extfieldsofstudy: Medicine Biology

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Endosomal Adaptor Proteins APPL1 and APPL2 Are Novel Activators of β-Catenin/TCF-mediated Transcription*

Canonical Wnt signaling regulates many aspects of cellular physiology and tissue homeostasis during development and in adult organisms. In molecular terms, stimulation by Wnt ligands leads to the stabilization of β-catenin, its translocation to the nucleus, and stimulation of TCF (T-cell factor)-dependent transcription of target genes. This process is controlled at various stages by a number of regulatory proteins, including transcriptional activators and repressors. Here we demonstrate that the endosomal proteins APPL1 and APPL2 are novel activators of β-catenin/TCF-mediated transcription. APPL proteins are multifunctional adaptors and effectors of the small GTPase Rab5, which localize to a subpopulation of early endosomes but are also capable of nucleocytoplasmic shuttling. Overexpression of APPL1 or APPL2 protein stimulates the activity of β-catenin/TCF-dependent reporter construct, whereas silencing of APPL1 reduces it. Both APPL proteins interact directly with Reptin, a transcriptional repressor binding to β-catenin and HDAC1 (histone deacetylase 1), and this interaction was mapped to the pleckstrin homology domain of APPL1. Moreover, APPL proteins are present in an endogenous complex containing Reptin, β-catenin, HDAC1, and HDAC2. Overexpression of either APPL protein relieves Reptin-dependent transcriptional repression and correlates with the reduced amounts of HDACs and β-catenin associated with Reptin as well as with the lower levels of Reptin and HDAC1 on the promoters of β-catenin target genes. We propose that APPL proteins exert their stimulatory effects on β-catenin/TCF-dependent transcription by decreasing the activity of a Reptin-containing repressive complex.

adaptor proteins, APPL1 and APPL2 (adaptor proteins containing pleckstrin homology domain, phosphotyrosine binding domain, and leucine zipper motif), which are effectors of the small GTPase Rab5, a key regulator of early steps of endocytosis (23). We have previously shown that APPL proteins localize predominantly in the cytoplasm on the surface of a distinct subpopulation of early endosomes and to a lower degree in the cell nucleus (23). They interact with a number of transmembrane receptors (TrkA, DCC, and receptors for follicle-stimulating hormone and adiponectin) (24 -27), signaling molecules (Akt, GIPC, phosphatidylinositol 3-kinase, and OCRL) (24,(27)(28)(29)(30), and nuclear proteins (NuRD (nucleosome remodeling and histone deacetylase complex)) (23). Consequently, APPL proteins not only participate in endosomal transport but appear also to be located at the crossroads of various signaling pathways regulating cell metabolism, proliferation, survival, or apoptosis.
Here we report that APPL1 and APPL2 proteins are novel positive regulators of ␤-catenin/TCF-dependent transcription. They interact directly with Reptin via their PH domains and relieve Reptin-mediated repression of ␤-catenin/TCF target genes by modulating the interactions within the ␤-catenin-Reptin-HDAC complex.

EXPERIMENTAL PROCEDURES
Cell Culture and Reagents-HEK293 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 2 mM L-glutamine, 100 units/ml penicillin, and 100 g/ml streptomycin. HeLa cells were grown in modified Eagle's medium supplemented as above. The BCA protein concentration measuring kit was from Pierce. Glutathione-Sepharose beads and pGEX-6P vectors were from GE Healthcare. Wnt3a-expressing mouse L cells and parental L cells were obtained from ATCC (CRL-2647 and CRL-2648, respectively), and conditioned media from these cell lines were obtained as recommended by the supplier. Oligonucleotides were custom-synthesized by the Institute of Biochemistry and Biophysics (Warsaw, Poland). The following antibodies were used: anti-Reptin (catalog number ab36569; Abcam), anti-HDAC1 (for immunoblotting (catalog number ab19845) and for chromatin immunoprecipitation (catalog number ab46985); Abcam), anti-Myc (catalog number 05-419; 9E10; Upstate Biotechnology, Inc.), anti-␤-catenin (catalog number 610154; BD Bioscience), anti-HA (catalog number sc-805; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-GAPDH (catalog number sc-25778; Santa Cruz Biotechnology), anti-His (catalog number 34660; Qiagen), anti-GST (catalog number 27-4577; GE Healthcare), and anti-HDAC2 (catalog number 05-814; Upstate Biotechnology). Polyclonal antibodies against recombinant APPL1 and APPL2 proteins were generated by immunization of rabbits according to standard procedures (23). Alexa-conjugated secondary antibodies used for immunofluorescence were from Invitrogen.
GST Pull-down and in Vitro Binding Assays-The full-length or truncated GST-APPL1 and GST-APPL2 fusion proteins were expressed and purified according to the manufacturer's instructions (GE Healthcare). Isopropyl-1-thio-␤-D-galactopyranoside (Sigma) at a concentration of 0.5 mM was used to induce the expression. The purified GST-APPL proteins bound to the glutathione-Sepharose beads were incubated overnight at 4°C with lysates of HEK293 cells transfected with HA-Reptin. After washing with 150 mM NaCl, 10 mM Tris, pH 8.0, 5 mM EDTA, 5 mM dithiothreitol, and 0.1% Triton X-100, the beads were collected and tested for the bound proteins by immunoblotting with anti-HA-specific antibodies.
In vitro translation reaction was carried out in a TNT coupled reticulocyte lysate using the Transcend non-radioactive translation detection system (Promega) according to the manufacturer's recommendations. For the in vitro binding reaction, 20 l of purified bead-bound GST or GST-APPL1 fragments were incubated for 4 h at 4°C in binding buffer 1 (20 mM Tris pH 8.0, 150 mM KCl, 1 mM EDTA, 4 mM MgCl 2 , 0.2% Nonidet P-40, 10% glycerol) with 3.5 l of in vitro translated Reptin. The samples were then washed three times in binding buffer 1. The bound proteins were liberated by boiling in Laemmli sample buffer and were analyzed by SDS-PAGE and immunoblotting with antibodies against Reptin.
Transient Transfection and Luciferase Assays-SuperTOPflash and SuperFOPflash luciferase reporters and plasmids encoding Renilla (pRL-SV40, pRL-TK) and ␤-catenin (pCIneo-␤-catenin) were kind gifts from Dr. Vladimir Korinek (Institute of Molecular Genetics, Prague). Typically, HEK293 cells were transfected at 50 -60% confluence with the appropriate plasmids using Lipofectamine2000 according to the manufacturer's instructions (Invitrogen). In silencing experiments, esiRNA against Reptin, APPL1, or EGFP (at a final concentration of 33 nM) were cotransfected with reporter plasmids by using Lipofectamine 2000 (Invitrogen) or SureFECT (SABiosciences). Transfected cells were collected and lysed with passive lysis buffer (Promega) after 48 -72 h. The resulting cell lysates were assayed using the dual luciferase assay kit according to the manufacturer's instructions (Promega). The firefly luciferase activity derived from the TCF-responsive reporter SuperTOPflash was normalized to its respective Renilla luciferase activity driven by the SV40 or TK promoters as a control for the transfection efficiency. For Wnt3a experiments, 30 h post-transfection, cells were treated with control conditioned or Wnt3a-conditioned medium and incubated for a further 18 h. Reporter gene activity was measured 48 h after the transfection.
Immunofluorescence-HeLa cells were plated on 12-mm coverslips in 24-well plates in 500 l of modified Eagle's medium with 10% of serum. At about 80% confluence, cells were transfected with Myc-Reptin, pEGFP-C3-Rab5-Q79L, and pCMV-Rab5-Q79L plasmids using FuGENE 6 (Roche Applied Science). 24 h post-transfection, cells were fixed with 3% paraformaldehyde, permeabilized with 0.1% Triton X-100, and processed for immunofluorescence with mouse monoclonal anti-Myc and rabbit anti-APPL1 antibodies, followed by appropriate secondary antibodies. Afterward, cells were examined with a laser-scanning confocal microscope (Leica TCS SP2 AOBS) using a ϫ63/1.4 numerical aperture oil immersion objective. Three confocal z-sections (8-bit, 1024 ϫ 1024-pixel resolution) per image were taken and processed with Leica software as maximal projections. The presented figure was assembled using Adobe Photoshop CS2.
Immunoprecipitation-HEK293 cells were homogenized in immunoprecipitation lysis buffer containing 50 mM Tris, pH 7.5, 150 mM NaCl, 2.5 mM EDTA, and 1% Nonidet P-40. Homogenates were incubated on ice for 15 min, and insoluble fractions were removed by centrifugation at 10,000 ϫ g for 10 min. Immunoprecipitation assays were performed using goat anti-mouse IgG or sheep anti-rabbit IgG Dynabeads (Invitrogen) according to the manufacturer's instructions. Collected beads were boiled in 50 l of Laemmli sample buffer for 10 min, and the precipitated proteins were analyzed by immunoblotting. Protein identification by mass spectrometry in APPL1 immunoprecipitates from HeLa cells was performed as described previously (23).
Assay for HDAC Activity-The assay was performed using the Fluor de Lys kit (Fluorogenic Histone Deacetylase Lysyl Substrate/Developer) according to the manufacturer's instructions (Biomol). Briefly, the Dynabeads containing Reptin immunocomplex were incubated with 150 l of 100 mol/liter acetylated substrates for a maximum of 30 min with gentle rocking at room temperature. As negative controls, the same amounts of rabbit IgG cross-linked Dynabeads undergoing immunoprecipitation assays were incubated with the substrates. Subsequently, equal aliquots from each condition were mixed with developer solution (100 M) and incubated for 30 min at room temperature. After excitation at 360 nm, emitted light was detected at 460 nm in a fluorometric reader (BMG Labtech). The fluorescence intensity (measured in relative fluorescence units) derived from the assay buffer was subtracted from each experimental sample.
Two-dimensional Blue Native/SDS-PAGE (2D BN/SDS-PAGE) Analysis-Blue Native/PAGE was performed according to a modification of the protocol by Schägger (34). All buffers were adjusted to pH 7.0 and filtered through 0.2-m filters. A 6 -13% gradient gel with a 3.5% stacking gel was poured in the glass plates using 1-mm spacers. The cathode buffer (15 mM BisTris/HCl, 50 mM Tricine) containing 0.02% (w/v) Coomassie Brilliant Blue G250 and the anode buffer (50 mM BisTris/HCl) were chilled to 4°C before loading. Electrophoresis was performed at 10 mA at 4°C for 1.5 h. Later, cathode buffer was replaced by the same buffer containing 0.005% Coomassie Brilliant Blue G250, and the electrophoresis was continued overnight at 8 mA. Since the dye front had run off of the gel, the lanes were cut out and incubated with 2% (w/v) SDS and 1% ␤-mercaptoethanol for 10 min at room temperature. For performing the second dimension electrophoresis, an 8% glycine-SDS gel with a 4% stacking gel was poured in glass plates (1.5-mm spacers). The excised lanes were then inserted between the glass plate assemblies and sealed with agarose (0.7% (w/v) agarose). The solution containing 1% SDS and 150 mM ␤-mercaptoethanol was added on top of the sealed strips and allowed to diffuse into the gel for 10 min. Gel plates were then placed in the electrophoretic tank and overlaid with 500 l of 2ϫ Laemmli buffer with 5% ␤-mercaptoethanol. Electrophoresis was performed at 20 mA for 1 h and then at 50 mA for 6 h.
Chromatin Immunoprecipitation Assay-HEK293 cells (10-cm confluent plate) treated with Wnt3a-conditioned medium were cross-linked by 1% paraformaldehyde for 10 min at 37°C. After removal of medium and washing with phosphate-buffered saline, extraction of chromatin was performed in lysis buffer I (5 mM Hepes, pH 7.5, 85 mM KCl, 0.5% Nonidet P-40, protease inhibitors), followed by incubation on ice for 20 min. After centrifugation at 1200 rpm for 5 min, the pellet was resuspended in 3 ml of lysis buffer II (10 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% deoxycholate, 1% Nonidet P-40, 0.1% SDS) and incubated on ice for 10 min. Fragmentation of chromatin was performed on ice by 10 rounds of sonication (10 s each) in a Sonics Vibra Cell sonicator with power setting at 50 -60%. 20 l of the sample was digested by proteinase K for 2-3 h and loaded on agarose gel for checking the chromatin fragment size, which was ϳ500 bp. Immunoprecipitation assays were performed using Dynabeads (Invitrogen) according to the manufacturer's instructions. To elute precipitated chromatin from the beads, 250 l of elution buffer (1% SDS, 0.1 M NaHCO 3 ) was added to the beads, and the mixture was incubated at room temperature for 15 min. Reversal of cross-link was made by adding 20 l of 5 M NaCl, 10 l of 500 mM EDTA, 20 l of 1 M Tris, pH 6.5, and incubating at 65°C overnight. Proteins in the DNA sample were removed by incubation with 2 l of proteinase K solution (20 mg/ml) for 2 h at 37°C. The sample was then extracted by PCR filter columns (A&A Biotech), and the DNA was dissolved in 30 l of TE buffer containing 10 g of DNase-free RNase A (Sigma), followed by incubation for 2 h at 37°C. PCR was performed from these immunoprecipitates, from chromatin processed in the absence of antibody or in the presence of unspecific IgG, as well as from input chromatin using the primer pair covering the ␤-catenin binding site of Wnt target gene promoters (cyclin D1 and Axin2). Primer sequences used are as follows: for cyclin D1, 5Ј-GCTTTCCATTCAGAGGTGTG-3Ј (forward primer) and 5Ј-CCGAAAATTCCAGCAGCAGC-3Ј (reverse primer) and for Axin2, 5Ј-CTGGAGCCGGCTGCGCTTTGA-TAA-3Ј (forward primer) and 5Ј-CGGCCCCGAAATCCATCG-CTCTGA-3Ј (reverse primer). PCR products were resolved by agarose gel electrophoresis and stained with ethidium bromide.

