Institute of Clinical Chemistry and Pathobiochemistry, Charité Campus Benjamin Franklin, Hindenburgdamm 30, 12200 Berlin, Germany
* Author for correspondence (e-mail: otmar.huber{at}charite.de)
Accepted 7 April 2005
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Summary |
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Key words: Histidine triad, Hint1, PKCI, TCF, ß-catenin, Wnt signaling
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Introduction |
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In this respect, Pontin (synonyms: Pontin52, TIP49a, RuvBl1, ECP51, Tih1) and Reptin (synonyms: Reptin52, TIP49b, RuvBl2, ECP54, Tih2) were previously identified as antagonistic regulators of TCFß-catenin transcriptional activity (Bauer et al., 2000; Bauer et al., 1998
; Rottbauer et al., 2002
). Both proteins are evolutionary highly conserved and their primary structures reveal characteristic Walker A and B motifs known to bind ATP/GTP. Overall both proteins exhibit some similarity to bacterial RuvB proteins, which are involved in DNA repair and act as branch migration motors at Holliday junctions. In enzymatic assays, Pontin and Reptin were reported to exhibit single-stranded DNA-stimulated ATPase and ATP-dependent helicase activity with opposite orientation (Kanemaki et al., 1999
; Makino et al., 1999
). Recent data now indicate that Pontin and Reptin act as essential components of chromatin remodeling complexes, such as the yeast Ino80 ATPase (Shen et al., 2000
) and the TIP60 histone acetyl transferase complex (Ikura et al., 2000
). Furthermore, both proteins associate with BAF53 (Park et al., 2002
) and p400 (Fuchs et al., 2001
), which were reported as components of numerous high molecular mass nuclear complexes. In Drosophila, Reptin was found in the Polycomb group complex 1, which maintains an inherited repressive state of genes (Saurin et al., 2001
). The role of Pontin and Reptin within these chromatin remodeling complexes currently is not clear. Recent data indicate that in yeast the Pontin and Reptin homologs are required for the correct assembly of the Ino80 complex (Jónsson et al., 2004
). The gene regulatory function of Reptin, moreover, was shown by its phosphorylation-dependent association with activating transcription factor 2 (ATF2) resulting in an inhibition of ATF2 transcriptional activity (Cho et al., 2001
). In addition, Pontin and Reptin are complexed with MYC, and mutated Pontin inhibits the MYC oncogenic function (Wood et al., 2000
). Recently, Pontin was shown to act as a regulator of ß-catenin-mediated neoplastic transformation by modulating the chromatin structure at target gene promoters (Feng et al., 2003
).
Gene disruption in Saccharomyces cerevisiae and Drosophila is lethal emphasizing the essential role of Pontin and Reptin in cell growth and early development (Bauer et al., 2000; Bauer et al., 1998
; Lim et al., 2000
). Analysis of zebrafish Liebeskummer (Lik) mutant embryos encoding an activated Reptin protein revealed that Reptin and Pontin antagonistically regulate cardiomyocyte proliferation and are involved in growth regulation of endoderm derived tissues by affecting ß-catenin-mediated signaling (Rottbauer et al., 2002
).
The Hint1/PKCI (histidine triad nucleotide-binding protein 1; synonyms: protein kinase C inhibitor 1, protein kinase C-interacting protein 1, adenosine 5'-monophosphoramidase) protein is a member of the Hint branch of the evolutionary conserved histidine triad (HIT) protein family, which is characterized by a common His-X-His-X-His-XX motif (X, hydrophobic amino acid). FHIT (fragile HIT), represents the second branch of HIT proteins and was shown to be inactive in numerous carcinomas (Huebner and Croce, 2003). All members analyzed so far show adenosylpolyphosphate-hydrolase activity with different specificities for substrates such as AMP-X, ADP, ATP, Ap3A and Ap4A (for a review, see Brenner, 2002
). Meanwhile, other family members of the Hint branch have been identified including Hint2, Hint3, Aprataxin (Date et al., 2001
; Moreira et al., 2001
) and the scavenger mRNA decapping enzyme DcpS (Liu et al., 2002
).
