK293T cells were transfected with 1 µg of TOPFLASH, 2 µg of RSV–β-Gal, 2 µg of β-catenin, and 3, 6 or 12 µg of pEFBOS-HBP1 or pmHMG (indicated by the ramp). Cells were harvested 48 h after transfection. Luciferase and β-Gal activity were measured. The results were normalized for transfection efficiency and are expressed as a ratio of luciferase to β-Gal activities. Anti-FLU western blotting for HBP protein level is shown for transfected samples below. (D) HBP1 does not bind the TOPFLASH LEF/TCF DNA binding site. GST–HBP1(HMG), GST–HBP1(pmHMG) and GST–TCF4(HMG) proteins were used to score the ability of GST–HBP1(HMG) to bind the LEF/TCF site present in TOPFLASH and the cyclin D1 promoter. Two hundred nanograms of each protein were used in each gel-shift reaction with radioactively labeled TOPFLASH probe or the high affinity (HA) HBP1 site probe as a control for HBP1 DNA binding. A 100-fold molar excess of wild-type and mutant competitors was used in all cases. GST–TCF4(HMG) was used as a positive control for binding TOPFLASH. (E) Repression of the n-myc promoter by HBP1 requires both DNA binding and a repression domain. C33A cells were trans fected with 3 µg of PUC–n-myc–CAT, 2 µg of RSV–β-Gal and 10 µg of pEFBOS-HBP1 or the indicated HBP1 mutant. Cells were harvested 48 h after transfection and results were scored as CAT activity normalized with RSV–β-Gal activity. (F) Repression of the cyclin D1 promoter by HBP1 requires a repression domain, but not an intact DNA binding domain. The –963 cyclin D1–luciferase construct was analyzed as in Figure 2B, except that β-catenin was omitted. Both wild-type and the indicated HBP1 mutants were used.

Fig. 4. HBP1 regulates LEF/TCF DNA binding activity. (A) HBP1 inhibits TCF4 and the TCF–β-catenin complex binding to DNA. HEK293T cells were transfected with 3 µg of Flag-TCF4, 7 µg of Flu–β-catenin, and 10 µg of HBP1 or the indicated HBP1 mutant. Cells were harvested 24 h after transfection. EMSA was performed using the 293T extract and the TOPFLASH probe. Both cold TOPFLASH and FOPFLASH were used as competitors at 100-fold molar excess. Where indicated by an asterisk, the Flag antibody was used to supershift the TCF4–β-catenin DNA binding complex. (B) Endogenous complex of HBP1 and TCF4. 293T and HCT116 cells were used to examine the endogenous interaction between HBP1 and TCF4. 293T and HCT116 cells were immunoprecipitated with anti-HBP1, anti-TCF4 or anti-IgG as a control. A SDS–PAGE gel was run, and to detect the presence of HBP1 the anti-HBP1 antibody was utilized in a western blot. As controls, 293T and HCT116 cellular extract untransfected (E), or 293T extract transfected (T) with HBP1 was used to visualize the mobility of HBP1. (C) The repression domain of HBP1 is necessary for TCF4 interaction. HEK293T cells were transfected with FLAG-TCF4 and pEFBOS-HBP1 or the indicated HBP1 mutant. Immunoprecipitations were performed using the FLAG antibody or normal mouse IgG (control). The immunoprecipitations were separated by SDS–PAGE and the associated HBP1 was detected by western blotting with the flu antibody (anti-HA.11; Babco). Total input of HBP1 and immunoprecipitated FLAG-TCF4 (lower panels) were also examined by western blotting as shown. The numbered lanes in the upper panel correlate with those in the lower panels for comparison.