RESULTS
Reptin Is Detected in Complex with APPL1 and APPL2-In search of proteins interacting with APPL proteins, we performed immunoprecipitation assays from lysates of HeLa cells followed by mass spectrometry analyses. Peptides corresponding to Reptin were detected in the APPL1 immunoprecipitates, along with components of the NuRD corepressor complex (23). In order to confirm the association between Reptin and APPL proteins, we performed coimmunoprecipitation experiments using lysates of HEK293 cells transiently expressing HA-Reptin together with APPL1-Myc. Reptin was readily detectable in the anti-Myc immunoprecipitate using anti-HA antibodies (Fig. 1A). Moreover, in the lysates of HEK293 cells transiently transfected with HA-Reptin and APPL2-Myc, anti-HA antibodies coimmunoprecipitated the overexpressed APPL2 (Fig. 1B). Furthermore, we detected endogenous APPL1 in immunoprecipitates obtained by a specific anti-Reptin antibody from lysates of untransfected HEK293 cells, arguing that the interaction of Reptin and APPL1 occurs also between endogenously expressed proteins (Fig. 1C). We could not detect the binding of endogenous APPL2 with Reptin due to the very low abundance of APPL2 protein in HEK293 cells.
In order to further confirm the specificity of interactions between Reptin and APPL1/2 proteins by an independent method, in vitro GST pull-down experiments were performed (Fig. 1D). Purified GST and full-length GST-APPL1 and GST-APPL2 fusion proteins were immobilized by binding to glutathione-Sepharose beads and incubated with equal amounts of extracts from HEK293 cells transfected with HA-Reptin. Bound proteins were analyzed by Western blotting with anti-HA antibodies. HA-Reptin was specifically retained by GST-APPL1 and GST-APPL2 but not by GST alone. Together, the results of immunoprecipitation and pull-down experiments demonstrate that Reptin is found in complex with both APPL1 and APPL2 proteins.
Given that Pontin and Reptin can act together in several processes as a double hexamer complex (19), we next sought to assess the binding of Pontin and APPL1. Accordingly, coimmunoprecipitation experiments were carried out by using lysates from transiently transfected HEK293 cells expressing Myc-Pontin and HA-APPL1. Although Reptin was readily found in the anti-Myc immunoprecipitates of Pontin, no APPL1 protein was detected (Fig. 1E), indicating that APPL1 does not bind to Pontin. This further suggested that APPL proteins might regulate a function of Reptin independent of its association with Pontin.
Mapping the Reptin Interaction Domain on APPL1-To define the region of APPL1 protein responsible for the interaction with Reptin, GST pull-down assays were performed using a series of GST-APPL1 deletion constructs encompassing the various functional domains (23,35) (Fig. 2A). The well established interaction between Reptin and Pontin was used as a positive control (Fig. 2B,  lane 3). Immunoblotting of the bound material revealed that Reptin was retained only by the N terminus of APPL1 (1-428 aa containing BAR and PH domains; Fig. 2B, lane 4). However, it did not bind to the C terminus comprising the PTB domain (429 -709 aa; lane 5). By further mapping, the minimal Reptin-binding region was identified to lie between aa 273 and 428, encompassing the PH domain ( Fig. 2B lane 6), indicating that this region of APPL1 is sufficient to bind with Reptin.
Reptin Interacts Directly with Both APPL Proteins-The results of immunoprecipitation and pull-down experiments established that Reptin and APPL proteins are found in a complex; however, these interactions could occur either directly or via other proteins present in the cell lysates. In order to distinguish between these possibilities, we performed two types of experiments. First, we produced Reptin by in vitro translation and incubated it with the 273-428 aa fragment of APPL1 immobilized on glutathione-Sepharose beads. After extensive washing, the material retained on beads was analyzed by Western blot with Reptinspecific antibodies. Reptin was detected bound to GST-APPL1-(273-428) beads, whereas no background binding of Reptin was visible in the case of GST beads (Fig. 3A), indicating that Reptin binds directly to APPL1 via a region encompassing the PH domain. Second, we performed in vitro binding assays of bacterially expressed, recombinant Reptin and APPL proteins as the most stringent tests for direct protein-protein interactions. To this end, we produced His 6 -tagged Reptin immobilized on Ni 2ϩ NTA beads. Reptin-containing or empty Ni 2ϩ NTA beads were subsequently incubated with the recombinant N termini of APPL proteins (1-428 aa for APPL1 and 1-377 aa for APPL2, both fragments comprising BAR and PH domains), which were purified as GST fusion proteins, followed by the cleavage of GST (Fig.  3B). Both N-terminal fragments of APPL1 and APPL2 were specifically retained on Reptin-containing Ni 2ϩ NTA beads, whereas no unspecific binding to empty beads was observed. Taken together, these data confirm the direct physical interactions of Reptin and APPL1 as well as Reptin and APPL2.
Reptin Is Not Detected on APPLpositive Early Endosomes-Having established that Reptin and APPL proteins bind directly, we next sought to determine a possible intracellular location of these interactions. APPL proteins are known to colocalize with each other on a distinct subpopulation of early endosomes, bearing the small GTPase Rab5 but largely devoid of other markers of canonical early endosomes, such as EEA1 (23). In addition to the membrane-bound endosomal pool, a fraction of APPL proteins is also found soluble in the cytosol or in the nucleus (23). Similarly, both nuclear and cytoplasmic localization have been reported for Reptin (17,36). Since available commercial antibodies do not detect endogenous Reptin in immunofluorescence applications, we therefore analyzed the distribution of Myc-tagged overexpressed Reptin with respect to the localization of APPL1 in HeLa cells (similarly to HEK293 cells, APPL2 is expressed at low levels in HeLa cells used in this study). Immunostaining of cells revealed that overexpressed Myc-Reptin is mainly detected both in the cell nuclei and cytoplasm (Fig.  4A). However, we could not detect any enrichment of Reptin on endosomes marked by APPL1 (Fig. 4, A-C). In order to visualize better the endosomal compartments, cells were cotransfected with Myc-Reptin and the Rab5-Q79L constitutively active mutant, which causes increased fusion and thus enlarged endosomes (31). No recruitment of Myc-Reptin was observed on endosomes that were mildly enlarged by expressing low levels of GFP-Rab5-Q79L (Fig. 4, D-F). In cells overexpressing high amounts of Rab5-Q79L and thus exhibiting giant endosomes (Fig. 4, G-I), Myc-Reptin was occasionally detected on the confined domains of endosomal membranes, which were also highly enriched in APPL1. These data suggest that a small pool of Reptin might be localized to endosomes, although normally the colocalization with APPL1 in this compartment is undetectable. Overall, immunofluorescence analyses demonstrated that, in agreement with the literature, both proteins are present in cytoplasmic and nuclear pools, thus permitting their interaction in vivo.
APPL1 Is a Part of a ␤-Catenin-Reptin-HDAC Ternary Complex-In order to elucidate the functional importance of the interactions between APPL proteins and Reptin, we focused on ␤-catenin-dependent transcription as one of the processes in which Reptin does not act in complex with Pontin (14). It was previously reported that Reptin-mediated repression activity at the KAI1 promoter requires ␤-catenin for binding the promoter and HDAC1 providing deacetylase activity (20). The ability of Reptin to bind both ␤-catenin and HDAC1 further suggested the formation of a ␤-catenin-Reptin-HDAC1 complex at the promoter. In order to analyze whether APPL1 bound to Reptin could also be detected in complex containing ␤-catenin and HDAC1, we performed coimmunoprecipitation with anti-Reptin antibody using HEK293 cell lysates (Fig. 5A). Interestingly, endogenous Reptin specifically retained APPL1 along with ␤-catenin, HDAC1, and HDAC2, suggesting that APPL proteins could potentially be a part of the ternary complex. However, in this conventional immunoprecipitation assay, we could not exclude the possibility that Reptin forms independent subcomplexes with APPL1 or with ␤-catenin-HDAC1.
In order to address this question directly, 2D BN/SDS-PAGE analysis was performed (34). In 2D BN/SDS-PAGE, the positions of protein spots migrating in one vertical lane are indicative of their presence in the same complex. We separated the endogenous multiprotein complexes from HEK293 cell lysates under native conditions by 2D BN/SDS-PAGE, followed by immunoblotting for Reptin, APPL1, ␤-catenin, HDAC1, and HDAC2. These analyses revealed two distinct complexes containing APPL1 protein (Fig. 5B), one of which included Reptin, ␤-catenin, HDAC1, and HDAC2. Interestingly, the majority of APPL1 protein appeared to be involved in another independent complex also containing HDAC1/2. Cumulatively, the results of coimmunoprecipitation and 2D BN/SDS-PAGE analyses indicate that APPL1 is present in a ternary complex comprising Reptin, ␤-catenin, and HDAC1/2.
APPL Proteins Are Positive Regulators of ␤-Catenin/TCF-dependent Transcription-We next wished to determine the potential functions underlying the interaction of APPL proteins with Reptin as well as with ␤-catenin and HDACs. Based on the evidence that Reptin binds to ␤-catenin in protein complexes that regulate the transcription of ␤-catenin/TCF target genes, luciferase reporter assays were performed to test whether APPL1 and APPL2 proteins affect ␤-catenin/TCF-dependent transcriptional activity. HEK293 cells were transiently transfected with expression vectors encoding APPL1 or APPL2 in different amounts (Fig. 6A). The luciferase activities derived from the cotransfected TCF-responsive reporter (SuperTOPflash, which contains optimal TCF-binding sites upstream of a minimal promoter element) (37) or the control reporter construct (SuperFOPflash having mutated TCF-binding sites) were assayed 48 h after transfection. As shown previously, ectopic expression of ␤-catenin activated TCF-dependent transcription (38, 39) (Fig. 6A). Importantly, overexpression of either APPL1 or APPL2 increased the levels of ␤-catenin-stimulated reporter activity in a dose-dependent manner (Fig. 6A). No modulation of the SuperFOPflash reporter activity by either APPL protein was detected. A similar dose-dependent increase in the reporter activity was observed upon APPL overexpression when Wnt3a-conditioned medium was used for stimulation of cells instead of ␤-catenin co-transfection (Fig. 6C). In both cases, however, the stimulatory effect of APPL2 was consistently lower than that of APPL1. Moreover, silencing of  in HEK293 cells by two independent esiRNAs targeting different regions of the gene (Fig. 6E) resulted in the inhibition of the reporter activity, both in ␤-cateninand Wnt3a-stimulated cells (Fig. 6, B and D). Since APPL2 is practically undetectable in HEK293 cells, we could not test the effects of its knockdown on transcription. Cumulatively, these results provided a first indication that APPL proteins can act as positive regulators of ␤-catenin/TCF-dependent transcription.
We also tested the effects of APPL overexpression in HEK293 cells without any stimulation. In this case, overexpression of APPL1 moderately increased the activity of SuperTOPflash reporter, whereas overexpression of APPL2 had no effect ( Fig. 6F). At present, we cannot provide an explanation for the observed differences between the two proteins. In HEK293 cells APPL1 is a predominant form, so it is plausible that only the endogenously present protein is active in driving the reporter stimulation.
We further verified these observations by checking the expression of known ␤-catenin/TCF target genes, such as cyclin D1 (40,41), c-myc (42), and c-jun (43), upon overexpression of APPL1 or APPL2 in unstimulated or Wnt3a-treated cells by semiquantitative reverse transcription-PCR. As shown in Fig.  6G, overexpression of APPL1 but not of APPL2 increased the transcript levels of c-jun, c-myc, and cyclin D1 in unstimulated cells, thus correlating with the results of the reporter assay (Fig. 6F). Instead, in cells treated with Wnt3a-conditioned medium, overexpression of either APPL1 or APPL2 proteins caused higher expression of the Wnt target genes (Fig. 6G), again in agreement with the luciferase assay (Fig. 6C). These data indicate that the positive effects of APPL proteins on transcription are not limited to the reporter construct but are also relevant in vivo for ␤-catenin/TCF target genes.
APPL Proteins Relieve Reptin-mediated Transcriptional Repression-We next investigated the effects of the simultaneous overexpression of APPL and Reptin on ␤-catenin/ TCF-mediated transcription. Reptin overexpression inhibited the activity of the ␤-catenin-stimulated reporter, as previously reported (14), and this repression was relieved in a dose-dependent manner by adding increasing amounts of APPL1 or APPL2 (Fig. 7A). In order to further examine the interplay between Reptin and APPL proteins in the regulation of transcription, we decided to reduce the levels of Reptin by esiRNA transfection and to measure the effects of APPL1 or APPL2 overexpression under such conditions. As shown in Fig. 7B, esiRNA against Reptin (esi-Reptin) transfected in HEK293 cells reduced the levels of the protein as compared with the control (esi-EGFP). As expected, the knockdown of Reptin stimulated reporter activity above basal levels (Fig. 7C). Introduction of nonspecific esiRNA against EGFP together with increasing amounts of APPL1 protein resulted in dose-dependent stimulation of ␤-catenin/TCF- and APPL2 (aa 1-377) were purified as GST fusion proteins, followed by tag cleavage with PreScission protease. They were incubated either with empty Ni 2ϩ -NTA beads or the Ni 2ϩ -NTA beads containing purified His 6tagged Reptin. After extensive washing of the resins, bound proteins were resolved by SDS-PAGE followed by blotting with antibodies against APPL1 or APPL2 proteins. mediated transcription identical to that of the APPL1 proteins alone (compare Figs. 6A and 7C). This control demonstrated that the transfection with esiRNA per se did not affect the activity of the ␤-catenin/TCF reporter. The addition of lower amounts of APPL1 plasmid (either 0.25 or 0.5 g) on top of esi-Reptin (0.25 g) did not stimulate transcription above levels observed for esi-Reptin alone (Fig. 7C). Surprisingly, upon higher overexpression of APPL1 (1 g of plasmid), ␤-catenin/TCF reporter activity was significantly increased (Fig. 7C). These data point out that a balance between the amounts of Reptin and APPL proteins appears to be important for regulating ␤-catenin/TCFdependent transcription. We further verified this conclusion in the experiments in which we simultaneously overexpressed high doses of APPL1 (1 g) together with decreasing amounts of esi-Reptin (0.25-0.1 g). Cotransfection of 1 g of APPL1 together with 0.25 g of esi-Reptin caused the maximal 3-fold increase in the activity of the ␤-catenin-stimulated reporter, which was decreased dose-dependently upon lowering the amounts of esi-Reptin (Fig. 7D). Taken together, these data establish that APPL proteins modulate ␤-catenin/ TCF-dependent transcription along with Reptin. As positive regulators of the pathway, APPL proteins can relieve Reptin-mediated repression of transcription, and the relative abundance of both Reptin and APPL proteins determines the final level of ␤-catenin/TCF-dependent transcriptional activity.