Less is known about the physiological and cellular functions of Hint1/PKCI. The identification of Hint1/PKCI interaction partners suggested that it might be involved in the regulation of transcriptional processes. In this respect, Hint1/PKCI was reported to interact with the transcription factor Microphthalmia (Mi), which plays an important role in mast cell and melanocyte growth and function (Razin et al., 1999). Hint1/PKCI was also shown to interact with the product of the ATDC gene, which previously was assumed to be an Ataxia-telangiectasia (AT) candidate gene (Brzoska et al., 1995
) and thus involved in the cellular response to ionizing radiation. Although the role of ATDC in this context is not clear, there is evidence that Hint1 plays a role in the radiation sensitivity of cells by repressing fos transcription (Choi et al., 2000
). The association of Hint1 with cyclin-dependent kinase 7 (CDK7), furthermore, supports a function in growth control and transcriptional regulation (Korsisaari and Mäkelä, 2000
). Recently the phenotypes of Hint1/PKCI knockout mice were reported (Korsisaari et al., 2003
; Su et al., 2003
). These mice develop normally and have a normal life span, however, N-nitrosomethylbenzylamine (NMBA) treatment induces squamous tumors with higher frequency than in wildtype mice (Su et al., 2003
).
Here, we report the identification of human Hint1 as a new direct interaction partner of human Pontin and Reptin and analyse the functional consequences of this interaction on the TCFß-catenin transcription complex. Hint1/PKCI disrupts the Pontin/Reptin complex and represses TCFß-catenin transcriptional activity. These data provide first insight into the molecular mechanism mediating the tumor suppressive role of Hint1/PKCI.
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Materials and Methods |
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Plasmids
Plasmids encoding myc-, FLAG-, MBP- or GST-tagged Pontin or Reptin were described previously (Bauer et al., 2000; Bauer et al., 1998
). Plasmids encoding Pontin deletion constructs containing residues 1-297, 1-213, 1-147, 147-456, 213-456 or 296-456 were generated by restriction enzyme cloning procedures. Reptin deletion constructs were generated by PCR and ligated into BamHICIP treated pGEX4T1 (Amersham Biosciences). Human Hint1 cDNA was amplified from total RNA of SW480 cells by RT-PCR using oligonucleotides 5'-CGC GGA TCC ATG GCA GAT GAG ATT GCC-3' (forward) and 5'-CGC GGA TCC TTA ACC AGG AGG CCA ATG CAT-3' (reverse). The PCR product was cloned into BamHI-CIP-treated plasmids pGEX-4T1, pMal (New England Biolabs) and pQE40 (Qiagen). Deletion constructs of Hint1 encoding amino acids 1-71 (Hint1
C) and 66-126 (Hint1
N) were generated by PCR using the following primer pairs: forward oligonucleotide (see above) and 5'-GCG GGA TCC TCA TTC ATC ATC ATC TTC TGC-3'; 5'-GCG GGA TCC GCC ACC ATG GCA GAA GAT GAT GAT GAA-3' and reverse oligonucleotide (see above), respectively. The PCR products were cloned into BamHICIP treated pGEX-4T1 vector. FLAG-tagged and myc-tagged variants of Hint1 were generated by amplification of the Hint1 cDNA with the oligonucleotide pairs 5'-GCG AAT TCA AAG CTT ATG GCA GAT GAG ATT GCC-3' and reverse oligonucleotide (see above) and forward oligonucleotide (see above) and 5'-CGC GGA TCC ACC AGG AGG CCA ATG CAT-3' respectively and ligation into HindIIIBamHICIP treated pFLAG-CMV4 (Sigma) and BamHICIP treated pCS2+myc6 (kindly provided by Ralf Rupp). The DNA sequences of all constructs were verified by sequencing. Plasmids pGEX4T1-LEF-1 and pGEX4T1-ß-catenin were described elsewhere (Huber et al., 1996
).
Antibodies
Monoclonal anti-FLAG M2 and anti-MBP (clone MBP-17) antibodies were obtained from Sigma, anti-Cyclin D1 (clone Ab-3) antibody from Oncogene (Calbiochem-Novabiochem GmbH), anti--Actinin (clone AT6/172) antibody from Upstate (Biomol), and anti-HA (clone 12CA5) antibody from Roche. HRPO-labeled anti-mouse and anti-rabbit antibodies were purchased from Dianova. Alexa FluorTM488 goat anti-guinea pig IgG and Alexa FluorTM594 goat anti-mouse IgG antibodies were obtained from Molecular Probes (MoBiTec). Rabbit anti-GST antibody was kindly provided by Jürgen Wienands. The polyclonal antibody against Pontin52 (
-23) was described previously (Bauer et al., 1998
).
