Research Article 3117 The histidine triad Hint1 interacts with Pontin and Reptin and inhibits TCF–β-catenin-mediated

Jörg Weiske and Otmar Huber* Institute of Clinical Chemistry and Pathobiochemistry, Charité Campus Benjamin Franklin, Hindenburgdamm 30, 12200 Berlin, Germany *Author for correspondence (e-mail: [email protected])

Accepted 7 April 2005 Journal of Cell Science 118, 3117-3129 Published by The Company of Biologists 2005 doi:10.1242/jcs.02437

Summary Pontin and Reptin previously were identified as nuclear β- assays. Consistent with these observations, Hint1/PKCI catenin interaction partners that antagonistically modulate represses expression of the endogenous target genes cyclin β-catenin transcriptional activity. In this study, D1 and axin2 whereas knockdown of Hint1/PKCI by RNA Hint1/PKCI, a member of the evolutionary conserved interference increases their expression. Disruption of the family of histidine triad , was characterised as a Pontin/Reptin complex appears to mediate this modulatory new interaction partner of Pontin and Reptin. Pull-down effect of Hint1/PKCI on TCF–β-catenin-mediated assays and co-immunoprecipitation experiments show that transcription. These data now provide a molecular Hint1/PKCI directly binds to Pontin and Reptin. The mechanism to explain the tumor suppressor function of Hint1/PKCI-binding site was mapped to amino acids 214- Hint1/PKCI recently suggested from the analysis of 295 and 218-289 in Pontin and Reptin, respectively. Hint1/PKCI knockout mice. Conversely, Pontin and Reptin bind to the N-terminus of Hint1/PKCI. Moreover, by its interaction with Pontin and Reptin, Hint1/PKCI is associated with the LEF-1/TCF–β- Supplementary material available online at catenin transcription complex. In this context, Hint1/PKCI http://jcs.biologists.org/cgi/content/full/118/14/3117/DC1 acts as a negative regulator of TCF–β-catenin transcriptional activity in Wnt-transfected cells and in Key words: Histidine triad, Hint1, PKCI, TCF, β-catenin, Wnt SW480 colon carcinoma cells as shown in reporter gene signaling Journal of Cell Science

Introduction 2002). Both proteins are evolutionary highly conserved and Tight regulation of the β-catenin signaling function in the Wnt their primary structures reveal characteristic Walker A and B pathway is of central importance during embryonic motifs known to bind ATP/GTP. Overall both proteins exhibit development and in the adult organism. De-regulation of the some similarity to bacterial RuvB proteins, which are involved pathway, detected in numerous cancers, results in an in DNA repair and act as branch migration motors at Holliday accumulation of β-catenin in the nucleus where it interacts with junctions. In enzymatic assays, Pontin and Reptin were transcription factors of the LEF-1/TCF family and induces reported to exhibit single-stranded DNA-stimulated ATPase transcription of Wnt target genes (Giles et al., 2003). This and ATP-dependent activity with opposite orientation requires efficient phosphorylation of β-catenin at its N- (Kanemaki et al., 1999; Makino et al., 1999). Recent data now terminus by a dual kinase mechanism involving casein kinase indicate that Pontin and Reptin act as essential components of 1 and glycogen-synthase kinase 3β (GSK3β), which are both remodeling complexes, such as the yeast Ino80 components of the β-catenin degradation complex (Polakis, ATPase (Shen et al., 2000) and the TIP60 histone acetyl 2002). During recent years, numerous additional factors transferase complex (Ikura et al., 2000). Furthermore, both including secreted extracellular proteins, kinases, phosphatases proteins associate with BAF53 (Park et al., 2002) and p400 and chromatin remodeling factors that modulate Wnt signaling (Fuchs et al., 2001), which were reported as components of at the levels of the Frizzled–LRP receptor complex, the β- numerous high molecular mass nuclear complexes. In catenin degradation complex and/or the TCF–β-catenin , Reptin was found in the Polycomb group complex transcription complex have been identified (for overview see 1, which maintains an inherited repressive state of genes http://www.stanford.edu/~rnusse/wntwindow.html). (Saurin et al., 2001). The role of Pontin and Reptin within these In this respect, Pontin (synonyms: Pontin52, TIP49a, chromatin remodeling complexes currently is not clear. Recent RuvBl1, ECP51, Tih1) and Reptin (synonyms: Reptin52, data indicate that in yeast the Pontin and Reptin homologs are TIP49b, RuvBl2, ECP54, Tih2) were previously identified as required for the correct assembly of the Ino80 complex antagonistic regulators of TCF–β-catenin transcriptional (Jónsson et al., 2004). The gene regulatory function of Reptin, activity (Bauer et al., 2000; Bauer et al., 1998; Rottbauer et al., moreover, was shown by its phosphorylation-dependent 3118 Journal of Cell Science 118 (14) association with activating transcription factor 2 (ATF2) molecular mechanism mediating the tumor suppressive role of resulting in an inhibition of ATF2 transcriptional activity (Cho Hint1/PKCI. 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 Materials and Methods β act as a regulator of -catenin-mediated neoplastic Cell culture transformation by modulating the chromatin structure at target HEK293, SW480 and HeLa cells were grown in supplemented gene promoters (Feng et al., 2003). DMEM as reported previously (Hämmerlein et al., 2005). C57MG Gene disruption in and and NIH3T3 cells stably expressing Wnt-1 (obtained from Jackie Drosophila is lethal emphasizing the essential role of Pontin Papkoff and Andreas Kispert, respectively) were cultivated in and Reptin in cell growth and early development (Bauer et al., supplemented DMEM as desribed above containing Geneticin (0.2 2000; Bauer et al., 1998; Lim et al., 2000). Analysis of mg/ml) (Invitrogen). H184A1 cells were grown as reported previously zebrafish Liebeskummer (Lik) mutant embryos encoding an (Bojarski et al., 2004). activated Reptin protein revealed that Reptin and Pontin antagonistically regulate cardiomyocyte proliferation and are Plasmids involved in growth regulation of endoderm derived tissues by β Plasmids encoding myc-, FLAG-, MBP- or GST-tagged Pontin or affecting -catenin-mediated signaling (Rottbauer et al., 2002). Reptin were described previously (Bauer et al., 2000; Bauer et al., The Hint1/PKCI (histidine triad nucleotide-binding protein 1998). Plasmids encoding Pontin deletion constructs containing 1; synonyms: protein kinase C inhibitor 1, protein kinase C- residues 1-297, 1-213, 1-147, 147-456, 213-456 or 296-456 were interacting protein 1, adenosine 5′-monophosphoramidase) generated by restriction enzyme cloning procedures. Reptin deletion protein is a member of the Hint branch of the evolutionary constructs were generated by PCR and ligated into BamHI–CIP conserved histidine triad (HIT) protein family, which is treated pGEX4T1 (Amersham Biosciences). Human Hint1 cDNA was characterized by a common His-X-His-X-His-XX motif (X, amplified from total RNA of SW480 cells by RT-PCR using hydrophobic amino acid). FHIT (fragile HIT), represents the oligonucleotides 5′-CGC GGA TCC ATG GCA GAT GAG ATT GCC-3′ (forward) and 5′-CGC GGA TCC TTA ACC AGG AGG second branch of HIT proteins and was shown to be inactive ′ in numerous carcinomas (Huebner and Croce, 2003). All CCA ATG CAT-3 (reverse). The PCR product was cloned into BamHI-CIP-treated plasmids pGEX-4T1, pMal (New England members analyzed so far show adenosylpolyphosphate- Biolabs) and pQE40 (Qiagen). Deletion constructs of Hint1 encoding hydrolase activity with different specificities for substrates amino acids 1-71 (Hint1∆C) and 66-126 (Hint1∆N) were generated such as AMP-X, ADP, ATP, Ap3A and Ap4A (for a review, see by PCR using the following primer pairs: forward oligonucleotide (see Brenner, 2002). Meanwhile, other family members of the Hint above) and 5′-GCG GGA TCC TCA TTC ATC ATC ATC TTC TGC- branch have been identified including Hint2, Hint3, Aprataxin 3′; 5′-GCG GGA TCC GCC ACC ATG GCA GAA GAT GAT GAT (Date et al., 2001; Moreira et al., 2001) and the scavenger GAA-3′ and reverse oligonucleotide (see above), respectively. The mRNA decapping enzyme DcpS (Liu et al., 2002). PCR products were cloned into BamHI–CIP treated pGEX-4T1 Less is known about the physiological and cellular functions vector. FLAG-tagged and myc-tagged variants of Hint1 were of Hint1/PKCI. The identification of Hint1/PKCI interaction generated by amplification of the Hint1 cDNA with the

