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First published online July 12, 2005
doi: 10.1242/10.1242/jcs.02437

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The histidine triad protein Hint1 interacts with Pontin and Reptin and inhibits TCF–ß-catenin-mediated transcription

Institute of Clinical Chemistry and Pathobiochemistry, Charité Campus Benjamin Franklin, Hindenburgdamm 30, 12200 Berlin, Germany

View larger version (35K): [in a new window]   Fig. 1. Hint1 directly binds to Pontin and Reptin. Purified recombinant GST-Pontin, -Reptin, -ß-catenin, -LEF-1 and -Hint1 fusion proteins were incubated with MPB-Hint1. Protein complexes were pulled down with GSH-agarose beads and analysed by western blotting with anti-MBP and anti-GST antibodies. MBP-Hint1 specifically binds to GST-Pontin (lane 4) and GST-Reptin (lane 6) but not to GST (lane 2), GST-ß-catenin (lane 8) or GST-LEF-1 (lane 10). GST-Hint1 associates with MBP-Hint1 (lane 12) as expected from the crystal structure published previously. In control experiments GST and GST-fusion proteins do not nonspecifically bind to MBP or MBP-Hint1 (lanes 1,2,3,5,7,9,11).

View larger version (48K): [in a new window]   Fig. 2. Mapping of the binding sites of Pontin and Reptin in Hint1 and vice versa. (A) C-terminal and N-terminal deletion constructs of Pontin were expressed as GST-fusion proteins as schematically presented and used to map the binding site of Hint1 in Pontin to amino acids 214-294 (lanes 2,4,10,12,14) in pull-down assays. (B) Hint1 binds to amino acids 218-289 in Reptin (lanes 4,10,14,16,18) as mapped with C- and N-terminally deleted MBP-Reptin fusion proteins. (C) MBP-Pontin and -Reptin bind to the N-terminus of Hint1 in pull-down assays with GST-Hint1C (amino acids 1-72) (lanes 5 and 8). The C-terminal half (amino acids 66-126) of Hint1 (GST-Hint1N) mediates dimerization (lane 12).

View larger version (56K): [in a new window]   Fig. 3. Pontin and Reptin form a complex with Hint1 in HEK293 cells. (A) HEK293 cells were transiently transfected with FLAG-tagged Pontin, FLAG-tagged Reptin and myc6-tagged Hint1 alone or in combination. Protein complexes were immunoprecipitated with anti-FLAG M2 monoclonal antibodies and analysed on western blots with anti-myc (9E10) monoclonal antibody. (B) In opposite type of experiments where cells were transfected with FLAG-tagged Hint1, myc6-tagged Pontin or myc6-tagged Reptin, co-precipitation was only detectable when cells were cotransfected with both Pontin-FLAG or Reptin-FLAG and Hint1-myc6 (lanes 8 and 10). After cotransfection of Hint1-FLAG and Hint1-myc6, Hint1 dimers were detected in western blots (lane 11). (C) Association of endogenous Hint1 and Pontin in HEK293 cell lysates as shown in co-immunoprecipitation experiments with anti-Pontin (-23) antibody and western blot detection with anti-Hint1 (-21). **Heavy and *light chain of the precipitating antibody.

View larger version (33K): [in a new window]   Fig. 4. Hint1 is associated with the LEF-1–ß-catenin transcription complex. (A) HEK293 cells were transiently transfected with FLAG-tagged ß-catenin and myc6-tagged Hint1 alone (lanes 1 and 2) or in combination (lane 3). After lysis immunoprecipitation was performed with anti-FLAG M2 and association of Hint1-myc6 was analysed by western blotting with anti-myc (9E10) antibody. (B) Hint1-myc6 associates with LEF-1–HA in HEK293 cell lysates as shown by co-immunoprecipitation with anti-HA (12CA5) and western blotting with anti-myc (9E10) antibody. **Heavy and *light chain of the precipitating antibody.

View larger version (93K): [in a new window]   Fig. 5. Hint1 and Pontin co-localize in the nucleus and are concentrated in speckled structures. Immunofluorescence images of H184A1 cells double-stained with mouse monoclonal anti-Pontin (5G3-11) (Aa, Ba') and guinea pig anti-Hint1 (-A15DC) (Ab, Bb') antibodies taken with a fluorescence (A) and a confocal (B) microscope. (Ac) DAPI staining; (Bc') merge of images Ba' and Bb'. Arrows mark co-localization of Pontin and Hint1 in speckled structures. Bar, 10 mm.