APPL1/APPL2 Proteins Reduce the Association between ␤-Catenin, Reptin, and HDACs and Affect Their Recruitment to the Promoters of Wnt
Target Genes-The observed functional cooperation between APPL proteins and Reptin in the regulation of transcription prompted us to investigate its possible molecular mechanisms. We hypothesized that in view of their direct physical interactions with Reptin, APPL proteins could modulate the composition  . APPL1 is a part of the ␤-catenin-Reptin-HDAC1/2 complex. A, HEK293 cell lysates were subjected to a coimmunoprecipitation assay (IP) using anti-Reptin or control IgG, and the resultant precipitates were subjected to immunoblotting (IB) with the indicated antibodies. TCL, total cell lysate. B, a 2D BN/SDS-PAGE analysis of HEK293 cell extracts. Multiprotein complexes were resolved on 6 -13% acrylamide gradient gel, followed by Tris-glycine-SDS-PAGE (8% gel). Immunoblotting was performed with specific antibodies recognizing the indicated proteins. and thus repressive activity of the Reptin-containing complex. Considering that APPL1 is also present in a Reptin-independent complex containing HDACs (Fig. 5B), overexpression of APPL proteins (which relieved Reptin-mediated transcriptional repression) could potentially interfere with the binding of HDACs to the Reptin-repressive complex. To investigate such a possibility, we assessed the composition of Reptin-containing complexes upon APPL1 or APPL2 overexpression. We performed immunoprecipitation of endogenous Reptin from lysates of HEK293 cells transfected with APPL1-Myc-or APPL2-Myc-expressing plasmids in comparison with cells transfected with empty vector (Fig. 8A). Interestingly, overexpression of either APPL protein clearly reduced the levels of HDAC1 and HDAC2 associated with Reptin. We further verified this effect in a converse experiment in which we analyzed immunoprecipitates of endogenous HDAC2 (Fig. 8B). Although endogenous Reptin is found in complex with HDAC2 in mock-transfected cells, its levels are reduced in the complex isolated from cells overexpressing YFP-APPL1. These results indicate that one potential mechanism underlying the observed transcriptional stimulation by APPL proteins, both without and upon Reptin overexpression, could involve the reduction of HDAC1/2 binding with Reptin. Intriguingly, ␤-catenin was also decreased in the Reptin immunoprecipitates of lysates containing overexpressed APPL1 or APPL2 proteins (Fig. 8A), although the total levels of ␤-catenin in cells were not changed. This indicates that an increase of ␤-catenin/TCF-dependent transcription via APPL proteins probably depends also on the relative abundance of ␤-catenin as well as HDAC1/2 in the Reptin complex.
We next tested whether the reduction of HDAC1/2 binding to Reptin upon APPL overproduction correlates with the changes in Reptin-associated deacetylase activity. We isolated Reptin-bound immunocomplexes from HEK293 cells transfected with APPL1-Myc, APPL2-Myc, or empty plasmid and measured the deacetylase activity (Fig. 8C). Compared with basal levels in mock-transfected cells, overexpressed APPL proteins reduced the levels of Reptin-associated HDAC activity. These results suggest that lowering the amounts of HDACs in the Reptin-containing complex upon APPL overproduction corresponds to the decreased enzymatic activity present in this complex.
To further verify the effects of APPL overexpression upon binding of ␤-catenin and Reptin, we performed ␤-catenin immunoprecipitation from Wnt3a-stimulated HEK293 cells. We could observe that overexpression of either APPL protein reduced the amounts of Reptin bound to ␤-catenin as compared with mock-transfected cells (Fig. 9A). Finally, we tested whether the levels of APPL proteins may affect the binding of ␤-catenin, Reptin, and HDAC1 to the promoters of Wnt target genes in Wnt3a-stimulated HEK293 cells. In chromatin immunoprecipitation experiments, overexpression of either APPL protein caused an increased association of ␤-catenin with the cyclin D1 and Axin2 promoters with respect to mock-transfected cells (Fig. 9B). In contrast, the levels of Reptin and HDAC1 on the same promoters were diminished (Fig. 9B), arguing that APPL overexpression affects the recruitment of repressive complexes to the Wnt target genes.