Journal of Cell Science ′ partners suggested that it might be involved in the regulation of oligonucleotide pairs 5 -GCG AAT TCA AAG CTT ATG GCA GAT GAG ATT GCC-3′ and reverse oligonucleotide (see above) and transcriptional processes. In this respect, Hint1/PKCI was forward oligonucleotide (see above) and 5′-CGC GGA TCC ACC reported to interact with the transcription factor Microphthalmia AGG AGG CCA ATG CAT-3′ respectively and ligation into (Mi), which plays an important role in mast cell and melanocyte HindIII–BamHI–CIP treated pFLAG-CMV4 (Sigma) and growth and function (Razin et al., 1999). Hint1/PKCI was also BamHI–CIP treated pCS2+myc6 (kindly provided by Ralf Rupp). The shown to interact with the product of the ATDC gene, which DNA sequences of all constructs were verified by sequencing. previously was assumed to be an Ataxia-telangiectasia (AT) Plasmids pGEX4T1-LEF-1 and pGEX4T1-β-catenin were described candidate gene (Brzoska et al., 1995) and thus involved in the elsewhere (Huber et al., 1996). cellular response to ionizing radiation. Although the role of ATDC in this context is not clear, there is evidence that Hint1 Antibodies plays a role in the radiation sensitivity of cells by repressing fos Monoclonal anti-FLAG M2 and anti-MBP (clone MBP-17) antibodies transcription (Choi et al., 2000). The association of Hint1 with were obtained from Sigma, anti-Cyclin D1 (clone Ab-3) antibody cyclin-dependent kinase 7 (CDK7), furthermore, supports a from Oncogene (Calbiochem-Novabiochem GmbH), anti-α-Actinin function in growth control and transcriptional regulation (clone AT6/172) antibody from Upstate (Biomol), and anti-HA (clone (Korsisaari and Mäkelä, 2000). Recently the phenotypes of 12CA5) antibody from Roche. HRPO-labeled anti-mouse and anti- Hint1/PKCI knockout mice were reported (Korsisaari et al., rabbit antibodies were purchased from Dianova. Alexa Fluor™488 2003; Su et al., 2003). These mice develop normally and have goat anti-guinea pig IgG and Alexa Fluor™594 goat anti-mouse IgG a normal life span, however, N-nitrosomethylbenzylamine antibodies were obtained from Molecular Probes (MoBiTec). Rabbit (NMBA) treatment induces squamous tumors with higher anti-GST antibody was kindly provided by Jürgen Wienands. The α frequency than in wildtype mice (Su et al., 2003). polyclonal antibody against Pontin52 ( -23) was described previously Here, we report the identification of human Hint1 as a new (Bauer et al., 1998). Monoclonal antibodies against Pontin and Reptin were generated direct interaction partner of human Pontin and Reptin and according to standard procedures. Hybridoma clones secreting analyse the functional consequences of this interaction on the antibodies directed against Pontin and Reptin were identified by TCF–β-catenin transcription complex. Hint1/PKCI disrupts ELISA screening. The specificity of the antibodies was confirmed by the Pontin/Reptin complex and represses TCF–β-catenin western blot analysis on purified recombinant proteins. The transcriptional activity. These data provide first insight into the monoclonal anti-Pontin (clone 5G3-11) and anti-Reptin (clone 2E9- Hint1 inhibits TCF–β-catenin activity 3119