Fig. 4. HBP1 regulates LEF/TCF DNA binding activity. (A) HBP1 inhibits TCF4 and the TCF–β-catenin complex binding to DNA. HEK293T cells were transfected with 3 µg of Flag-TCF4, 7 µg of Flu–β-catenin, and 10 µg of HBP1 or the indicated HBP1 mutant. Cells were harvested 24 h after transfection. EMSA was performed using the 293T extract and the TOPFLASH probe. Both cold TOPFLASH and FOPFLASH were used as competitors at 100-fold molar excess. Where indicated by an asterisk, the Flag antibody was used to supershift the TCF4–β-catenin DNA binding complex. (B) Endogenous complex of HBP1 and TCF4. 293T and HCT116 cells were used to examine the endogenous interaction between HBP1 and TCF4. 293T and HCT116 cells were immunoprecipitated with anti-HBP1, anti-TCF4 or anti-IgG as a control. A SDS–PAGE gel was run, and to detect the presence of HBP1 the anti-HBP1 antibody was utilized in a western blot. As controls, 293T and HCT116 cellular extract untransfected (E), or 293T extract transfected (T) with HBP1 was used to visualize the mobility of HBP1. (C) The repression domain of HBP1 is necessary for TCF4 interaction. HEK293T cells were transfected with FLAG-TCF4 and pEFBOS-HBP1 or the indicated HBP1 mutant. Immunoprecipitations were performed using the FLAG antibody or normal mouse IgG (control). The immunoprecipitations were separated by SDS–PAGE and the associated HBP1 was detected by western blotting with the flu antibody (anti-HA.11; Babco). Total input of HBP1 and immunoprecipitated FLAG-TCF4 (lower panels) were also examined by western blotting as shown. The numbered lanes in the upper panel correlate with those in the lower panels for comparison.

Fig. 4. HBP1 regulates LEF/TCF DNA binding activity. (A) HBP1 inhibits TCF4 and the TCF–β-catenin complex binding to DNA. HEK293T cells were transfected with 3 µg of Flag-TCF4, 7 µg of Flu–β-catenin, and 10 µg of HBP1 or the indicated HBP1 mutant. Cells were harvested 24 h after transfection. EMSA was performed using the 293T extract and the TOPFLASH probe. Both cold TOPFLASH and FOPFLASH were used as competitors at 100-fold molar excess. Where indicated by an asterisk, the Flag antibody was used to supershift the TCF4–β-catenin DNA binding complex. (B) Endogenous complex of HBP1 and TCF4. 293T and HCT116 cells were used to examine the endogenous interaction between HBP1 and TCF4. 293T and HCT116 cells were immunoprecipitated with anti-HBP1, anti-TCF4 or anti-IgG as a control. A SDS–PAGE gel was run, and to detect the presence of HBP1 the anti-HBP1 antibody was utilized in a western blot. As controls, 293T and HCT116 cellular extract untransfected (E), or 293T extract transfected (T) with HBP1 was used to visualize the mobility of HBP1. (C) The repression domain of HBP1 is necessary for TCF4 interaction. HEK293T cells were transfected with FLAG-TCF4 and pEFBOS-HBP1 or the indicated HBP1 mutant. Immunoprecipitations were performed using the FLAG antibody or normal mouse IgG (control). The immunoprecipitations were separated by SDS–PAGE and the associated HBP1 was detected by western blotting with the flu antibody (anti-HA.11; Babco). Total input of HBP1 and immunoprecipitated FLAG-TCF4 (lower panels) were also examined by western blotting as shown. The numbered lanes in the upper panel correlate with those in the lower panels for comparison.

Fig. 5. HBP1 interacts with two separate regions of TCF4. (A) HEK293T cells were transfected with pEFBOS-HBP1 and FLAG-TCF4 or the indicated FLAG-TCF4 mutant. Immunoprecipitations were performed using the FLAG antibody or normal mouse IgG (control). The immunoprecipitations were separated by SDS–PAGE and the associated HBP1 was detected by western blotting with the flu antibody (anti-HA.11; Babco). (B) Total input of HBP1 (lower panel) and immunoprecipitated FLAG-TCF4 or the indicated mutant (upper panel) was also examined by western blotting as shown. The numbered lanes correlate with the numbered lanes in (A) for comparison. (C) Schematic diagram of TCF4 mutants. (D) Summary of interactions on both TCF4 and HBP1. The rat and human HBP1 contain 513 and 509 amino acids, respectively. However, the indicated motifs lie at identical positions in both species.

Fig. 5. HBP1 interacts with two separate regions of TCF4. (A) HEK293T cells were transfected with pEFBOS-HBP1 and FLAG-TCF4 or the indicated FLAG-TCF4 mutant. Immunoprecipitations were performed using the FLAG antibody or normal mouse IgG (control). The immunoprecipitations were separated by SDS–PAGE and the associated HBP1 was detected by western blotting with the flu antibody (anti-HA.11; Babco). (B) Total input of HBP1 (lower panel) and immunoprecipitated FLAG-TCF4 or the indicated mutant (upper panel) was also examined by western blotting as shown. The numbered lanes correlate with the numbered lanes in (A) for comparison. (C) Schematic diagram of TCF4 mutants. (D) Summary of interactions on both TCF4 and HBP1. The rat and human HBP1 contain 513 and 509 amino acids, respectively. However, the indicated motifs lie at identical positions in both species.