Monoclonal antibodies against Pontin and Reptin were generated according to standard procedures. Hybridoma clones secreting antibodies directed against Pontin and Reptin were identified by ELISA screening. The specificity of the antibodies was confirmed by western blot analysis on purified recombinant proteins. The monoclonal anti-Pontin (clone 5G3-11) and anti-Reptin (clone 2E9-5) antibodies were purified on Protein A-sepharose (Amersham Biosciences). Both 5G3-11 and 2E9-5 monoclonal antibodies are specific and do not cross-react with Reptin and Pontin, respectively, as shown in western blot analyses. Rabbit anti-Hint1 antibody was generated by three sequential immunizations of rabbits with purified recombinant Hint1-His6 protein mixed 1:1 with complete adjuvant (BioGenes GmbH). Antibodies directed against the peptide Acteyl-ADEIAKAQVARPGGDC-CONH2 (A15DC) of human Hint1 were generated at Pineda Antikörper Service by immunization of two rabbits and a guinea pig.
RNA interference
Hairpin oligonucleotides to suppress Hint1 expression were designed according to the recommendations given by Miyagishi et al. (Miyagishi et al., 2004) and cloned into the BamHIHindIII sites of the vector pRNAT-H1.1/Neo (GenScript Corp.). Oligonucleotide sequences were: ON430, 5'-GAT CCG TGT CTT GCT TTC TAT GAT ATT GTG CTG TCA TGT CAT GGA AAG CAA GGC ACT TTT TTG GAA A-3'; ON431, 5'-AGC TTT TCC AAA AAA GTG CCT TGC TTT CCA TGA CAT GAC AGC ACA ATA TCA TAG AAA GCA AGA CAC G-3'; ON432, 5'-GAT CCG ATT ATC TGT AAG GAG ATA CCT GTG CTG TCG GTA TTT CCT TGC GGA TGA TCT TTT TTG GAA A-3'; ON433, 5'-AGC TTT TCC AAA AAA GAT CAT CCG CAA GGA AAT ACC GAC AGC ACA GGT ATC TCC TTA CAG ATA ATC G-3'. Interferring activity of these oligonucleotides was tested in a reporter assay using pJOsiCheck (see supplementary material, Fig. S2), an improved variant of psiCheck (Promega). Further details are available on request. HeLa cells expressing vector-based short hairpin RNA were used for reporter gene assays and RT-PCR analysis and were transiently or stably transfected with Fugene (Roche) according to the manufacturer's recommendation.
Transient transfections and reporter gene assays
HEK293 cells (5 x105) were transfected with the calcium-phosphate method. The following amounts of expression vectors were used for transfections: 1.0 µg of the Siamois-luciferase (S5, S0) or Topflash/Fopflash (pGL3-OT/OF) reporter construct (kindley provided by David Kimelman and Bert Vogelstein, respectively), 0.7 µg of Reptin and Pontin, 0.5 µg of hTCF4 (kindly provided by Hans Clevers) and 0.5 µg of ß-catenin expression vectors. To normalize transfection efficiency 0.1 µg pCH110 (ß-gal) was cotransfected. The amount of DNA for each transfection was adjusted by addition of empty pCS2+ vector. Luciferase and ß-galactosidase activities were measured with the luciferase reporter gene assay (constant light signal) and the chemoluminescent ß-Gal reporter gene assay (Roche) in a Lumat LB9507 luminometer (Berthold Technologies) 42 hours after transfection. Each value was obtained by double measurement and subsequent normalization of luciferase activities with ß-galactosidase activities.
Transfection of C57MG cells and NIH3T3 cells was performed with LipofectaminePlus (Invitrogen) according to the manufacturer's recommendations. SW480 cells were transfected with Metafectene (Biontex GmbH). Cells were transfected for 24 h with 1.0 µg Siamois-luciferase reporter construct (S5, S0) and 0.1 µg pHRL-TK (Renilla luciferase) (Promega). Reporter gene assays were performed with the Dual-Luciferase Reporter Assay System (Promega). Average values of four independent transfection experiments are presented for all reporter gene assays.