DISCUSSION
Here we report that the endosomal adaptor proteins APPL1 and APPL2 are novel activators of ␤-catenin/TCF-dependent transcription. Both APPL proteins exhibit direct interactions with the transcriptional repressor Reptin and can counter its inhibitory effects on transcription. Furthermore, APPL proteins are components of a complex containing Reptin, HDAC1, HDAC2, and ␤-catenin. Upon overproduction, APPL proteins affect the composition of this complex, reducing the amounts of HDACs and ␤-catenin associated with Reptin. Moreover, they increase the recruitment of ␤-catenin and reduce the amounts of Reptin and HDAC1 at the promoter of Wnt target genes. We propose that APPL proteins exert their stimulatory effects on ␤-catenin/TCF-dependent transcription by decreasing the activity of Reptin-containing repressive complexes.
APPL Proteins as Activators of ␤-Catenin/TCF-dependent Transcription-APPL proteins are multifunctional adaptors primarily localized to a subpopulation of early endosomes. However, a pool of APPL proteins is also capable of undergoing nuclear translocation. In this respect, they resemble several components of the Wnt/␤-catenin pathway, which, although initially characterized for their cytoplasmic functions, do undergo nucleocytoplasmic shuttling. This is the case for Dishevelled, Dapper1, APC, Axin, GSK3␤, Bcl-9/Legless, and ␤TrCP in addition to ␤-catenin itself (44 -50). It has long been known that the levels of nuclear ␤-catenin are pivotal for its effects on transcription. However, more recent data argue that the ratios between the amounts of ␤-catenin-interacting proteins and regulators in the nucleus may be of equal importance (3).
We demonstrate that APPL proteins interact directly with Reptin, a transcriptional repressor in the Wnt/␤-catenin pathway. Reptin is known to bind ␤-catenin (14) and HDAC1 (20), and the latter interaction is crucial for providing the repressive activity of the Reptin-containing complex, as shown for the KAI1 promoter (20). We present evidence that APPL proteins are present in a complex containing Reptin, ␤-catenin, HDAC1, and HDAC2. Moreover, they are capable of modulating the composition and thus probably the activity of this complex. Based on our data, we envisage that increased levels of either APPL protein sequester Reptin and HDACs away from ␤-catenin-containing transcriptional complexes. Further studies are required to delineate the exact order of events and the possible interactions of APPL proteins within this complex. They may be of intricate nature, particularly because APPL proteins also bind the NuRD corepressor complex containing class I HDACs (23), and our data indicate that APPL1 may weakly interact in vitro with HDAC1 and HDAC2. 4 Taking into account a large number of interactions exhibited by Reptin (17), it is likely that the Reptin-associated repressive complex could involve additional components, which may contribute to its activity via interactions with other key regulatory proteins. Importantly, in a recent genome-wide RNA interference screen for the regulators of the Wnt/␤-catenin pathway, APPL2 scored as a positive regulator (51). This result, based on SuperTOPflash reporter activity in response to Wnt3a stimulation, represents an independent confirmation of our data that identify APPL proteins as activators of ␤-catenin/TCFdependent transcription.
In general, the mechanisms of regulating the activity of transcriptional repressors by modulating the amounts of associated HDACs, as we propose for APPL proteins, appear to be common in the Wnt/␤-catenin pathway. Dapper1, originally identified as a protein binding to Dishevelled (52), can associate with HDAC1 and enhance the interaction of Lef1 with HDAC1 (at the expense of Lef1 binding to ␤-catenin), thus maintaining Lef1 in the repressive state (47), as previously proposed (53). A transcriptional corepressor TIS7 inhibits the expression of ␤-catenin target genes, such as c-myc or osteopontin, by inducing the interaction of ␤-catenin with enzymatically active HDACs (54). These reports indicate that the balance between the stimulatory and inhibitory transcriptional activities of complexes containing ␤-catenin can be modulated by changing the amounts of associated repressive HDACs. This could be achieved by altering the local concentration and/or availability of ␤-catenin or HDAC-interacting partners and could result from the activity of various signaling pathways.