Fig. 5. HBP1 interacts with two separate regions of TCF4. (A) HEK293T cells were transfected with pEFBOS-HBP1 and FLAG-TCF4 or the indicated FLAG-TCF4 mutant. Immunoprecipitations were performed using the FLAG antibody or normal mouse IgG (control). The immunoprecipitations were separated by SDS–PAGE and the associated HBP1 was detected by western blotting with the flu antibody (anti-HA.11; Babco). (B) Total input of HBP1 (lower panel) and immunoprecipitated FLAG-TCF4 or the indicated mutant (upper panel) was also examined by western blotting as shown. The numbered lanes correlate with the numbered lanes in (A) for comparison. (C) Schematic diagram of TCF4 mutants. (D) Summary of interactions on both TCF4 and HBP1. The rat and human HBP1 contain 513 and 509 amino acids, respectively. However, the indicated motifs lie at identical positions in both species.

Fig. 5. HBP1 interacts with two separate regions of TCF4. (A) HEK293T cells were transfected with pEFBOS-HBP1 and FLAG-TCF4 or the indicated FLAG-TCF4 mutant. Immunoprecipitations were performed using the FLAG antibody or normal mouse IgG (control). The immunoprecipitations were separated by SDS–PAGE and the associated HBP1 was detected by western blotting with the flu antibody (anti-HA.11; Babco). (B) Total input of HBP1 (lower panel) and immunoprecipitated FLAG-TCF4 or the indicated mutant (upper panel) was also examined by western blotting as shown. The numbered lanes correlate with the numbered lanes in (A) for comparison. (C) Schematic diagram of TCF4 mutants. (D) Summary of interactions on both TCF4 and HBP1. The rat and human HBP1 contain 513 and 509 amino acids, respectively. However, the indicated motifs lie at identical positions in both species.

Fig. 6. HBP1 suppresses colony formation in colon carcinoma cells. Caco-2a cells were transfected with 2 µg of pEFBOSHygro alone, or with 9 µg of pEFBOS-HBP1, or the indicated mutant. Transfected cells were selected in hygromycin media and after 10 days of selection stained with crystal violet. Positive colonies were counted, quantitated and normalized to the HYGRO-only transfection. The mean and SEM from five different experiments are plotted.

Fig. 7. The proposed role of HBP1 in the Wnt signaling pathway. As described in the background, the Wnt signaling pathway is conserved in evolution and has an intricate role in cancer. When Wnt proteins bind to Frizzled receptors, a signaling cascade is triggered where the β-catenin protein becomes stabilized and can enter the nucleus to activate transcription of target genes through binding target HMG-box proteins. Lef/TCF proteins directly bind the DNA, and when bound to β-catenin activate transcription of target genes such as cyclin D1 and c-myc (reviewed in Bienz and Clevers, 2000; Polakis, 2000). In normal tissues, the tumor suppressor protein, APC, along with a complex containing axin and GSK-3β, triggers the degradation of β-catenin when Wnt signaling is turned off. All three components are important for preventing accumulation of β-catenin; loss-of-function mutations in APC and axin are associated with colorectal and hepatocellular cancers. In addition, stabilizing mutations in β-catenin have been identified in many human cancers. With the literature of the Wnt pathway as a backdrop, the work in this paper supports a model in which HBP1 is a suppressor of Wnt signaling and is a negative regulator of proliferation in normal tissues. HBP1 can block Wnt signaling from four points in the pathway: Wnt itself, GSK3β inhibition, β-catenin and LEF/TCF. The endogenous cyclin D1 and c-myc target genes are also inhibited by HBP1 expression in a cell with a constitutive Wnt signaling pathway. Our data indicate that the probable mechanism is the inhibition of DNA binding by LEF and TCF through a physical interaction with HBP1. The same regions of HBP1 that are necessary for suppression of Wnt signaling are also required for growth inhibition in cells with a constitutive Wnt pathway. Thus, HBP1 may have a role in tumor suppression by inhibiting the Wnt signaling pathway.