Immunoprecipitation
HEK293 cells were transiently transfected with pCS2+Pontin52-FLAG (2.5 µg), pCS2+Reptin52-FLAG (2.5 µg), pCS2+Hint1-myc6 (2.5 µg), pCS2+Pontin52-myc6 (2.5 µg), pCS2+Reptin52-myc6 (2.5 µg), pFLAG-CMV4-Hint1 (2.5 µg), pCIneo-LEF-HA (3.0 µg) and pCS2+ß-cat-FLAG (3.0 µg). After 48 hours cells were incubated with ice-cold lysis buffer [50 mM Tris pH 6.8, 150 mM NaCl, 2 mM ZnCl2, 300 mM sucrose, 0.25% (v/v) Triton X-100 and CompleteTM-protease inhibitor mix (Roche)] for 15 minutes at 4°C. Immunoprecipitation from precleared cell lysates (150 µg protein) was performed with 4 µg of the appropriate antibody pre-bound to Protein-A beads as described previously (Weiske et al., 2001). To detect endogenous protein complexes, HEK293 cells were lysed with buffer E (50 mM Tris pH 6.8, 100 mM NaCl, 2 mM ZnCl2, 300 mM Sucrose, 0.25% (v/v) Triton X-100 and CompleteTM-protease inhibitor mix) and immunoprecipitations were performed with 20 µl rabbit anti-Pontin (anti-23) antibody (Bauer et al., 1998
) from precleared cell lysates (200 µg total protein) as described above.
Pull-down and competition assays
GST and MBP fusion proteins were expressed in Escherichia coli and purified by affinity chromatography on glutathione-conjugated (GSH)-agarose beads (Sigma) or amylose resin (New England Biolabs). For pull-down assays, 5 µg of GST or GST fusion proteins were incubated with 5 µg MBP fusion proteins in binding buffer (150 mM NaCl, 50 mM Tris pH 6.8, 2 mM ZnCl2, 300 mM sucrose, 0.25% (v/v) Triton X-100) for 30 minutes at 4°C. After centrifugation (5 minutes, 4°C, 20,800 g) 35 µl of GSH-beads were added to the supernatants and incubated for 1 hour at 4°C under constant agitation. GSH-beads were pelleted at 2700 g, 4°C for 1 minute and washed five times with binding buffer. The bound complexes were eluted in 2 x SDS sample buffer and subsequently analysed by SDS-PAGE and western blotting.
For competition assays GST-Pontin or GST-ß-catenin fusion proteins were incubated with Pontin-His6 for 30 minutes at 4°C in buffer C (150 mM NaCl, 50 mM Tris pH 6.8, 2 mM ZnCl2, 300 mM Sucrose, 0.1% (v/v) Triton X-100). After centrifugation (5 minutes, 4°C, 20,800 g) the supernatants were incubated with 35 µl GSH-agarose beads for 30 minutes at 4°C. After three washes with buffer C, Hint1-His6 protein was added to the complexes and incubated for 30 minutes at 4°C. GSH-beads were again pelleted by centrifugation, washed three times with buffer C and resuspended in 2 x SDS sample buffer.
Western blot analysis
Western blot analysis was performed as described previously (Weiske et al., 2001). Antibodies were diluted in TST (1.5 µg/ml anti-Flag M2, 0.5 µg/ml anti-GST, 0.4 µg/ml anti-HA (clone 12CA5), 0.5 µg/ml anti-Cyclin D1 (clone Ab-3) and 1.0 µg/ml anti-Pontin [affinity purified
-23 (Bauer et al., 1998
)]; anti-Myc and affinity-purified rabbit anti-Hint1 (
-21) antibodies were diluted 1:1000; anti-MBP (clone MBP-17) and anti-
-Actinin (clone AT6/172) were diluted 1:4000 and 1:1000, respectively.
