Download The histidine triad protein Hint1 interacts with Pontin and Reptin and

Research Article
3117
The histidine triad protein Hint1 interacts with Pontin
and Reptin and inhibits TCF–β-catenin-mediated
transcription
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])
Journal of Cell Science
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 βcatenin interaction partners that antagonistically modulate
β-catenin transcriptional activity. In this study,
Hint1/PKCI, a member of the evolutionary conserved
family of histidine triad proteins, was characterised as a
new interaction partner of Pontin and Reptin. Pull-down
assays and co-immunoprecipitation experiments show that
Hint1/PKCI directly binds to Pontin and Reptin. The
Hint1/PKCI-binding site was mapped to amino acids 214295 and 218-289 in Pontin and Reptin, respectively.
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–βcatenin transcription complex. In this context, Hint1/PKCI
acts as a negative regulator of TCF–β-catenin
transcriptional activity in Wnt-transfected cells and in
SW480 colon carcinoma cells as shown in reporter gene
Introduction
Tight regulation of the β-catenin signaling function in the Wnt
pathway is of central importance during embryonic
development and in the adult organism. De-regulation of the
pathway, detected in numerous cancers, results in an
accumulation of β-catenin in the nucleus where it interacts with
transcription factors of the LEF-1/TCF family and induces
transcription of Wnt target genes (Giles et al., 2003). This
requires efficient phosphorylation of β-catenin at its Nterminus by a dual kinase mechanism involving casein kinase
1 and glycogen-synthase kinase 3β (GSK3β), which are both
components of the β-catenin degradation complex (Polakis,
2002). During recent years, numerous additional factors
including secreted extracellular proteins, kinases, phosphatases
and chromatin remodeling factors that modulate Wnt signaling
at the levels of the Frizzled–LRP receptor complex, the βcatenin degradation complex and/or the TCF–β-catenin
transcription complex have been identified (for overview see
http://www.stanford.edu/~rnusse/wntwindow.html).
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 TCF–β-catenin transcriptional
activity (Bauer et al., 2000; Bauer et al., 1998; Rottbauer et al.,
assays. Consistent with these observations, Hint1/PKCI
represses expression of the endogenous target genes cyclin
D1 and axin2 whereas knockdown of Hint1/PKCI by RNA
interference increases their expression. Disruption of the
Pontin/Reptin complex appears to mediate this modulatory
effect of Hint1/PKCI on TCF–β-catenin-mediated
transcription. These data now provide a molecular
mechanism to explain the tumor suppressor function of
Hint1/PKCI recently suggested from the analysis of
Hint1/PKCI knockout mice.
Supplementary material available online at
http://jcs.biologists.org/cgi/content/full/118/14/3117/DC1
Key words: Histidine triad, Hint1, PKCI, TCF, β-catenin, Wnt
signaling
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
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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 β-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 β-catenin-mediated signaling (Rottbauer et al., 2002).
The Hint1/PKCI (histidine triad nucleotide-binding protein
1; synonyms: protein kinase C inhibitor 1, protein kinase Cinteracting 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 adenosylpolyphosphatehydrolase 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
TCF–β-catenin transcription complex. Hint1/PKCI disrupts
the Pontin/Reptin complex and represses TCF–β-catenin
transcriptional activity. These data provide first insight into the
molecular mechanism mediating the tumor suppressive role of
Hint1/PKCI.
Materials and Methods
Cell culture
HEK293, SW480 and HeLa cells were grown in supplemented
DMEM as reported previously (Hämmerlein et al., 2005). C57MG
and NIH3T3 cells stably expressing Wnt-1 (obtained from Jackie
Papkoff and Andreas Kispert, respectively) were cultivated in
supplemented DMEM as desribed above containing Geneticin (0.2
mg/ml) (Invitrogen). H184A1 cells were grown as reported previously
(Bojarski et al., 2004).
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 BamHI–CIP
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 TGC3′; 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 BamHI–CIP 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
HindIII–BamHI–CIP treated pFLAG-CMV4 (Sigma) and
BamHI–CIP treated pCS2+myc6 (kindly provided by Ralf Rupp). The
DNA sequences of all constructs were verified by sequencing.