5) antibodies were purified on Protein A-sepharose (Amersham µg), pFLAG-CMV4-Hint1 (2.5 µg), pCIneo-LEF-HA (3.0 µg) and Biosciences). Both 5G3-11 and 2E9-5 monoclonal antibodies are pCS2+β-cat-FLAG (3.0 µg). After 48 hours cells were incubated with specific and do not cross-react with Reptin and Pontin, respectively, ice-cold lysis buffer [50 mM Tris pH 6.8, 150 mM NaCl, 2 mM ZnCl2, as shown in western blot analyses. Rabbit anti-Hint1 antibody was 300 mM sucrose, 0.25% (v/v) Triton X-100 and Complete™-protease generated by three sequential immunizations of rabbits with purified inhibitor mix (Roche)] for 15 minutes at 4°C. Immunoprecipitation recombinant Hint1-His6 protein mixed 1:1 with complete adjuvant from precleared cell lysates (150 µg protein) was performed with 4 (BioGenes GmbH). Antibodies directed against the peptide Acteyl- µg of the appropriate antibody pre-bound to Protein-A beads as ADEIAKAQVARPGGDC-CONH2 (A15DC) of human Hint1 were described previously (Weiske et al., 2001). To detect endogenous generated at Pineda Antikörper Service by immunization of two protein complexes, HEK293 cells were lysed with buffer E (50 mM rabbits and a guinea pig. Tris pH 6.8, 100 mM NaCl, 2 mM ZnCl2, 300 mM Sucrose, 0.25% (v/v) Triton X-100 and Complete™-protease inhibitor mix) and immunoprecipitations were performed with 20 µl rabbit anti-Pontin RNA interference (anti-23) antibody (Bauer et al., 1998) from precleared cell lysates Hairpin oligonucleotides to suppress Hint1 expression were designed (200 µg total protein) as described above. according to the recommendations given by Miyagishi et al. (Miyagishi et al., 2004) and cloned into the BamHI–HindIII sites of the vector pRNAT-H1.1/Neo (GenScript Corp.). Oligonucleotide Pull-down and competition assays sequences were: ON430, 5′-GAT CCG TGT CTT GCT TTC TAT GST and MBP fusion proteins were expressed in Escherichia coli and GAT ATT GTG CTG TCA TGT CAT GGA AAG CAA GGC ACT purified by affinity chromatography on glutathione-conjugated TTT TTG GAA A-3′; ON431, 5′-AGC TTT TCC AAA AAA GTG (GSH)-agarose beads (Sigma) or amylose resin (New England CCT TGC TTT CCA TGA CAT GAC AGC ACA ATA TCA TAG Biolabs). For pull-down assays, 5 µg of GST or GST fusion proteins AAA GCA AGA CAC G-3′; ON432, 5′-GAT CCG ATT ATC TGT were incubated with 5 µg MBP fusion proteins in binding buffer (150 AAG GAG ATA CCT GTG CTG TCG GTA TTT CCT TGC GGA mM NaCl, 50 mM Tris pH 6.8, 2 mM ZnCl2, 300 mM sucrose, 0.25% TGA TCT TTT TTG GAA A-3′; ON433, 5′-AGC TTT TCC AAA (v/v) Triton X-100) for 30 minutes at 4°C. After centrifugation (5 AAA GAT CAT CCG CAA GGA AAT ACC GAC AGC ACA GGT minutes, 4°C, 20,800 g) 35 µl of GSH-beads were added to the ATC TCC TTA CAG ATA ATC G-3′. Interferring activity of these supernatants and incubated for 1 hour at 4°C under constant agitation. oligonucleotides was tested in a reporter assay using pJOsiCheck (see GSH-beads were pelleted at 2700 g, 4°C for 1 minute and washed five supplementary material, Fig. S2), an improved variant of psiCheck times with binding buffer. The bound complexes were eluted in 2 (Promega). Further details are available on request. HeLa cells SDS sample buffer and subsequently analysed by SDS-PAGE and expressing vector-based short hairpin RNA were used for reporter western blotting. gene assays and RT-PCR analysis and were transiently or stably For competition assays GST-Pontin or GST-β-catenin fusion transfected with Fugene (Roche) according to the manufacturer’s proteins were incubated with Pontin-His6 for 30 minutes at 4°C in recommendation. 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- Transient transfections and reporter gene assays agarose beads for 30 minutes at 4°C. After three washes with buffer 5 HEK293 cells (510 ) were transfected with the calcium-phosphate C, Hint1-His6 protein was added to the complexes and incubated for method. The following amounts of expression vectors were used for 30 minutes at 4°C. GSH-beads were again pelleted by centrifugation,