Immunofluorescence microscopy
Cells were grown on gelatine-coated glass coverslides for 48 hours. For immunofluorescenece microscopy cells were washed with PBS, permeabilized with 0.5% (v/v) Triton X-100 for 20 minutes on ice, and fixed in 3.7% (w/v) paraformaldehyde for a further 20 minutes. Subsequently cells were gently washed again in PBS, blocked with 0.1% (v/v) goat serum in PBS for 30 minutes at room temperature and incubated with mouse anti-Pontin (5G3-11) (1:100) for 30 minutes at room temperature. After three washes with PBS cells were incubated with polyclonal guinea pig anti-Hint1 (1:1000) for 30 minutes at room temperature, washed three times and treated with Alexa FluorTM488 goat anti-guinea pig IgG (1:1000) and Alexa FluorTM594 goat anti-mouse IgG (1:1000) for a further 30 minutes. To stain the nuclei cells were treated with DAPI (0.1 µg/ml) for 5 minutes at room temperature. Coverslides were mounted with ProTaqs Mount Fluor (Biocyc). Analysis and photography were performed on an Olympus BX-60 microscope with a Plan-Neofluar objective (63 x magnification, 1.25 numerical aperture, 0.17 mm working distance). JPEG images were generated using the Soft Imaging System (SIS) Color View 12 and the analysis 3.0 software. Confocal immunofluorescence microscopy was performed at room temperature on a Leica TCS SP2 3 confocal microscope with a HCX PL Apo 40 x 1,25 oil objective with 4 x zoom at excitation wavelengths 488 nm and 543 nm, respectively. Data acquisition was performed with the Leica Confocal Software Pack (version 2.00) and data collected from 4 x serial sections. Figures were prepared with the Adobe Photoshop 4.0 software without any adjustments.
Cyclin D1 and Axin2 expression
Total RNA was isolated from Hint1- and mock-transfected SW480 cells with the RNeasy Midi Kit (Qiagen) and used for OneStep RT-PCR reactions (Qiagen) to amplify human cyclin D1 and ß-actin cDNA with oligonucleotide pairs 5'-GAC CAT CCC CCT GAC GGC CGA G-3' (forward), 5'-CCG CAC GTC GGT GGG TGT GC-3' (reverse) and 5'-TCC TGG GCA TGG AGT CCT GTG-3' (forward), 5'-CGC CTA GAA GCA TTT GCG GTG-3' (reverse), respectively, according to the manufacturer's instructions (95°C, 20 seconds; 60°C, 20 seconds; 72°C, 60 seconds). After 20, 25, 30 and 35 cycles samples were removed from the PCR reactions and analysed by agarose gel electrophoresis. Quantification and data analysis was performed on a FujiFilm LAS-1000 imager with the Image Gauge version 3.2 software. Values represent relative intensities of cyclin D1 versus ß-actin signals. Mean values based on RT-PCRs performed on RNAs from three independent preparations are presented. Cyclin D1 protein levels in Hint-1- and mock-transfected SW480 cells were detected by analysis of 30 mg total protein by SDS-PAGE and subsequent western blotting.
Quantitative RT-PCR for axin2 was performed on a LightCycler system (Roche) with the Quanti-TectTM SYBR Green RT PCR Kit (Qiagen) and oligonucleotides 5'-GAA ACT GGC AGG TGT CCA CGC-3' and 5'-GCG GGA TCC TCA ATC GAT CCG CTC CAC TTT GCC-3' according to the manufacturer's instructions (95°C, 20 seconds; 62°C, 20 seconds; 72°C, 60 seconds, 35x).
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Results |
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Pontin/Reptin and Hint1 are components of high molecular mass LEF-1ß-catenin transcription complexes
To verify the cellular interaction of Pontin and Reptin with Hint1, co-immunoprecipitation experiments were performed. HEK293 cells were transiently transfected with FLAG-tagged Pontin or Reptin and Hint1-myc6. In immunoprecipitations with the anti-FLAG-M2 antibody, Hint1-myc6 was readily detectable in the Pontin-FLAG and Reptin-FLAG immunocomplexes (Fig. 3A). Complex formation was also detectable in the opposite type of experiments when cells were transfected with FLAG-Hint1 and Pontin-myc6 or Reptin-myc6. Moreover, Hint1 dimerization was demonstrated in cells cotransfected with FLAG-Hint1 and Hint1-myc6 (Fig. 3B). In immunoprecipitations with the polyclonal anti-Pontin (-23) antibody described previously (Bauer et al., 1998
), the endogenous protein complex was isolated and co-precipitating Hint1 protein was detected on western blots with an affinity-purified polyclonal anti-Hint1 (
-21) antibody that was generated against recombinant His6-tagged Hint1 protein (Fig. 3C).