Plasmids pGEX4T1-LEF-1 and pGEX4T1-β-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-α-Actinin
(clone AT6/172) antibody from Upstate (Biomol), and anti-HA (clone
12CA5) antibody from Roche. HRPO-labeled anti-mouse and antirabbit antibodies were purchased from Dianova. Alexa Fluor™488
goat anti-guinea pig IgG and Alexa Fluor™594 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 (α-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-
Hint1 inhibits TCF–β-catenin activity
Journal of Cell Science
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 ActeylADEIAKAQVARPGGDC-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 BamHI–HindIII 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 (5105) 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 β-catenin expression vectors. To
normalize transfection efficiency 0.1 µg pCH110 (β-gal) was
cotransfected. The amount of DNA for each transfection was adjusted
by addition of empty pCS2+ vector. Luciferase and β-galactosidase
activities were measured with the luciferase reporter gene assay
(constant light signal) and the chemoluminescent β-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 β-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 Siamoisluciferase 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+Pontin52FLAG (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
3119
µg), pFLAG-CMV4-Hint1 (2.5 µg), pCIneo-LEF-HA (3.0 µg) and
pCS2+β-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 Complete™-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 Complete™-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
SDS sample buffer and subsequently analysed by SDS-PAGE and
western blotting.
For competition assays GST-Pontin or GST-β-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 GSHagarose 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 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 α-23 (Bauer et al., 1998)]; anti-Myc and affinity-purified
rabbit anti-Hint1 (α-21) antibodies were diluted 1:1000; anti-MBP
(clone MBP-17) and anti-α-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 Fluor™488 goat anti-guinea pig IgG (1:1000) and Alexa
Fluor™594 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
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Journal of Cell Science 118 (14)
Journal of Cell Science
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 RTPCR 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 βactin 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, 35).
Results
Pontin and Reptin directly interact with Hint1
In a yeast two-hybrid screen using full-length Pontin as bait
(Bauer et al., 2000), Hint1/PKCI was identified as a new
interaction partner of Pontin. In the following we use the
nomenclature Hint1 for this gene to be consistent with the
official nomenclature. To verify that Hint1 is indeed associated
with Pontin, Hint1 full-length cDNA was amplified by RT-PCR
from total RNA isolated from SW480 colon carcinoma cells
and cloned into bacterial and eukaryotic expression vectors. A
direct interaction of Pontin with Hint1 was demonstrated by in
vitro pull-down experiments with purified recombinant
glutathione-S-transferase (GST)-Pontin and maltose binding
protein (MBP)-Hint1 fusion proteins. Hint1 also specifically
binds to GST-Reptin but not to GST, GST-β-catenin nor GSTLEF-1 (Fig. 1). In the opposite type of experiments, GST-Hint1
was used to pull-down MBP-Pontin or MBP-Reptin,
confirming the direct physical interaction (not shown).
Consistent with data from X-ray structural analyses (Lima et
al., 1996) GST-Hint1 was shown to dimerize with MBP-Hint1
(Fig. 1).
To map the Hint1-binding site in Pontin, a series of N- and
Fig. 1. Hint1 directly binds to Pontin and Reptin. Purified
recombinant GST-Pontin, -Reptin, -β-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-β-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).
C-terminal deletion mutants of GST-Pontin were generated.
MPB-Hint1 efficiently interacted with GST-Pontin1-297 but
no longer with GST-Pontin1-213 suggesting that the Hint1
binding site is located between amino acid 214 and 297 in
Pontin (Fig. 2A). This was confirmed in experiments with Nterminally deleted GST-Pontin constructs. Deletion of the Nterminal 146 or 210 amino acids did not affect binding of
MBP-Hint1. However, when the N-terminal 295 amino acids
were deleted, MBP-Hint1 no longer bound (Fig. 2A). In a
similar approach, the Hint1-binding site in Reptin was mapped
to amino acids 218-289 localized between the Walker A and B
motives as in Pontin (Fig. 2B). To identify the binding site of
Pontin and Reptin in Hint1, the N-terminal and C-terminal half
of Hint1 were expressed as GST fusion proteins. The GSTHint1∆C (amino acids 1-72) construct efficiently associated
with MPB-Pontin and MBP-Reptin but did not form dimers
with MBP-Hint1, whereas the GST-Hint1∆N (amino acids 66126) construct did not bind MBP-Pontin or MBP-Reptin but
interacted with MBP-Hint1 (Fig. 2C). This again is consistent
with structural data showing that the C-terminal domains of
Hint1 are involved in dimerization (Lima et al., 1996).