Journal of Cell Science transfections: 1.0 µg of the Siamois-luciferase (S5, S0) or washed three times with buffer C and resuspended in 2 SDS sample Topflash/Fopflash (pGL3-OT/OF) reporter construct (kindley buffer. 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 β-catenin expression vectors. To Western blot analysis normalize transfection efficiency 0.1 µg pCH110 (β-gal) was Western blot analysis was performed as described previously (Weiske cotransfected. The amount of DNA for each transfection was adjusted et al., 2001). Antibodies were diluted in TST (1.5 µg/ml anti-Flag M2, by addition of empty pCS2+ vector. Luciferase and β-galactosidase 0.5 µg/ml anti-GST, 0.4 µg/ml anti-HA (clone 12CA5), 0.5 µg/ml activities were measured with the luciferase reporter gene assay anti-Cyclin D1 (clone Ab-3) and 1.0 µg/ml anti-Pontin [affinity (constant light signal) and the chemoluminescent β-Gal reporter gene purified α-23 (Bauer et al., 1998)]; anti-Myc and affinity-purified assay (Roche) in a Lumat LB9507 luminometer (Berthold rabbit anti-Hint1 (α-21) antibodies were diluted 1:1000; anti-MBP Technologies) 42 hours after transfection. Each value was obtained by (clone MBP-17) and anti-α-Actinin (clone AT6/172) were diluted double measurement and subsequent normalization of luciferase 1:4000 and 1:1000, respectively. activities with β-galactosidase activities. Transfection of C57MG cells and NIH3T3 cells was performed with LipofectaminePlus (Invitrogen) according to the manufacturer’s Immunofluorescence microscopy recommendations. SW480 cells were transfected with Metafectene Cells were grown on gelatine-coated glass coverslides for 48 hours. (Biontex GmbH). Cells were transfected for 24 h with 1.0 µg Siamois- For immunofluorescenece microscopy cells were washed with PBS, luciferase reporter construct (S5, S0) and 0.1 µg pHRL-TK (Renilla permeabilized with 0.5% (v/v) Triton X-100 for 20 minutes on ice, luciferase) (Promega). Reporter gene assays were performed with the and fixed in 3.7% (w/v) paraformaldehyde for a further 20 minutes. Dual-Luciferase Reporter Assay System (Promega). Average values Subsequently cells were gently washed again in PBS, blocked with of four independent transfection experiments are presented for all 0.1% (v/v) goat serum in PBS for 30 minutes at room temperature reporter gene assays. 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 Immunoprecipitation minutes at room temperature, washed three times and treated with HEK293 cells were transiently transfected with pCS2+Pontin52- Alexa Fluor™488 goat anti-guinea pig IgG (1:1000) and Alexa ™ FLAG (2.5 µg), pCS2+Reptin52-FLAG (2.5 µg), pCS2+Hint1-myc6 Fluor 594 goat anti-mouse IgG (1:1000) for a further 30 minutes. To (2.5 µg), pCS2+Pontin52-myc6 (2.5 µg), pCS2+Reptin52-myc6 (2.5 stain the nuclei cells were treated with DAPI (0.1 µg/ml) for 5 minutes 3120 Journal of Cell Science 118 (14)

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 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 1,25 oil objective with 4 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 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 β-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 β- actin signals. Mean values based on RT-PCRs performed on RNAs from three independent preparations are presented. Cyclin D1 protein Fig. 1. Hint1 directly binds to Pontin and Reptin. Purified levels in Hint-1- and mock-transfected SW480 cells were detected by recombinant GST-Pontin, -Reptin, -β-catenin, -LEF-1 and -Hint1 analysis of 30 mg total protein by SDS-PAGE and subsequent western fusion proteins were incubated with MPB-Hint1. Protein complexes blotting. were pulled down with GSH-agarose beads and analysed by western Quantitative RT-PCR for axin2 was performed on a LightCycler blotting with anti-MBP and anti-GST antibodies. MBP-Hint1 TM system (Roche) with the Quanti-Tect SYBR Green RT PCR Kit specifically binds to GST-Pontin (lane 4) and GST-Reptin (lane 6) (Qiagen) and oligonucleotides 5′-GAA ACT GGC AGG TGT CCA but not to GST (lane 2), GST-β-catenin (lane 8) or GST-LEF-1 (lane CGC-3′ and 5′-GCG GGA TCC TCA ATC GAT CCG CTC CAC TTT 10). GST-Hint1 associates with MBP-Hint1 (lane 12) as expected Journal of Cell Science GCC-3′ according to the manufacturer’s instructions (95°C, 20 from the crystal structure published previously. In control seconds; 62°C, 20 seconds; 72°C, 60 seconds, 35). experiments GST and GST-fusion proteins do not nonspecifically bind to MBP or MBP-Hint1 (lanes 1,2,3,5,7,9,11).