APPL Proteins as Dual Function Adaptors Acting in Endocytosis and
Signaling-It is becoming clear that endocytic proteins may affect signal transduction processes at multiple  1 g) or SuperFOPflash with mutated TCF-binding sites (0.1 g) were cotransfected with plasmids encoding Renilla luciferase (0.1 g), ␤-catenin (0.3 g), or APPL1 or APPL2 (0.25, 0.5, and 1 g), as indicated. After 48 h, the transfected cells were lysed, and the lysates were subjected to the luciferase activity assay. Data are normalized to the luciferase activity stimulated by ␤-catenin alone, which was arbitrarily set to 1 unit of relative activity. B, reporter gene activity was measured in ␤-cateninstimulated HEK293 cells 72 h after transient transfection of two different esiRNA (0.075 g, corresponding to 33 nM in a 96-well plate) against APPL1 (esi-APPL1-1 and esi-APPL1-2). Nonspecific esiRNA against EGFP (esi-EGFP) was used as a control. Values are normalized to the luciferase activity derived from esi-EGFP-treated cells stimulated by ␤-catenin. C, HEK293 cells transfected with the indicated plasmids were stimulated by Wnt3a-conditioned medium for 18 h, and reporter gene activity was measured 48 h after transfection. Data are normalized to the luciferase activity stimulated by Wnt3a, which was arbitrarily set to 1 unit of relative activity. D, HEK293 cells transfected with two independent esiRNA against APPL1 (esi-APPL1-1 and esi-APPL1-2) were stimulated by Wnt3a-conditioned medium for 18 h, and reporter gene activity was measured 72 h after transfection. Nonspecific esiRNA against EGFP (esi-EGFP) was used as a control. Values are normalized to the luciferase activity derived from esi-EGFP-treated, Wnt3a-stimulated cells. E, the efficiency of APPL1 knockdown was tested by Western blotting (0.075 g of esiRNA, corresponding to 33 nM in a 96-well plate). GAPDH levels indicate equal amounts of lysates loaded in each lane. F, APPL proteins were overexpressed in HEK293 cells, and reporter gene activity was measured 48 h after transfection without any stimulation. Data are normalized to the luciferase activity in unstimulated cells transfected with an empty pcDNA3.1 plasmid, which was arbitrarily set to 1 unit of relative activity. G, HEK293 cells transiently transfected with either empty plasmid (pcDNA 3.1; lane 1) or plasmids expressing APPL1 (lane 2) and APPL2 (lane 3) were either left untreated (ϪWnt3a panel) or stimulated with Wnt3a-conditioned medium for 8 h (ϩWnt3a panel). Thirty-eight hours post-transfection, cells were trypsinized, total RNA was isolated, and semiquantitative reverse transcription-PCR was performed with gene-specific primers, as indicated. Amplified samples were loaded onto 2% agarose gel and stained with ethidium bromide. Data are normalized to the luciferase activity stimulated by ␤-catenin alone, which was arbitrarily set to 1 unit of relative activity. A, plasmids encoding SuperTOPflash reporter (0.1 g), Renilla luciferase (0.1 g), ␤-catenin (0.3 g), Reptin (0.5 g), or APPL1 or APPL2 (0.05, 0.25, 0.5, and 1 g) were cotransfected into HEK293 cells, as indicated. After 48 h, the transfected cells were lysed, and the lysates were subjected to the luciferase activity assay. B, the knockdown of Reptin by esiRNA (0.25 g, corresponding to 33 nM in a 24-well plate) in HEK293 cells was analyzed by Western blot. Nonspecific esiRNA against EGFP was used as a control. GAPDH levels indicate equal amounts of lysates loaded in each lane. C, reporter gene activity was measured in HEK293 cells 72 h after transient transfection of the increasing amounts of APPL1 protein (0.25, 0.5, and 1 g) together with 0.25 g of esiRNA against EGFP or Reptin. D, APPL1 (1 g) was cotransfected together with decreasing amounts of either esi-Reptin or esi-EGFP (0.25, 0.2, 0.15, and 0.1 g). HEK293 cells were lysed 72 h after transient transfection, and reporter gene activity was measured. levels (55,56). Endocytosis has long been known to regulate the levels of plasma membrane receptors and to contribute to the down-regulation of signaling. More recently, however, it has become evident that in several cases signaling initiated at the plasma membrane can continue intracellularly from receptors internalized into the endosomal compartments (57)(58)(59). Endosomes can thus serve as platforms for active signal propagation, and many endocytic proteins appear to be bifunctional, providing a coordinated control between membrane transport and signaling events (58,60). Intriguingly, several endocytic proteins, including APPL1/2, ␤-arrestins, HIP1, Dab1/2, Eps15, Epsin1, CALM, and the components of ESCRTs (endosomal sorting complexes required for transport) shuttle to the nucleus, where they may interact with nuclear partners and modulate the levels and specificity of gene transcription (22). In most cases, it is not clear whether these proteins act as messengers transmitting signals from the cytoplasm into the nucleus or whether they "moonlight" and perform independent functions.
APPL proteins are effectors of the small GTPase Rab5, which is a master regulator of early steps in endocytosis (23). Interestingly, Rab5 itself seems to affect ␤-catenin/TCF-dependent transcription, although the results of two reports on this issue are conflicting (61,62). Here we tested whether APPL1 localized to endosomes might be involved in binding to Reptin; however, the observed lack of significant recruitment of Reptin to APPL-positive early endosomes argues against this possibility. We have previously shown that the binding of APPL1 to the endosomal membranes is strictly dependent on its interaction with Rab5, which occurs via the BAR and PH domains of APPL1 (23). In the present study, the association between APPL1 and Reptin has been mapped to the PH domain of APPL1. Interestingly, a majority of the known proteins interact with APPL1 at its C-terminal PTB domain (63), and so far, only Rab5 and Reptin appear to bind via its PH domain. This may imply that the association of APPL proteins with Reptin and Rab5 could be mutually exclusive. It is thus possible that the functions of were prepared, and a coimmunoprecipitation assay (IP) was performed with either anti-Reptin or control IgG. The resultant precipitates were analyzed by immunoblotting (IB) against the indicated antibodies (A) or were subjected to fluorometric assays for measuring the respective HDAC activities (C). Each HDAC activity assay was performed in duplicate, and relative fluorescence unit (RFU) values shown are the averages Ϯ S.D. from which the RFU values of assay buffer were subtracted. B, HEK293 cells were cotransfected with plasmids encoding YFP-tagged APPL1 or empty vector. Cell extracts were prepared and immunoprecipitated using anti-HDAC2. Bound proteins were separated and visualized by immunoblotting using anti-HDAC2, anti-HDAC1, anti-Reptin, and anti-APPL1 antibodies. A, overexpression of APPL proteins reduces the amount of Reptin bound to ␤-catenin. HEK293 cells were transiently transfected with APPL1 or APPL2. Thirty-six hours post-transfection, cells were treated with Wnt3a-conditioned medium for 4 h. Subsequently, cell extracts were prepared, and a coimmunoprecipitation assay (IP) was performed with anti-␤-catenin or control IgG. The resulting precipitates were analyzed by immunoblotting (IB) with the indicated antibodies. B, APPL proteins increase the recruitment of ␤-catenin and reduce the amounts of Reptin and HDAC1 at the Wnt target gene promoters. HEK293 cells were transfected with either empty pcDNA 3.1 (lane 1) or plasmids expressing APPL1 (lane 2) and APPL2 (lane 3). Thirty-six hours posttransfection, cells were treated with Wnt3a-conditioned medium for 4 h and then subjected to chromatin immunoprecipitation using anti-␤-catenin, anti-HDAC1, and anti-Reptin antibodies. PCR was performed from these immunoprecipitates by using the primer pair covering the ␤-catenin binding sites at the promoters of cyclin D1 and Axin2. PCR products were resolved by agarose gel and stained with ethidium bromide. Lane c, PCR mixture without template.
APPL proteins in Wnt signaling and in endocytosis are independent of each other; however, finding out whether the proper distribution of APPL proteins between endosomes and the nucleus is important for Wnt signaling will await more detailed studies. Moreover, a precise determination of internalization routes and transport kinetics of the Wnt/Frizzled/LRP5/6 ligand-receptor complex, as well as the identification of molecules governing these endocytic processes will be instrumental in understanding the relationship between endocytosis, endocytic proteins, and Wnt signaling.