Hint1 inhibits TCF–β-catenin activity
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Journal of Cell Science
Fig. 2. Mapping of the
binding sites of Pontin and
Reptin in Hint1 and vice
versa. (A) C-terminal and Nterminal 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 Nterminally deleted MBPReptin fusion proteins.
(C) MBP-Pontin and -Reptin
bind to the N-terminus of
Hint1 in pull-down assays
with GST-Hint1∆C (amino
acids 1-72) (lanes 5 and 8).
The C-terminal half (amino
acids 66-126) of Hint1 (GSTHint1∆N) mediates
dimerization (lane 12).
Pontin/Reptin and Hint1 are components of high
molecular mass LEF-1–β-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 (α-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 (α21) antibody that was generated against recombinant His6tagged Hint1 protein (Fig. 3C).
Previous data indicate that the β-catenin signaling
activity is modulated by the direct interaction of β-catenin
with Pontin and/or Reptin. The direct interaction of Hint1
with Pontin and Reptin now suggests that Hint1 is a
component of a high molecular mass nucleoprotein
complex assembled around the LEF-1/TCF–β-catenin
transcription complex. To test this, co-immunoprecipitation
experiments were performed with cell lysates obtained
from transiently transfected HEK293 cells expressing βcatenin-FLAG and Hint1-myc6. Indeed, Hint1-myc6 was
readily detectable in anti-FLAG-M2 immunoprecipitates
(Fig.
4A).
Moreover,
anti-HA
antibody
coimmunoprecipitates Hint1-myc6 from HEK293 cell lysates
transiently transfected with LEF-1–HA and Hint1-myc6 (Fig.
4B). These observations suggested that Hint1 is associated
with the LEF-1–β-catenin transcription complex as a
component of a high molecular mass nucleoprotein complex
and may modulate LEF-1/TCF–β-catenin transcriptional
activity.
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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 FLAGtagged Pontin, FLAG-tagged
Reptin and myc6-tagged Hint1
alone or in combination. Protein
complexes were
immunoprecipitated with antiFLAG 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, coprecipitation was only detectable
when cells were cotransfected with
both Pontin-FLAG or ReptinFLAG and Hint1-myc6 (lanes 8 and
10). After cotransfection of Hint1FLAG 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 (α23) antibody and western blot
detection with anti-Hint1 (α-21).
**Heavy and *light chain of the
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 Hint1myc6 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
Journal of Cell Science
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
According to the association with the LEF-1–β-catenin
transcription complex, Hint1 and Pontin were expected to
localize in the nucleus. To confirm this immunofluorescence,
microscopy was performed in H184A1 cells. Endogenous
Pontin showed strong overall nuclear staining with prominent
dot-like structures. In addition, faint staining was also
detectable in the cytoplasm. With our fixation protocol,
endogenous Hint1 was predominantly localized in the nucleus
and, consistent with previous observations (Klein et al., 1998),
some staining in the cytoplasm was also detectable (Fig. 5A).
Specificity of the signals was confirmed by preincubation of
the respective antibodies with recombinant proteins resulting
in a nearly complete loss of the signals (not shown). To analyse
the nuclear localization in more detail, confocal
immunofluorescence microscopy was performed. In these
images, Hint1 is concentrated in speckled structures and Pontin
was found to co-localize with some of these Hint speckles (Fig.
5B).
Hint1 is a negative regulator of the TCF–β-catenin
transcriptional activity
To analyse whether Hint1 indeed modulates the transcriptional
activity of the TCF–β-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-4–β-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 pGL3OF 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-4–β-catenin
activity, cotransfection of Hint1 reduced the reporter gene
activity to the level of TCF-4–β-catenin alone. In the presence
of Reptin, which by itself reduces TCF-4–β-catenin
transcriptional activity, Hint1 repressed transcription further to
nearly basal levels (Fig. 6B). In these reporter assays Hint1 did
not affect CMV promoter-driven β-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 βcatenin (Aoki et al., 1999), again Hint1 did not repress activity
of the Siamois-luciferase reporter construct. In addition, a
LEF-1 construct with deleted β-catenin binding site (∆N-LEF1) did not activate transcription and was not affected by Hint1 (Fig. 6C). Taken together, this indicates that the repressive
effect of Hint1 on TCF-4–β-catenin-mediated transcription is
depending on β-catenin and does not represent a general
repressive effect on transcription.