Results Pontin and Reptin directly interact with Hint1 C-terminal deletion mutants of GST-Pontin were generated. In a yeast two-hybrid screen using full-length Pontin as bait MPB-Hint1 efficiently interacted with GST-Pontin1-297 but (Bauer et al., 2000), Hint1/PKCI was identified as a new no longer with GST-Pontin1-213 suggesting that the Hint1 interaction partner of Pontin. In the following we use the binding site is located between amino acid 214 and 297 in nomenclature Hint1 for this gene to be consistent with the Pontin (Fig. 2A). This was confirmed in experiments with N- official nomenclature. To verify that Hint1 is indeed associated terminally deleted GST-Pontin constructs. Deletion of the N- with Pontin, Hint1 full-length cDNA was amplified by RT-PCR terminal 146 or 210 amino acids did not affect binding of from total RNA isolated from SW480 colon carcinoma cells MBP-Hint1. However, when the N-terminal 295 amino acids and cloned into bacterial and eukaryotic expression vectors. A were deleted, MBP-Hint1 no longer bound (Fig. 2A). In a direct interaction of Pontin with Hint1 was demonstrated by in similar approach, the Hint1-binding site in Reptin was mapped vitro pull-down experiments with purified recombinant to amino acids 218-289 localized between the Walker A and B glutathione-S-transferase (GST)-Pontin and maltose binding motives as in Pontin (Fig. 2B). To identify the binding site of protein (MBP)-Hint1 fusion proteins. Hint1 also specifically Pontin and Reptin in Hint1, the N-terminal and C-terminal half binds to GST-Reptin but not to GST, GST-β-catenin nor GST- of Hint1 were expressed as GST fusion proteins. The GST- LEF-1 (Fig. 1). In the opposite type of experiments, GST-Hint1 Hint1∆C (amino acids 1-72) construct efficiently associated was used to pull-down MBP-Pontin or MBP-Reptin, with MPB-Pontin and MBP-Reptin but did not form dimers confirming the direct physical interaction (not shown). with MBP-Hint1, whereas the GST-Hint1∆N (amino acids 66- Consistent with data from X-ray structural analyses (Lima et 126) construct did not bind MBP-Pontin or MBP-Reptin but al., 1996) GST-Hint1 was shown to dimerize with MBP-Hint1 interacted with MBP-Hint1 (Fig. 2C). This again is consistent (Fig. 1). with structural data showing that the C-terminal domains of To map the Hint1-binding site in Pontin, a series of N- and Hint1 are involved in dimerization (Lima et al., 1996). Hint1 inhibits TCF–β-catenin activity 3121

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-Hint1∆C (amino Journal of Cell Science acids 1-72) (lanes 5 and 8). The C-terminal half (amino acids 66-126) of Hint1 (GST- Hint1∆N) mediates dimerization (lane 12).

Pontin/Reptin and Hint1 are components of high Previous data indicate that the β-catenin signaling molecular mass LEF-1–β-catenin transcription complexes activity is modulated by the direct interaction of β-catenin To verify the cellular interaction of Pontin and Reptin with with Pontin and/or Reptin. The direct interaction of Hint1 Hint1, co-immunoprecipitation experiments were performed. with Pontin and Reptin now suggests that Hint1 is a HEK293 cells were transiently transfected with FLAG-tagged component of a high molecular mass nucleoprotein Pontin or Reptin and Hint1-myc6. In immunoprecipitations with complex assembled around the LEF-1/TCF–β-catenin the anti-FLAG-M2 antibody, Hint1-myc6 was readily detectable transcription complex. To test this, co-immunoprecipitation in the Pontin-FLAG and Reptin-FLAG immunocomplexes (Fig. experiments were performed with cell lysates obtained 3A). Complex formation was also detectable in the opposite type from transiently transfected HEK293 cells expressing β- of experiments when cells were transfected with FLAG-Hint1 catenin-FLAG and Hint1-myc6. Indeed, Hint1-myc6 was and Pontin-myc6 or Reptin-myc6. Moreover, Hint1 dimerization readily detectable in anti-FLAG-M2 immunoprecipitates was demonstrated in cells cotransfected with FLAG-Hint1 and (Fig. 4A). Moreover, anti-HA antibody co- Hint1-myc6 (Fig. 3B). In immunoprecipitations with the immunoprecipitates Hint1-myc6 from HEK293 cell lysates polyclonal anti-Pontin (α-23) antibody described previously transiently transfected with LEF-1–HA and Hint1-myc6 (Fig. (Bauer et al., 1998), the endogenous protein complex was 4B). These observations suggested that Hint1 is associated isolated and co-precipitating Hint1 protein was detected on with the LEF-1–β-catenin transcription complex as a western blots with an affinity-purified polyclonal anti-Hint1 (α- component of a high molecular mass nucleoprotein complex 21) antibody that was generated against recombinant His6- and may modulate LEF-1/TCF–β-catenin transcriptional tagged Hint1 protein (Fig. 3C). activity. 3122 Journal of Cell Science 118 (14)

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 (α- 23) antibody and western blot detection with anti-Hint1 (α-21). **Heavy and *light chain of the

Journal of Cell Science precipitating antibody.

Fig. 4. Hint1 is associated with the LEF-1–β-catenin transcription complex. (A) HEK293 cells were transiently transfected with FLAG-tagged β-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. Hint1 inhibits TCF–β-catenin activity 3123

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 (α-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.