extfieldsofstudy: Biology Medicine

text:
The Complex Regulation of HIC (Human I-mfa Domain Containing Protein) Expression

Human I-mfa domain containing protein (HIC) differentially regulates transcription from viral promoters. HIC affects the Wnt pathway, the JNK/SAPK pathway and the activity of positive transcription elongation factor-b (P-TEFb). Studies exploring HIC function in mammalian cells used ectopically expressed HIC due to undetected endogenous HIC protein. HIC mRNA contains exceptionally long 5′ and 3′ untranslated regions (UTRs) compared to the average length of mRNA UTRs. Here we show that HIC protein is subject to strict repression at multiple levels. The HIC mRNA UTRs reduce the expression of HIC or of a reporter protein: The HIC 3′-UTR decreases both HIC and reporter mRNA levels, whereas upstream open reading frames located in the 5′-UTR repress the translation of HIC or of the reporter protein. In addition, ectopically expressed HIC protein is degraded by the proteasome, with a half-life of approximately 1 h, suggesting that upon activation, HIC expression in cells may be transient. The strict regulation of HIC expression at the levels of mRNA stability, translation efficiency and protein stability suggests that expression of the HIC protein and its involvement in the various pathways is required only under specific cellular conditions.

Introduction
The C-terminal region of HIC contains an 81 amino acid domain, which shares 77% identity and 81% similarity with the cysteine-rich C-terminal domain of the protein I-mfa. Hence, the name HIC for Human I-mfa domain Containing protein. I-mfa (Inhibitor of MyoD Family A) inhibits the MyoD family of myogenic transcription factors [1], the Mash2 transcription factor involved in trophoblast differentiation, and TCF3 [2] and LEF1 [3], mediators of the Wnt pathway. Despite of the high homology between HIC and I-mfa, they appear to have different functions.
HIC was first identified as a protein that differentially regulates Tat-mediated and Tax-mediated expression of the human T-cell leukemia virus type I long terminal repeat (HTLV-I LTR) and the human immunodeficiency virus type I long terminal repeat (HIV-I LTR) [4][5][6]. HIC has also been reported to affect the Wnt pathway [3], the JNK/SAPK pathway [3] and the activity of positive transcription elongation factor-b (P-TEFb) [4,7,8]. Cigognini et al. recently studied chromosome 7 deletions in myeloid disorders [9]. 27% of acute myeloid leukemia (AML) and myelodysplastic syndrome (MSD) patients presented a chromosome 7 abnormality. The marker that showed the most frequent loss of heterozygosity is adjacent to HIC, hence, HIC has been proposed to be a candidate tumor suppressor gene. Although several studies have demonstrated that HIC is involved in a number of important signalling pathways and cellular processes, the exact role of HIC and the mechanism by which it affects the different pathways is still obscure. To date, studies exploring the role of HIC have been performed on over-expressed protein. No report of endogenous HIC protein has been published.
The mRNA encoding HIC contains a 590 nt 59-untranslated region (UTR), a 741 nt coding sequence, and a 3276 nt 39-UTR. Such UTRs are extremely long compared to the average length of UTRs of cellular mRNAs. The average length of the 59-UTR in human mRNAs is 125-210 nt [10,11] and the average length of the 39-UTR is 1027 [10]. Long UTRs are usually involved in posttranscriptional regulation of mRNAs.
Post-transcriptional regulation of gene expression provides a key mechanism by which cells can rapidly change gene expression patterns in response to a variety of extracellular signals and disparate biological processes. mRNA-binding proteins interact with unique sequences in mRNAs to coordinately regulate their localization, translation and/or degradation. A common feature of many rapidly degraded mRNAs is the presence of AU-rich elements (AREs) in their 39-UTRs. The sequence of this cis-element is variable, but frequently contains one or moreAUUUA pentameric motifs within or near a U-rich region [12]. Interactions between AREs and their specific binding proteins have diverse effects on target mRNAs. 59-UTRs like 39-UTRs, are deeply involved in posttranscriptional regulation of gene expression through specific mRNA motifs and RNA binding proteins. An increasing number of reports describe regulation of translation of specific mRNAs in response to specific stimuli. These mRNAs often contain a 59-UTR considerably longer than the average cellular 59-UTR [13,14], may contain AUG codons upstream of the initiation codon for the main open reading frame, and have complex secondary structures [15].
Here we show that the expression of the HIC protein is subject to strict repression, reducing its expression to undetectable levels. We demonstrate that the HIC mRNA UTRs reduce the expression of HIC or of a reporter gene in transfected cells. The HIC 59-UTR represses translation of HIC or of the reporter gene in a mechanism involving upstream open reading frames (uORFs), whereas the HIC 39-UTR decreases the mRNA level. Ectopically expressed HIC protein is degraded by the proteasome with a halflife of approximately 1 h, suggesting that HIC protein expression in cells is transient even under conditions that elevate its translation in cells.

Expression of HIC mRNA and Protein
HIC mRNA is expressed in the spleen, thymus, prostate, uterus, small intestine, peripheral blood leukocytes, but not in the testis and colon [5]. To compare expression of HIC mRNA in various human cell lines, we performed Northern blot analysis. A band of the expected size (,4600 nt) was detected in Saos-2, Karpas 299, HeLa and HF1 cells, but not in A431 or K562 cells ( Figure 1A). We could not detect endogenous HIC protein in any of these cell lines by Western blots using a rabbit polyclonal antibody that we generated against HIC (not shown and Fig. 1B, first lane). This antibody did however detect ectopic HIC over-expressed from a bicistronic vector encoding both GFP and the HIC ORF ( Figure 1B). Western blots for samples expressing ectopic HIC revealed a 32-kDa doublet band of the expected molecular weight, and a higher molecular weight doublet band. The various bands may represent covalently modified protein ( Figure 1B).

HIC Untranslated Regions (UTRs) Inhibit the Expression of a Reporter Gene
We sought to examine whether the HIC mRNA UTRs affect the expression of a luciferase reporter protein in cells. We prepared constructs encoding the Firefly luciferase (FFL) reporter gene fused to the 59-UTR of HIC (59-UTR-FFL), the full length 39-UTR (FFL-39-UTR), or just 237 nt of the 59 end of the 39-UTR (FFL-39-UTR-237). Saos-2 cells were transfected with each of the constructs. To control transfection efficiency all cells were cotransfected with a plasmid encoding Renilla luciferase (RL). 48 h after transfection the activities of FFL and RL were measured and FFL activity was normalized to RL activity. Fusion of the HIC 39-UTR downstream to FFL reduced FFL activity by 65% (Figure 2A). Fusion of the first 237 nucleotides of the 39-UTR did not have a significant effect on FFL expression. Fusion of the HIC 59-UTR upstream to FFL reduced FFL activity by 75%. Similar results were obtained in all other cell lines examined ( Figure S1).
To determine whether the decrease in FFL activity was due to reduced FFL mRNA or protein level, we purified RNA from cells expressing the UTR-FFL constructs and analyzed it on Northern blots using probes for FFL, RL and b-actin mRNAs. FFL mRNA levels were normalized to RL, as a measure of transfection efficiency and to b-actin, as a control for the amount of mRNA. mRNAs encoding FFL, RL and b-actin were detected in all samples ( Figure 2B). Quantification of the intensities of the bands showed similar amounts of mRNA encoding FFL in cells transfected with constructs coding for the FFL gene, the FFL gene fused to the first 237 nucleotides of HIC 39-UTR or the FFL gene fused to HIC 59-UTR. In contrast, mRNA encoding FFL was barely detected in cells transfected with the FFL fused to the full length HIC mRNA 39-UTR ( Fig. 1B and C). Since all mRNAs were transcribed from the same CMV promoter, these results suggest that the low level of the 39-UTR-expressing mRNA is caused by decreased mRNA stability. Both the 39-and 59-UTRs of HIC reduced FFL activity in cells ( Fig. 2A). The fact that the HIC 39-UTR but not the HIC 59-UTR reduced FFL mRNA levels implies that the UTRs inhibit HIC protein expression by two different mechanisms. The HIC 39-UTR causes a decrease in the mRNA level, most likely due to decreased mRNA stability, while the 59-UTR inhibits mRNA translation.