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
reporter gene constructs in NIH3T3 and C57MG cells, stably
transfected with Wnt-1, was compared with control cells not
expressing Wnt-1. In these cells the Wnt pathway is activated
by an autocrine loop resulting in activation of TCF–β-catenindependent target gene transcription. Consistent with the results
shown above Hint1 expression in Wnt-1-transfected NIH3T3
and C57MG cells repressed transcriptional activity, whereas no
changes were observed in control cells (Fig. 6D,E). Finally, in
SW480 cells, which are mutant in APC and therefore express
high levels of nuclear and transcriptionally active β-catenin,
transfection of Hint1 similarly repressed reporter gene activity
in a dose-dependent manner (Fig. 6F).
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
3124
Journal of Cell Science 118 (14)
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 TCF–β-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
rel. activity
rel. activity
rel. activity
rel. activity
rel. activity
Fig. 6. Hint1 represses TCFA 32
B 90
pGL3-OT
4–β-catenin transcriptional
HEK 293
HEK 293
Siamois-luciferase
pGL3-OF
80
28
activity. Reporter gene assays
70
were performed in HEK293
24
60
cells transiently transfected
20
50
with different combinations of
16
40
plasmids as indicated. Data
30
represent the mean of at least
12
four independent transfections
20
8
each measured in duplicate.
10
4
(A) The improved
TCF-4 - + + + + + + + + + + + + + + +
Topflash/Fopflash reporter
β-catenin - + + + + + + + + + + + + + + +
- + - + + + +
TCF-4 - + - constructs pGL3-OT/OF were
Reptin - - - - - - + + + + + - - - - β-catenin - - + + + + +
- + used to measure the effects of
Pontin - - - - - - - - - - - + + + + +
Pontin - - + - +
- - + - + Hint1
Hint1 - + +
Hint1 on TCF-4–β-catenin
- - - + + + + transcriptional activity. Cells
were transfected with 1.0 µg
pGL3-OT/OF, 0.7 µg
D
C
pCS2+Reptin52 or
8
S5
pCS2+Pontin52, 0.5 µg
HEK 293
NIH + Wnt1
S0
25
7
NIH
pcDNAI-hTCF4, 0.5 µg
6
pCS2+β-catenin, 1 µg
20
pCS2+Hint1 and 0.1 µg
5
pCH110. (B) The repressive
4
15
effect of Hint-1 on TCF-4–β3
catenin mediated transcription
10
2
was also detectable with the
1
Siamois luciferase reporter
5
construct (1.0 µg). Increasing
S5 +
+
+
+
amounts of Hint1 (0.5 µg, 1.0
S0 +
+
+
+
+
+
+
+
LEF-1
µg, 1.5 µg, 2.0 µg) expression
Hint1 +
+
+
+
LEF-VP16 - - - - + + plasmid were transfected with
∆N-LEF-1 - - - + + + +
constant amounts of TCF-4, ββ-catenin - - - + + + - + - +
catenin and Pontin or Reptin
Hint1 - - + - + +
- + - + plasmids. Transfection of an
equal amount of empty pCS2+
vector was used as a control.
F
E
(C) LEF-VP16 (1.0 µg) driven
10
16
C57 + Wnt1
S5
SW480
transcription is not repressed by
9
C57
14
S0
Hint1. ∆N-LEF-1 (1.0 µg)
8
12
without β-catenin binding site
7
10
does not activate transcription
6
8
of the reporter gene.
5
(D) Reporter gene activity was
4
6
measured in NIH3T3 cells
3
4
stably expressing Wnt-1 and
2
2
control cells after transient
1
transfection with Hint1 (2 µg).
Hint1
+
S5 +
+
+
(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.
rel. activity
Journal of Cell Science
D1 antibody (Fig. 7C) and by reporter assays with Cyclin D1
promoter-luciferase constructs (Tetsu and McCormick, 1999)
(Fig. 7D).