Hint1 and Pontin co-localize in the cell nucleus activity to the level of TCF-4–β-catenin alone. In the presence According to the association with the LEF-1–β-catenin of Reptin, which by itself reduces TCF-4–β-catenin transcription complex, Hint1 and Pontin were expected to transcriptional activity, Hint1 repressed transcription further to localize in the nucleus. To confirm this immunofluorescence, nearly basal levels (Fig. 6B). In these reporter assays Hint1 did microscopy was performed in H184A1 cells. Endogenous not affect CMV promoter-driven β-galactosidase activity used Pontin showed strong overall nuclear staining with prominent to normalize transfection efficiency (not shown). Moreover, in dot-like structures. In addition, faint staining was also HEK293 cells transfected with LEF-VP16, a construct where detectable in the cytoplasm. With our fixation protocol, LEF-1 is directly linked to the transcriptional activation endogenous Hint1 was predominantly localized in the nucleus domain of the herpes simplex virus protein VP16 constitutively and, consistent with previous observations (Klein et al., 1998), inducing transcription of target genes in the absence of β- some staining in the cytoplasm was also detectable (Fig. 5A). catenin (Aoki et al., 1999), again Hint1 did not repress activity Specificity of the signals was confirmed by preincubation of of the Siamois-luciferase reporter construct. In addition, a the respective antibodies with recombinant proteins resulting LEF-1 construct with deleted β-catenin binding site (∆N-LEF- Journal of Cell Science in a nearly complete loss of the signals (not shown). To analyse 1) did not activate transcription and was not affected by Hint- the nuclear localization in more detail, confocal 1 (Fig. 6C). Taken together, this indicates that the repressive immunofluorescence microscopy was performed. In these effect of Hint1 on TCF-4–β-catenin-mediated transcription is images, Hint1 is concentrated in speckled structures and Pontin depending on β-catenin and does not represent a general was found to co-localize with some of these Hint speckles (Fig. repressive effect on transcription. 5B). Next we wanted to confirm these results in a more physiological system. Therefore, the effect of Hint1 expression on the transcriptional activation of the Siamois-luciferase Hint1 is a negative regulator of the TCF–β-catenin reporter gene constructs in NIH3T3 and C57MG cells, stably transcriptional activity transfected with Wnt-1, was compared with control cells not To analyse whether Hint1 indeed modulates the transcriptional expressing Wnt-1. In these cells the Wnt pathway is activated activity of the TCF–β-catenin complex, reporter gene assays by an autocrine loop resulting in activation of TCF–β-catenin- were performed using Siamois-luciferase (Brannon et al., dependent target gene transcription. Consistent with the results 1997) or the pGL3-OT/OF-luciferase (He et al., 1998) reporter shown above Hint1 expression in Wnt-1-transfected NIH3T3 constructs. As previously shown, co-expression of Pontin and C57MG cells repressed transcriptional activity, whereas no increased TCF-4–β-catenin transcriptional activity (Bauer et changes were observed in control cells (Fig. 6D,E). Finally, in al., 2000). When Hint1 was co-expressed the luciferase activity SW480 cells, which are mutant in APC and therefore express was reduced. Hint1 also represses transcriptional activity high levels of nuclear and transcriptionally active β-catenin, independent of the co-expression of Pontin by binding to transfection of Hint1 similarly repressed reporter gene activity endogenous protein. No effects were detectable with the pGL3- in a dose-dependent manner (Fig. 6F). 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 Hint1 represses Cyclin D1 expression reduction of reporter gene activity. In the presence of Pontin, The results presented so far are consistent with Hint1 as a which induces a further increase of the TCF-4–β-catenin negative regulator of Wnt target gene expression. In a next step activity, cotransfection of Hint1 reduced the reporter gene we analysed whether Hint1 modulates expression of an 3124 Journal of Cell Science 118 (14) endogenous target gene of the Wnt pathway. In this respect the D1 antibody (Fig. 7C) and by reporter assays with Cyclin D1 expression of the cell-cycle regulator Cyclin D1, a known promoter-luciferase constructs (Tetsu and McCormick, 1999) direct target gene of the TCF–β-catenin complex (Shtutman et (Fig. 7D). al., 1999; Tetsu and McCormick, 1999) was examined. If Hint1 is indeed a repressor of LEF-1/TCF–β-catenin- Transient transfection of Hint1 cDNA in SW480 cells reduced mediated transcription, silencing of its expression should cyclin D1 mRNA expression levels by approximately 50%, as enhance transcriptional activity. Short hairpin RNAs (shRNA) shown by RT-PCR, compared with mock-transfected cells (Fig. were designed and analysed for their interfering activity by 7A,B). This repression of Cyclin D1 expression was confirmed reporter screening (see supplementary material Figs S1 and at the protein level by western blot analysis with an anti-Cyclin S2). Vectors expressing an interfering shRNA (shRNA-

Fig. 6. Hint1 represses TCF- β A 32 B 90 4– -catenin transcriptional HEK 293 pGL3-OT HEK 293 Siamois-luciferase activity. Reporter gene assays 28 pGL3-OF 80 70 were performed in HEK293 24 cells transiently transfected 60 with different combinations of 20 50 plasmids as indicated. Data 16 40

rel. activity rel. 30 represent the mean of at least activity rel. 12 four independent transfections 20 8 each measured in duplicate. 10 (A) The improved 4 + + + + Topflash/Fopflash reporter TCF-4 - + + + + + + + + + + + TCF-4 - + - - - - + - + + + + β-catenin - + + + + + + + + + + + + + + + constructs pGL3-OT/OF were β-catenin - - + - - - - + + + + + Reptin ------+ + + + + - - - - - used to measure the effects of Pontin - - - + - + - - - + - + Pontin ------+ + + + + Hint1 on TCF-4–β-catenin Hint1 - - - - + + + + - - + + Hint1 transcriptional activity. Cells were transfected with 1.0 µg pGL3-OT/OF, 0.7 µg pCS2+Reptin52 or C D 8 pCS2+Pontin52, 0.5 µg HEK 293 S5 NIH + Wnt1 pcDNAI-hTCF4, 0.5 µg 25 S0 7 NIH pCS2+β-catenin, 1 µg 6 20 pCS2+Hint1 and 0.1 µg 5 pCH110. (B) The repressive 15 4 effect of Hint-1 on TCF-4–β- 3 rel. activity rel.