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Hint1 is a negative regulator of the TCFß-catenin transcriptional activity
To analyse whether Hint1 indeed modulates the transcriptional activity of the TCFß-catenin complex, reporter gene assays were performed using Siamois-luciferase (Brannon et al., 1997) or the pGL3-OT/OF-luciferase (He et al., 1998
) reporter constructs. As previously shown, co-expression of Pontin increased TCF-4ß-catenin transcriptional activity (Bauer et al., 2000
). When Hint1 was co-expressed the luciferase activity was reduced. Hint1 also represses transcriptional activity independent of the co-expression of Pontin by binding to endogenous protein. No effects were detectable with the pGL3-OF control reporter construct containing a promoter with mutated LEF/TCF binding sites (Fig. 6A). Addition of increasing amounts of Hint1 resulted in a dose-dependent reduction of reporter gene activity. In the presence of Pontin, which induces a further increase of the TCF-4ß-catenin activity, cotransfection of Hint1 reduced the reporter gene activity to the level of TCF-4ß-catenin alone. In the presence of Reptin, which by itself reduces TCF-4ß-catenin transcriptional activity, Hint1 repressed transcription further to nearly basal levels (Fig. 6B). In these reporter assays Hint1 did not affect CMV promoter-driven ß-galactosidase activity used to normalize transfection efficiency (not shown). Moreover, in HEK293 cells transfected with LEF-VP16, a construct where LEF-1 is directly linked to the transcriptional activation domain of the herpes simplex virus protein VP16 constitutively inducing transcription of target genes in the absence of ß-catenin (Aoki et al., 1999
), again Hint1 did not repress activity of the Siamois-luciferase reporter construct. In addition, a LEF-1 construct with deleted ß-catenin binding site (
N-LEF-1) did not activate transcription and was not affected by Hint-1 (Fig. 6C). Taken together, this indicates that the repressive effect of Hint1 on TCF-4ß-catenin-mediated transcription is depending on ß-catenin and does not represent a general repressive effect on transcription.
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Hint1 represses Cyclin D1 expression
The results presented so far are consistent with Hint1 as a negative regulator of Wnt target gene expression. In a next step we analysed whether Hint1 modulates expression of an endogenous target gene of the Wnt pathway. In this respect the expression of the cell-cycle regulator Cyclin D1, a known direct target gene of the TCFß-catenin complex (Shtutman et al., 1999; Tetsu and McCormick, 1999
) was examined. Transient transfection of Hint1 cDNA in SW480 cells reduced cyclin D1 mRNA expression levels by approximately 50%, as shown by RT-PCR, compared with mock-transfected cells (Fig. 7A,B). This repression of Cyclin D1 expression was confirmed at the protein level by western blot analysis with an anti-Cyclin D1 antibody (Fig. 7C) and by reporter assays with Cyclin D1 promoter-luciferase constructs (Tetsu and McCormick, 1999
) (Fig. 7D).
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If Hint1 is indeed a repressor of LEF-1/TCFß-catenin-mediated transcription, silencing of its expression should enhance transcriptional activity. Short hairpin RNAs (shRNA) were designed and analysed for their interfering activity by reporter screening (see supplementary material Figs S1 and S2). Vectors expressing an interfering shRNA (shRNA-Hint1430/431) or an inactive shRNA (shRNA-Hint1432/433) were stably expressed in HeLa cells and the knockdown of Hint1 was verified by immunoblotting (Fig. 8A). When we compared the activation of ß-catenin-mediated transcription in these cells with cyclin D1-luciferase reporter gene assays, Hela cells transfected with shRNA-Hint1430/431 showed a dose-dependent increase in transcriptional activity. Cells transfected with the non-functional shRNA-Hint1432/433 did not differ from control cells. In a further control, shRNA-ß-catenin decreased reporter gene activity. Moreover, when Hela cells stably transfected with the non-functional shRNA-Hint1432/433 were in addition transfected transiently with the knockdown shRNA-Hint1430/431 vector, again enhanced luciferase activity was detectable (Fig. 8B). Expression of endogenous target genes in HeLa cells transiently transfected with shRNA-Hint1430/431 was analyzed by quantitative RT-PCR. As shown for Axin2, expression levels were upregulated 1.95-fold in shRNA-Hint1430/431-transfected cells in contrast with cells transfected with Hint1 cDNA exhibiting a 54% downregulation (Fig. 8C). Similar upregulation of Cyclin D1 expression in shRNA-Hint1430/431-transfected HeLa cells was detected by RT-PCR (not shown).