The 59-UTR Represses HIC Translation in a Mechanism Involving Upstream Open Reading Frames (uORFs)
To determine whether the UTRs affect HIC expression in a manner similar to their effect on reporter FFL expression, we prepared a set of expression constructs encoding different parts of the HIC gene ( Figure 3A). All constructs contained the HIC open reading frame (ORF). The HIC-FL construct contained the full length 4607 nt HIC cDNA. The HIC-ORF construct contained only the HIC coding region. The HIC-1.7 kb construct included the coding sequence as well as parts of the 59 and 39-UTRs. The HIC-59-UTR-ORF construct consisted of the 59-UTR and the coding region, and the HIC-ORF-39-UTR construct included the coding region and 39-UTR ( Figure 3A).
We first examined HIC translation in an in vitro translation system. Equal amounts of in vitro transcribed RNA from the various constructs illustrated in figure 3A were translated using a We next examined the effects of the UTRs on HIC protein levels in cells. HEK-293 cells were transfected with expression plasmids encoding the various HIC constructs (illustrated in Figure 3A). To increase the sensitivity of HIC protein detection, cells were metabolically labeled using L-[ 35 S]-methionine and L-[ 35 S]-cysteine for 11 h. HIC was immunoprecipitated from the cell extracts, subjected to SDS-PAGE and visualized by autoradiography. HIC was immunoprecipitated from cells over-expressing the HIC-ORF ( Figure 3B) and, to a lesser degree, from cells transfected with the HIC-ORF-39-UTR construct. No HIC protein was detected in cells expressing the full length 59-UTR (HIC-59UTR-ORF and HIC-full length) or from cells expressing the 39 end of the 59-UTR (HIC-1.7 kb). Furthermore, no endogenous HIC was immunoprecipitated. This data is consistent with the in vitro translation experiment, confirming that the translation of HIC is repressed by the HIC mRNA 59-UTR in transfected cells.
Although differing in their translation efficiency, mRNAs that included the HIC-59-UTR as well as mRNAs that did not include the 59-UTR were detected in the polysomal fraction in a polysomal profile analysis ( Figure 3C). Therefore, the HIC mRNA is exported to the cytoplasm and not sequestered in the nucleus. Since the HIC protein is not translated in the cells, ribosomes may also be attached to the non-coding parts of HIC mRNA.
The sequence of the HIC mRNA 59-UTR includes three short upstream open reading frames (uORFs) of 13, 8 and 25 amino acid lengths ( Figure 4A). Short uORFs in the 59-UTR affect translation efficiency of many eukaryotic genes [16][17][18][19][20][21][22]. To determine whether the potential uORFs in HIC 59-UTR are involved in inhibition of its translation, we mutated the initiation codons (AUGs) of the uORFs, rendering them non-functional for initiation of translation in a set of constructs encoding the HIC 59-UTR fused to a FFL reporter gene. The 59UTR was cloned into an NcoI site in which the ATG start codon is included, thus all the nucleotides upstream to the ATG belong to the HIC sequence. In each construct, a single upstream AUG (uAUG) initiation codon or combinations of two or three uAUGs were mutated to AUC codons. Saos-2 cells were co-transfected with the various constructs and an RL expressing plasmid as a control for transfection efficiency. Dual luciferase activity was measured 24 h after transfection. Fusion of the HIC 59-UTR upstream to FFL decreased its activity more than 5-fold ( Figure 4B). A mutation in the initiation codon of uORF1 or uORF2 alone did not affect the activity of FFL. However, a mutation that abolished the initiation codon of uORF3 increased FFL activity 1.8-fold. Although neither uORF1 nor uORF2 elimination significantly affected the translation of the reporter gene when each was mutated separately, both mutations had an effect when mutated along with another uORF (uORF1 in combination with uORF3 or uORF2+3 and uORF2 in combination with uORF3 or uORF1+3). FFL activity was further increased (2.4-fold) by mutations in the initiation codons of both uORF2 and uORF3 suggesting an additive effect of the two mutations. Moreover, mutations in all three uORFs, increased FFL activity to a higher extant than the mutations in uORF1 and uORF3 only. Therefore, uORF2 may have a positive role in the inhibition of HIC translation. Surprisingly, mutations in the initiation codons for both uORF1 and uORF3 abolished the increase in FFL activity induced by mutation of uORF3 alone, suggesting that intact uORF1 leads to enhanced translation. Similarly, mutations in all three uORFs increased FFL activity to a lower extent than the mutations in uORF2 and uORF3 only. Therefore, uORF1 may have a positive role in the regulation of HIC translation. We conclude that translation of HIC is repressed by the 59-UTR through uORF3 and uORF2. In contrast, uORF1 may have a positive role in translation of HIC mRNA.

Stability of the HIC Protein
To study HIC protein stability, we over-expressed HIC in HEK-293 cells using a construct encoding the ORF of HIC (without the UTRs) fused to myc and his tags. Expression of the HIC-myc tag-his tag following transfection of plasmid DNA was relatively low ( Figure 5A) (compared to the expression using viral infection shown in figure 1). Transfected cells were treated with the translation inhibitor cycloheximide, harvested at various time points, and the level of the myc-tagged HIC protein was determined by Western blot. The half-life of exogenous HIC in HEK-293 cells was approximately 1 h ( Figure 5A). Although the level of HIC was low when expressed from a plasmid vector, 18 h treatment with MG132, a proteasome inhibitor, significantly increased the amount of HIC detected in the cell extracts ( Figure 5B). The elevation of HIC levels in cells treated with MG132 suggests that HIC is degraded by the proteasome. Therefore, expression of the HIC protein may be transient and limited by its short half life, as well as by the regulation of its translation and RNA stability.