catenin mediated transcription activity rel. 10 Journal of Cell Science was also detectable with the 2 Siamois luciferase reporter 5 1 construct (1.0 µg). Increasing µ S5 + - + - + - + - amounts of Hint1 (0.5 g, 1.0 S0 - + - + - + - + µg, 1.5 µg, 2.0 µg) expression LEF-1 - + + - + + ------LEF-VP16 ------+ + - - - - Hint1 - - + + - - + + plasmid were transfected with ∆ β N-LEF-1 ------+ + + + constant amounts of TCF-4, - β-catenin - - - + + + - - - + - + catenin and Pontin or Reptin Hint1 - - + - - + - + - - + + plasmids. Transfection of an equal amount of empty pCS2+ vector was used as a control. E F (C) LEF-VP16 (1.0 µg) driven 10 C57 + Wnt1 16 transcription is not repressed by 9 SW480 S5 C57 14 S0 Hint1. ∆N-LEF-1 (1.0 µg) 8 12 without β-catenin binding site 7 10 does not activate transcription 6 of the reporter gene. 5 8 rel. activity rel. (D) Reporter gene activity was 4 activity rel. 6 measured in NIH3T3 cells 3 4 stably expressing Wnt-1 and 2 2 control cells after transient 1 transfection with Hint1 (2 µg). S5 + - + - + - + - Hint1 (E) Wnt-1 expressing C57MG S0 - + - + - + - + cells show reduced reporter Hint1 - - + + - - + + 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–β-catenin transcriptional activity was analysed with the Siamois luciferase reporter system. In A-C pCH110 (β-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. Hint1 inhibits TCF–β-catenin activity 3125

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. β-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. α-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.

Hint1430/431) or an inactive shRNA (shRNA-Hint1432/433) were non-functional shRNA-Hint1432/433 did not differ from control stably expressed in HeLa cells and the knockdown of Hint1 cells. In a further control, shRNA-β-catenin decreased reporter was verified by immunoblotting (Fig. 8A). When we compared gene activity. Moreover, when Hela cells stably transfected the activation of β-catenin-mediated transcription in these cells with the non-functional shRNA-Hint1432/433 were in addition with cyclin D1-luciferase reporter gene assays, Hela cells transfected transiently with the knockdown shRNA- transfected with shRNA-Hint1430/431 showed a dose-dependent Hint1430/431 vector, again enhanced luciferase activity was increase in transcriptional activity. Cells transfected with the 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- Journal of Cell Science 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).

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 β-catenin or

Fig. 8. ShRNA expression interferes with Hint1 expression and enhances TCF–β-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 (α-21) antibody. (B) Increasing amounts of interfering shRNA-Hint1430/431 increase TCF–β-catenin-mediated transcription whereas control shRNA-Hint1432/433 did not show an effect. By contrast, shRNA directed against β-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. 3126 Journal of Cell Science 118 (14)

LEF-1. This suggests that the binding of Hint1 to Pontin and/or Pontin–Reptin-His6 or GST-Reptin–Reptin-His6 complexes Reptin may affect the homo- or heteromeric Pontin–Reptin were analysed (not shown). In contrast, addition of Hint1 did interaction. To address whether this may represent a molecular not disrupt a GST–β-catenin–Pontin-His6 complex but results mechanism that contributes to the Hint1 function, competition in formation of a ternary GST–β-catenin–Pontin-His6–Hint1- assays were performed. A preformed complex of purified His6 complex (Fig. 9B). These in vitro experiments provide recombinant GST-Pontin and Pontin-His6 was isolated with good evidence that Hint1 by competing for the Pontin/Reptin GSH-agarose beads. Excess Pontin-His6 was washed away and interaction site may modulate the high molecular mass LEF- increasing amounts of Hint1-His6 were added. Subsequently the 1/TCF–β-catenin transcription complex. Consistent with this, protein complexes were pulled down again. After washing, pull-down experiments localized the Pontin/Reptin interaction protein complexes bound to the GSH-beads were analysed by sites between amino acids 215-296 and amino acids 218-289, western blotting. Indeed, increasing amounts of Hint1 resulted respectively (Fig. 9C). This observation indicates that the Hint1 in a concomitant reduction of Pontin-His6 associated with GST- association site is identical or overlaps with the homomeric and Pontin and increased binding of Hint1. Consistent with this, heteromeric Pontin–Reptin interaction sites suggesting that increasing amounts of Pontin-His6 were detected in the Hint1 may affect Pontin/Reptin homomeric/heteromeric supernatant (Fig. 9A). Similar results were obtained when GST- complex formation or stability.