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Hint1 disrupts the Pontin/Reptin complexes
As shown by the in vitro binding studies (Fig. 1), Hint1 directly binds to Pontin and Reptin but not to ß-catenin or LEF-1. This suggests that the binding of Hint1 to Pontin and/or Reptin may affect the homo- or heteromeric PontinReptin interaction. To address whether this may represent a molecular mechanism that contributes to the Hint1 function, competition assays were performed. A preformed complex of purified recombinant GST-Pontin and Pontin-His6 was isolated with GSH-agarose beads. Excess Pontin-His6 was washed away and increasing amounts of Hint1-His6 were added. Subsequently the protein complexes were pulled down again. After washing, protein complexes bound to the GSH-beads were analysed by western blotting. Indeed, increasing amounts of Hint1 resulted in a concomitant reduction of Pontin-His6 associated with GST-Pontin and increased binding of Hint1. Consistent with this, increasing amounts of Pontin-His6 were detected in the supernatant (Fig. 9A). Similar results were obtained when GST-PontinReptin-His6 or GST-ReptinReptin-His6 complexes were analysed (not shown). In contrast, addition of Hint1 did not disrupt a GSTß-cateninPontin-His6 complex but results in formation of a ternary GSTß-cateninPontin-His6Hint1-His6 complex (Fig. 9B). These in vitro experiments provide good evidence that Hint1 by competing for the Pontin/Reptin interaction site may modulate the high molecular mass LEF-1/TCFß-catenin transcription complex. Consistent with this, pull-down experiments localized the Pontin/Reptin interaction sites between amino acids 215-296 and amino acids 218-289, respectively (Fig. 9C). This observation indicates that the Hint1 association site is identical or overlaps with the homomeric and heteromeric PontinReptin interaction sites suggesting that Hint1 may affect Pontin/Reptin homomeric/heteromeric complex formation or stability.
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Discussion |
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Co-immunopreciptitation experiments confirmed that Hint 1 forms a complex with Pontin and Reptin in cells. Moreover, ß-catenin and LEF-1 were able to pull down Hint1. However, this interaction appears to be indirect and mediated by Pontin or Reptin since Hint1 did not associate directly with ß-catenin or LEF-1 in pull-down experiments with recombinant proteins. This suggested that Hint1 modulates ß-catenin mediated transcriptional regulation. Indeed, reporter gene assays with the Siamois and Topflash/Fopflash luciferase reporter constructs both revealed a dose-dependent repressive activity of Hint-1 on LEF-1/TCFß-catenin-induced luciferase activity, whereas knockdown of Hint1 by RNA interference increased transcriptional activity. Consistent with these observations, Hint1 overexpression reduced reporter gene activity in NIH3T3 and C57MG cells stimulated by autocrine Wnt signal and in SW480 colon carcinoma cells. Further support for the repressive effect of Hint1 on Wnt target gene transcription was provided by analysis of the endogenous expression of Cyclin D1 (Shtutman et al., 1999; Tetsu and McCormick, 1999
) and Axin2 (Jho et al., 2002
; Lustig et al., 2002
), two known TCFß-catenin target genes.