View larger version (29K): [in a new window]   Fig. 6. Hint1 represses TCF-4–ß-catenin transcriptional activity. Reporter gene assays were performed in HEK293 cells transiently transfected with different combinations of plasmids as indicated. Data represent the mean of at least four independent transfections each measured in duplicate. (A) The improved Topflash/Fopflash reporter constructs pGL3-OT/OF were used to measure the effects of Hint1 on TCF-4–ß-catenin transcriptional activity. Cells were transfected with 1.0 µg pGL3-OT/OF, 0.7 µg pCS2+Reptin52 or pCS2+Pontin52, 0.5 µg pcDNAI-hTCF4, 0.5 µg pCS2+ß-catenin, 1 µg pCS2+Hint1 and 0.1 µg pCH110. (B) The repressive effect of Hint-1 on TCF-4–ß-catenin mediated transcription was also detectable with the Siamois luciferase reporter construct (1.0 µg). Increasing amounts of Hint1 (0.5 µg, 1.0 µg, 1.5 µg, 2.0 µg) expression plasmid were transfected with constant amounts of TCF-4, ß-catenin and Pontin or Reptin plasmids. Transfection of an equal amount of empty pCS2+ vector was used as a control. (C) LEF-VP16 (1.0 µg) driven transcription is not repressed by Hint1. N-LEF-1 (1.0 µg) without ß-catenin binding site does not activate transcription of the reporter gene. (D) Reporter gene activity was measured in NIH3T3 cells stably expressing Wnt-1 and control cells after transient transfection with Hint1 (2 µg). (E) Wnt-1 expressing C57MG cells show reduced reporter gene activity when transfected with Hint1 (2 µg). (F) SW480 colon carcinoma cells were transiently transfected with increasing amounts (0.5 µg, 1.0 µg, 1.5 µg, 2.0 µg) of Hint1 and TCF-4–ß-catenin transcriptional activity was analysed with the Siamois luciferase reporter system. In A-C pCH110 (ß-galactosidase) and in D-F pHRL-TK (Renilla luciferase) was used for normalization. S5, wild-type Siamois promoter; S0, Siamois promoter with mutated LEF-1/TCF binding sites.

View larger version (27K): [in a new window]   Fig. 7. Hint1 represses endogenous Cyclin D1 expression in SW480 cells. (A) Total RNA was isolated from SW480 cells transiently transfected with Hint1 expression plasmid or pCS2+ as a control. One-step RT-PCR was performed with total RNA and PCR products were analysed after the indicated number of cycles by agarose gel electophoresis. ß-Actin was used as a loading control. The presented experiment is representative of four independent transfections. (B) The amount of PCR products was quantified on a FujiFilm LAS-1000 system. The average of four independent RT-PCR experiments with total RNA from independent transfections is presented. (C) Western blot analysis of Cyclin D1 in cell lysates of SW480 cells transiently transfected with Hint1 expression plasmid. -Actinin was used as a loading control. (D) Dose-dependent repression of Cyclin D1 promoter in reporter gene assays with cyclin D1-luciferase constructs containing wild-type promoter or promoter with mutated TCF binding-sites.

View larger version (35K): [in a new window]   Fig. 8. ShRNA expression interferes with Hint1 expression and enhances TCF–ß-catenin transcriptional activity. (A) Lysates from HeLa cells stably transfected with interfering shRNA-Hint1430/431 or non-functional shRNA-Hint1432/433 were analyzed for Hint1 expression by western blotting with anti-Hint1 (-21) antibody. (B) Increasing amounts of interfering shRNA-Hint1430/431 increase TCF–ß-catenin-mediated transcription whereas control shRNA-Hint1432/433 did not show an effect. By contrast, shRNA directed against ß-catenin reduces reporter gene activity. When HeLa cells stably transfected with control shRNA-Hint1432/433 were transiently transfected with interfering shRNA-Hint1430/431 again transcriptional activity was increased whereas vice versa no effect was detectable. (C) Quantitative RT-PCR analysis of Axin2 expression in HeLa cells transiently transfected with Hint1 or interfering shRNA-Hint1430/431.

View larger version (54K): [in a new window]   Fig. 9. Hint1 disrupts the Pontin-Pontin interaction. (A) Purified recombinant GST-Pontin and Pontin-His6 fusion proteins were mixed to allow association. Protein complexes were isolated with GSH-agarose beads and subsequently incubated with increasing amounts of Hint1-His6 protein. After a second pull-down, proteins bound to the GSH-agarose beads or in the supernatant were analysed by western blotting with anti-GST, anti-Pontin (-23) and anti-Hint1 (-21) antibodies. (B) A similar experiment was performed with GST-ß-catenin and Pontin-His6. After formation and isolation of GST-ß-catenin–Pontin-His6 complexes, increasing amounts of Hint1-His6 were added. Precipitated protein complexes and supernatants were analysed by western blotting as described above. Lanes 1,2: control showing that Pontin-His6 and Hint1-His6 does not nonspecifically bind to GST alone. Lanes 4-6: addition of increasing amounts of Hint1-His6 protein. (C) Mapping of the Pontin-binding sites in Pontin (amino acids 214-294) and Reptin (amino acids 218-289).