Fig. 9. Hint1 disrupts the Pontin- Pontin interaction. (A) Purified recombinant GST-Pontin and Pontin-His6 fusion proteins were mixed to allow association. Journal of Cell Science 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 (α-23) and anti-Hint1 (α-21) antibodies. (B) A similar experiment was performed with GST-β-catenin and Pontin-His6. After formation and isolation of GST-β-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). Hint1 inhibits TCF–β-catenin activity 3127 Discussion Hint1 itself is not a key regulator of Cdk7 activity (Korsisaari Here we show that the histidine triad protein superfamily et al., 2003). As Pontin and Reptin are components of the member Hint1 is a novel binding partner of Pontin and Reptin. chromatin remodeling machinery it is assumed that Hint1 by Pull-down assays with purified recombinant proteins interacting with these proteins may assemble in high molecular confirmed direct binding of Hint1 to Pontin and Reptin. The mass chromatin remodeling complexes. Currently it is not crucial region for binding of Hint1 was localized to amino acid known how Hint1 may functionally modulate chromatin residues 214-295 and 218-289 in Pontin and Reptin, remodeling. One possibility is that the nucleotidyl-hydrolase respectively, between the Walker A (amino acids 70-77) and activity of Hint1 may be involved. From our in vitro the Walker B (amino acids 302-305) motif. Structural analysis experiments showing that Hint1 disrupts the homo- and of Thermotoga maritima RuvB suggested that this site is heteromeric interactions of Pontin and Reptin by competition, located in a part of the protein that represents a unique insertion we currently favor a mechanism that depends on Hint1-induced in eukaryotic RuvB homologs (Pontin and Reptin). This conformational and/or compositional changes in transcription insertion may substitute the winged-helix DNA-binding fold regulatory complexes. In this respect it was recently reported detected in the C-terminal domain III in the bacterial RuvB that the exotic nucleotide Ap4A by binding to Hint1 disrupts protein, which is missing in the eukaryotic homolog (Putnam the Hint1-Mi transcription factor (MITF) complex, thereby et al., 2001). According to this model, our data suggest that liberating MITF to transcribe target genes (Lee et al., 2004; this insertion is directly involved in the Pontin-Pontin and Weinstein and Li, 2004). Pontin-Reptin interaction and that binding of Hint1 to this Analysis of Hint1 knockout mice suggested that Hint1 acts inserted domain disrupts subunit interaction. If this insertion in as a tumor suppressor. NMBA-treatment of these mice revealed addition contains a DNA-binding fold, the association of Hint1 a significant increase in squamous tumors of the forestomach may also affect the DNA interaction and helicase function of compared to wild-type mice. Moreover, embryonic fibroblasts Pontin. Interestingly, the Liebeskummer mutation in zebrafish isolated from this knockout mice exhibit increased growth rates was also mapped within this inserted domain in Reptin and underwent spontaneous immortalization (Su et al., 2003). (Rottbauer et al., 2002), emphasizing the functional importance Our observation that Hint1 has a repressive function in TCF–β- of this domain. catenin-mediated transcription now provides a molecular Co-immunopreciptitation experiments confirmed that Hint 1 mechanism for this and is consistent with a tumor suppressive forms a complex with Pontin and Reptin in cells. Moreover, β- function of Hint1. Whether and how a potential interaction catenin and LEF-1 were able to pull down Hint1. However, this with PKC (Klein et al., 1998) or effects on β-catenin accessory interaction appears to be indirect and mediated by Pontin or proteins such as Legless and Pygopus are involved in the Hint1 Reptin since Hint1 did not associate directly with β-catenin or function is currently unknown. Interestingly, the binding sites LEF-1 in pull-down experiments with recombinant proteins. of Pontin and Legless in β-catenin were localized to This suggested that Hint1 modulates β-catenin mediated overlapping regions (Bauer et al., 1998; Kramps et al., 2002). transcriptional regulation. Indeed, reporter gene assays with Despite the widespread expression of Hint1, mouse the Siamois and Topflash/Fopflash luciferase reporter development was not impaired in Hint1–/– mice (Korsisaari et Journal of Cell Science constructs both revealed a dose-dependent repressive activity al., 2003; Su et al., 2003). This either can be explained by a of Hint-1 on LEF-1/TCF–β-catenin-induced luciferase activity, functional compensation mediated by Hint2 or Hint3, two whereas knockdown of Hint1 by RNA interference increased recently identified homologs of Hint1. This redundancy, transcriptional activity. Consistent with these observations, however, apparently is not associated with dramatic Hint1 overexpression reduced reporter gene activity in upregulation of Hint2 or Hint3 transcription (Korsisaari et al., NIH3T3 and C57MG cells stimulated by autocrine Wnt signal 2003). Hint1 may be required for the maintenance of the and in SW480 colon carcinoma cells. Further support for the specific functions of cell types in adult tissues where it is highly repressive effect of Hint1 on Wnt target gene transcription was expressed, such as in cells of the stratified epithelium of the provided by analysis of the endogenous expression of Cyclin stomach, epithelial cells of the small intestine and colon, D1 (Shtutman et al., 1999; Tetsu and McCormick, 1999) and proximal tubules of the kidney and adult testis. Axin2 (Jho et al., 2002; Lustig et al., 2002), two known TCF–β- During recent years another HIT family member, the FHIT catenin target genes. (fragile HIT) gene, has attracted much attention. FHIT is A transcriptional repressor function for Hint1 was also encoded on chromosome 3p14.2 at a locus that is subjected to reported in conjunction with other transcription factors. In this frequent fragmentation. Loss of FHIT was detected in context Hint 1 was shown to repress Micropthalmia (Mi)- and numerous carcinomas (reviewed by Huebner and Croce, 2003). FOS-dependent transcription (Choi et al., 2000; Razin et al., Re-expression of FHIT in deficient lines suppressed 1999). Moreover, Hint1 by its interaction with Pontin and tumor formation in mice and defined it as a tumor suppressor Reptin may also modulate MYC-dependent transcription. The gene (Dumon et al., 2001; Ishii et al., 2001; Ji et al., 1999; MYC-binding site in Pontin was mapped to amino acids 136- Siprashvili et al., 1997). FHIT–/– mice develop normally but are 187 (Wood et al., 2000) and thus does not overlap with the highly susceptible to tumor induction in response to NMBA Hint1 binding domain (amino acids 214-294) in Pontin. treatment (Fong et al., 2000; Zanesi et al., 2001). Further Therefore, formation of a ternary complex between MYC, analyses indicate that FHIT mediates its tumor suppressive Pontin and Hint1 appears to be possible. In addition, Pontin function by induction of apoptosis (Dumon et al., 2001; Ji et and/or Reptin by binding to Hint1 may modulate Cdk7 activity al., 1999; Roz et al., 2002; Sevignani et al., 2003). In (Korsisaari and Mäkelä, 2000). Analysis of embryonic comparison with Hint1 knockout mice, deficiency of FHIT fibroblasts from Hint1-deficient mice, however, suggested that apparently causes greater susceptibility to tumor formation. 3128 Journal of Cell Science 118 (14)

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