A transcriptional repressor function for Hint1 was also reported in conjunction with other transcription factors. In this context Hint 1 was shown to repress Micropthalmia (Mi)- and FOS-dependent transcription (Choi et al., 2000; Razin et al., 1999
). Moreover, Hint1 by its interaction with Pontin and Reptin may also modulate MYC-dependent transcription. The MYC-binding site in Pontin was mapped to amino acids 136-187 (Wood et al., 2000
) and thus does not overlap with the Hint1 binding domain (amino acids 214-294) in Pontin. Therefore, formation of a ternary complex between MYC, Pontin and Hint1 appears to be possible. In addition, Pontin and/or Reptin by binding to Hint1 may modulate Cdk7 activity (Korsisaari and Mäkelä, 2000
). Analysis of embryonic fibroblasts from Hint1-deficient mice, however, suggested that Hint1 itself is not a key regulator of Cdk7 activity (Korsisaari et al., 2003
). As Pontin and Reptin are components of the chromatin remodeling machinery it is assumed that Hint1 by interacting with these proteins may assemble in high molecular mass chromatin remodeling complexes. Currently it is not known how Hint1 may functionally modulate chromatin remodeling. One possibility is that the nucleotidyl-hydrolase activity of Hint1 may be involved. From our in vitro experiments showing that Hint1 disrupts the homo- and heteromeric interactions of Pontin and Reptin by competition, we currently favor a mechanism that depends on Hint1-induced conformational and/or compositional changes in transcription regulatory complexes. In this respect it was recently reported that the exotic nucleotide Ap4A by binding to Hint1 disrupts the Hint1-Mi transcription factor (MITF) complex, thereby liberating MITF to transcribe target genes (Lee et al., 2004
; Weinstein and Li, 2004
).
Analysis of Hint1 knockout mice suggested that Hint1 acts as a tumor suppressor. NMBA-treatment of these mice revealed a significant increase in squamous tumors of the forestomach compared to wild-type mice. Moreover, embryonic fibroblasts isolated from this knockout mice exhibit increased growth rates and underwent spontaneous immortalization (Su et al., 2003). Our observation that Hint1 has a repressive function in TCFß-catenin-mediated transcription now provides a molecular mechanism for this and is consistent with a tumor suppressive function of Hint1. Whether and how a potential interaction with PKC (Klein et al., 1998
) or effects on ß-catenin accessory proteins such as Legless and Pygopus are involved in the Hint1 function is currently unknown. Interestingly, the binding sites of Pontin and Legless in ß-catenin were localized to overlapping regions (Bauer et al., 1998
; Kramps et al., 2002
). Despite the widespread expression of Hint1, mouse development was not impaired in Hint1/ mice (Korsisaari et al., 2003
; Su et al., 2003
). This either can be explained by a functional compensation mediated by Hint2 or Hint3, two recently identified homologs of Hint1. This redundancy, however, apparently is not associated with dramatic upregulation of Hint2 or Hint3 transcription (Korsisaari et al., 2003
). Hint1 may be required for the maintenance of the specific functions of cell types in adult tissues where it is highly expressed, such as in cells of the stratified epithelium of the stomach, epithelial cells of the small intestine and colon, proximal tubules of the kidney and adult testis.
During recent years another HIT family member, the FHIT (fragile HIT) gene, has attracted much attention. FHIT is encoded on chromosome 3p14.2 at a locus that is subjected to frequent fragmentation. Loss of FHIT was detected in numerous carcinomas (reviewed by Huebner and Croce, 2003). Re-expression of FHIT in deficient cancer cell lines suppressed tumor formation in mice and defined it as a tumor suppressor gene (Dumon et al., 2001
; Ishii et al., 2001
; Ji et al., 1999
; Siprashvili et al., 1997
). FHIT/ mice develop normally but are highly susceptible to tumor induction in response to NMBA treatment (Fong et al., 2000
; Zanesi et al., 2001
). Further analyses indicate that FHIT mediates its tumor suppressive function by induction of apoptosis (Dumon et al., 2001
; Ji et al., 1999
; Roz et al., 2002
; Sevignani et al., 2003
). In comparison with Hint1 knockout mice, deficiency of FHIT apparently causes greater susceptibility to tumor formation. These observations suggest that the molecular basis of FHIT and Hint1 function is somehow different.
Taken together, our investigations reveal a new Hint1-mediated mode of functional modulation of ß-catenin activity and support a tumor suppressive function of Hint1. During recent years it became increasingly apparent that the downstream effects of signaling pathways are modulated in cells by a specific set of transcription factors and their co-regulators (Kim et al., 2005; Kioussi et al., 2002
). In this respect, Hint1 in association with Pontin and Reptin appears to act as a co-regulator repressing TCFß-catenin-mediated transcription suggesting a functional relationship to Wnt signal transduction.
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