If Hint1 is indeed a repressor of LEF-1/TCF–β-cateninmediated 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-
Hint1 inhibits TCF–β-catenin activity
3125
Journal of Cell Science
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
stably expressed in HeLa cells and the knockdown of Hint1
was verified by immunoblotting (Fig. 8A). When we compared
the activation of β-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-β-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 shRNAHint1430/431 vector, again enhanced luciferase activity was
detectable (Fig. 8B). Expression of endogenous target genes in
HeLa cells transiently transfected with shRNAHint1430/431 was analyzed by quantitative RT-PCR. As
shown for Axin2, expression levels were upregulated 1.95fold 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 shRNAHint1430/431 or non-functional shRNA-Hint1432/433 were
analyzed for Hint1 expression by western blotting with antiHint1 (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)
Journal of Cell Science
LEF-1. This suggests that the binding of Hint1 to Pontin and/or
Reptin may affect the homo- or heteromeric Pontin–Reptin
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 GSTPontin 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-
Fig. 9. Hint1 disrupts the PontinPontin 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 GSHagarose 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).
Pontin–Reptin-His6 or GST-Reptin–Reptin-His6 complexes
were analysed (not shown). In contrast, addition of Hint1 did
not disrupt a GST–β-catenin–Pontin-His6 complex but results
in formation of a ternary GST–β-catenin–Pontin-His6–Hint1His6 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 LEF1/TCF–β-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 Pontin–Reptin interaction sites suggesting that
Hint1 may affect Pontin/Reptin homomeric/heteromeric
complex formation or stability.
Journal of Cell Science
Hint1 inhibits TCF–β-catenin activity
Discussion
Here we show that the histidine triad protein superfamily
member Hint1 is a novel binding partner of Pontin and Reptin.
Pull-down assays with purified recombinant proteins
confirmed direct binding of Hint1 to Pontin and Reptin. The
crucial region for binding of Hint1 was localized to amino acid
residues 214-295 and 218-289 in Pontin and Reptin,
respectively, between the Walker A (amino acids 70-77) and
the Walker B (amino acids 302-305) motif. Structural analysis
of Thermotoga maritima RuvB suggested that this site is
located in a part of the protein that represents a unique insertion
in eukaryotic RuvB homologs (Pontin and Reptin). This
insertion may substitute the winged-helix DNA-binding fold
detected in the C-terminal domain III in the bacterial RuvB
protein, which is missing in the eukaryotic homolog (Putnam
et al., 2001). According to this model, our data suggest that
this insertion is directly involved in the Pontin-Pontin and
Pontin-Reptin interaction and that binding of Hint1 to this
inserted domain disrupts subunit interaction. If this insertion in
addition contains a DNA-binding fold, the association of Hint1
may also affect the DNA interaction and helicase function of
Pontin. Interestingly, the Liebeskummer mutation in zebrafish
was also mapped within this inserted domain in Reptin
(Rottbauer et al., 2002), emphasizing the functional importance
of this domain.
Co-immunopreciptitation experiments confirmed that Hint 1
forms a complex with Pontin and Reptin in cells. Moreover, βcatenin 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 β-catenin or
LEF-1 in pull-down experiments with recombinant proteins.
This suggested that Hint1 modulates β-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/TCF–β-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 TCF–βcatenin 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 136187 (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
3127
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 TCF–βcatenin-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 β-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 β-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.
3128
Journal of Cell Science 118 (14)
These observations suggest that the molecular basis of FHIT
and Hint1 function is somehow different.
Taken together, our investigations reveal a new Hint1mediated mode of functional modulation of β-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 coregulators (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 TCF–β-catenin-mediated
transcription suggesting a functional relationship to Wnt signal
transduction.
Journal of Cell Science
We thank H. Clevers, D. Kimelman, B. Vogelstein, O. Tetsu, F.
McCormick, A. Kispert, J. Papkoff for plasmids and cell lines, A.
Bauer for support in performing the two-hybrid screen, W. Reutter
and C. Heidrich for help in generating monoclonal antibodies, D.
Blottner for confocal immunofluorescence microscopy and M.
Sutherland for critically reading the manuscript. We also want to thank
K. Taira and M. Miyagishi for their help in the design of short hairpin
oligonucleotides for RNA interference. Technical assistance of B.
Kosel is gratefully acknowledged. This project was funded by the
Deutsche Forschungsgemeinschaft (SFB366/C12) and the
Sonnenfeld-Stiftung.
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