Discussion
Gene expression is a dynamic and tightly regulated process. A large number of transcription factors and other proteins regulating DNA methylation and chromatin structure are involved in transcriptional regulation. The transcribed mRNAs are regulated by multiple posttranscriptional mechanisms, including mRNA processing, editing, transport and stability [23][24][25]. Moreover, many mRNAs show lack of correlation between mRNA and protein levels [26][27][28], indicating that these genes are regulated at the level of translation. Changes in transcription rate as well as variations in mRNA decay rate, translation efficiency and protein stability play pivotal roles in regulation of protein expression.
HIC mRNA is expressed in several cell lines and tissues, but to date, no expression of endogenous HIC protein has been detected. Previous studies exploring the function of HIC in mammalian cells used ectopically expressed HIC. We have shown here that the expression of HIC is subject to complex regulation. The inability to detect HIC protein in these cells may be attributed to a large degree to repression of its expression. This includes repression at the levels of mRNA stability and of translation, mediated by HIC mRNA 39-and 59-UTRs. In addition, any HIC protein that is translated is rapidly degraded by the proteosome. We have demonstrated that the HIC 59-UTR represses HIC translation, whereas the HIC 39-UTR decreases its mRNA level (Figure 2, 3).
uORFs have been found to repress the translation of a number of mRNAs [16][17][18][19][20][21][22]. The presence of a few uORFs, or of an uORF which flanks or is proximal to the main ORF, greatly reduces the probability of reinitiation at the main ORF [29,30]. We found that the third uORF plays the most significant role in the inhibition of translation by the HIC mRNA 59-UTR (Figure 4), and that only when the initiation codon of this uORF was mutated, the effects of mutations in uORF1 and uORF2 could be detected. The second uORF was found to contribute to the inhibition of translation. In contrast, a mutation that abolished the first uORF decreased the effect of mutations in the following uORFs.
The finding that the first uORF has an opposite effect on translation efficiency implies that it may facilitate the reinitiation of translation in the downstream ORFs. A similar observation was reported for the translation of ATF4 (activating transcription factor 4), which is regulated by two uORFs with opposing roles [21,31]. Lu et al. [21] and Vattem and Wek [31] suggested that uORF1 facilitates ribosome scanning and increases the reinitiation efficiency at a downstream ORF, uORF2, in unstressed cells or at the more distant main ORF in stressed cells.
The diverse impact of the different uORFs can be attributed to several factors, such as the distance between the main ORF and the uORF and the AUG context of the different ORFs. The efficiency of reinitiation improves as the spacing between two ORFs is lengthened [29,30]. The distance between the termination codon of the third HIC uORF and the initiation codon of the HIC ORF is less than 40 nt. It is therefore likely that ribosomes initiating translation at the third uORF are precluded from translating HIC. The distances between the other ORFs are longer: 214 nt between uORF1 and uORF2 and 60 nt between uORF2 and uORF3 ( Figure 4A). This is consistent with our finding that their inhibitory influence on translation of the main downstream ORF is minor.
A mutation in uORF1 or uORF2 alone did not affect the activity of FFL. This could be explained by the strong inhibitory effect of uORF3. Mutations in uORF1 or uORF2 may probably affect the translation efficiency of the following uORF, but not the translation of the main ORF. Translation of the main ORF would still be limited by uORF3, which is very proximate to the main ORF.
Although mutations in the initiation codons of HIC 59-UTR enhanced translation of FFL, no combination of the mutations in the UORF was able to completely restore the activity of FFL. Thus, while the uORFs certainly have translational regulatory function, other factors found in the 59-UTR of HIC may also repress HIC translation. Another uAUG followed by a stop codon at position 127 of the 59-UTR may also contribute to the inhibition of HIC translation.
In mammals, the optimal consensus sequence for initiation is GCCA/GCCAUGG, with a purine at position 23 and a G at position +4 (underlined) being the most important nucleotides [32]. It is likely that the suboptimal context for the HIC translation initiation codon (CGGCCCAUGU) as well as the very long 59 UTR containing other features (e.g. a complex RNA secondary structure) also contribute to the low translation efficiency induced by the 59-UTR of HIC mRNA.
Fusion of the HIC 39-UTR to a FFL reporter gene decreased FFL mRNA levels in the cells ( Figure 2B). Since both the control FFL and the FFL fused to the 39-UTR were expressed from a CMV promoter, we attribute the change in mRNA levels to altered mRNA stability. When over-expressed in cells, much more HIC protein was immunoprecipitated from cells transfected with a construct expressing HIC-ORF compared to the construct expressing HIC-ORF-39-UTR ( Figure 3C). Consistent with our results, Young et al. expressed HIC from a construct that contained HIC-ORF only or from a construct that contained the HIC-ORF and the 39-UTR (both lacking the HIC 59-UTR), and detected a much lower HIC protein level when the HIC construct included the 39-UTR [33]. The half-life of an mRNA depends on sequences within the transcript itself, usually located in the 39 UTR, and on RNA binding factors that interact with those sequences [34,35]. Adenylate-uridylate rich elements (AREs) located in the 39-UTR regulate the stability of various mRNAs encoding a wide repertoire of functionally diverse proteins [36][37][38]. AREs are found within Urich regions and frequently contain one or more AUUUA motifs [36,39]. The HIC 39-UTR contains 18 repeats of the AUUUA consensus sequence, 16 of which are dispersed and two of which overlap. 35% of the HIC 39-UTR is comprised of Uridylate nucleotides, some of which are clustered in long stretches (none of the clusters is included in the HIC-1.7 kb construct). Although the presence of AUUUA motifs in an AU-rich region does not guarantee the function of this motif in mRNA stability [40], it would be interesting to determine whether the numerous AUUUA repeats confer instability to HIC mRNA. However, HIC mRNA instability does not seem to be the major regulator of HIC repression. Endogenous HIC mRNA is expressed in various cell lines and tissues, but HIC protein is not detected in any of them. Therefore, expression of HIC protein expression is probably controlled primarily at the levels of translation and protein stability.
The level of ectopically expressed HIC protein was greatly increased by treatment with the proteasome inhibitor MG132, indicating that HIC is degraded by the proteasome ( Figure 5A). The half-life of HIC is approximately 1 h ( Figure 5B). Under conditions that stimulate translation, induction of endogenous HIC expression may still be transient due to the relatively short half-life of HIC. Thus, HIC expression is also regulated posttranslationally. Under normal cellular conditions HIC is hardly translated and degradation by the proteasome may only act as a ''backup'' mechanism to prevent its expression. Nevertheless, under other yet unknown conditions in which HIC is translated, the regulation of HIC degradation may be more crucial and significant to the protein level.
HIC protein has been shown to interact with the cyclinT1 subunit of P-TEFb and with HIV-I Tat [4,8], and inhibit Tat and p-TEFb dependent transcription from the HIV promoter [4]. In contrast, expression of HIC mRNA has been shown to activate transcription from the HIV-I promoter in a p-TEFb dependent manner [33]. Young et. al. sought to resolve this discrepancy and found that the 39 end of HIC mRNA binds p-TEFb and activates it by displacing 7SK, an inhibitory small nuclear RNA (snRNA) [33]. Thus, HIC plays opposing roles in the regulation of P-TEFb: ectopically expressed HIC protein represses transcription from the HIV-I promoter while HIC mRNA elevates HIV-I promoter dependent translation. Activation of p-TEFb by HIC mRNA is presumably also required for general cellular transcription in the cell. We have evidence for a role for HIC in the stress response (submitted for publication), suggesting that HIC protein may be translated when the cell is subjected to certain forms of stress and/or as a protective mechanism against viral infection. Although the last 314 nt are sufficient for HIC mRNA to activate P-TEFb, an mRNA of 4607 nt is transcribed [33]. Inhibition of HIC translation during normal cell growth, by the long 59UTR may also be required in order to keep HIC mRNA available and functional as an RNA molecule. At times of stress, reduced activation of HIC mRNA may be coordinated with induction of translation of the HIC protein.
We have shown here that the expression of ectopically expressed HIC protein is strongly repressed at the levels of mRNA stability, translation and protein stability. We suggest that endogenous HIC is down-regulated through similar mechanisms under normal cellular conditions. The tight suppression of HIC protein at multiple levels of gene expression may be withdrawn under certain, yet unidentified, cellular conditions. Further understanding of the complex regulation of HIC expression will contribute to the understanding of the cellular roles of HIC and the cellular pathways in which it is involved.

Materials and Methods
Plasmids were prepared as detailed in Table 1. Adenoviruses Expressing HIC Adenoviruses expressing HIC were prepared as described in [42]. In brief, the HIC ORF was cloned into pAdTrack-CMV vector. pAdTrack-CMV-encoding HIC was digested with PmeI and inserted into pAdeasy using recombination in E.coli BJ5183.

Cell Culture
All tissue culture reagents were purchased from Biological Industries Beit-Haemek LTD (Israel). MG132 was purchased from Calbiochem (USA). Cycloheximide was purchased from Sigma.

Cell Transfection and Infection
Cells were transfected using polyethyleneimine (PEI) as described previously [43]. Adenoviral infection was performed as described in [42].
Northern Blot Analysis 122610 6 cells were seeded in 10 mm plates. 24-48 h after the plating, cells were transfected with the indicated plasmids. RNA was extracted from the cells using Tri-reagent (Sigma) or the RNeasy kit (Qiagen). 20 mg RNA were resolved on a 1% agarose gel containing 8% formaldehyde and capillary blotted onto a nylon membrane. The blot was then hybridized overnight at 42uC with 32 P-labeled DNA probe, prepared with the Rediprime kit (GE Healthcare, USA) and exposed to film. For densitometry, subsaturation exposures were analyzed using the NIH image 1.61 software.

Immunoblotting
Equal amounts of protein in Laemmli sample buffer [44] were resolved by SDS-PAGE and electroblotted onto nitrocellulose membranes.
Antibodies: Anti-myc tag (9E10) was purchased from Santa Cruz Biotechnology Inc. (USA), Anti-tubulin was from Sigma. The anti-HIC serum was generated by immunization of rabbits with the peptide SGAGEALAPGPVG, comprising the first 13 amino acids of HIC.
Immunoprecipitation of Radiolabeled HIC 10 6 HEK-293 cells were seeded in 10 cm plates. The following day, cells were transfected with constructs encoding various parts of the HIC gene. 24 h after transfection, cells were labelled with 100 mCi/ml of Pro-mix (L-[ 35 S]-methionine and L-[ 35 S]-cysteine) (Amersham Pharmacia) for 11 h in DMEM lacking methionine and cysteine (Gibco) supplemented with 10% dialyzed FCS. Cells were washed 3 times with PBS and lysed in 0.1% SDS, 0.5% deoxycholate, 1% NP-40 in PBS supplemented with protease inhibitors (Sigma). Protein content was quantified using the micro Bradford assay [45].
HIC was immunoprecipitated from 1 mg protein lysate using anti-HIC epitope antibody (described above) coupled to protein G Sepharose (GE healthcare, USA). 25 ml of protein G were coupled to 20 ml anti-HIC serum by incubating the mixture for 2 h at 4uC in PBS containing 5% low-fat milk. Excess antibody was removed by washing the beads extensively with PBS. Lysates were incubated with coupled beads for 16 h at 4uC. Immunocomplexes were washed 4 times with lysis buffer. Then, 26Laemmli sample buffer was added to the beads and the samples were boiled for 10 min. Immunocomplexes were resolved by SDS-PAGE and electroblotted. Nitrocellulose membranes were exposed to autoradiography.

In Vitro Translation
RNAs were transcribed in vitro with T7 polymerase from linearized pcDNA3-HIC constructs using the Riboprobe in vitro Transcription System (Promega, USA). 0.5 mg of each purified transcript was translated using a rabbit reticulocyte lysate system (Promega, USA), according to the manufacturer's instructions, in the presence of 20 mCi Pro-mix (L-[ 35 S]-methionine and L-[ 35 S]cysteine (Amersham Pharmacia Biotech) per reaction. Samples were resolved by SDS-PAGE, electroblotted onto nitrocellulose membranes, and membranes were exposed to autoradiography.

Reporter Assays
Cells were seeded in 12-well plates (6610 4 /well). 48 h after seeding cells were transfected with the indicated plasmids. A plasmid encoding Renilla luciferase (pRL-PGK) was added to each transfection mixture as a control for transfection efficiency. 24-48 h after transfection cells were lysed with passive lysis buffer (Promega, USA). Firefly and Renilla luciferase activities were measured using a dual luciferase assay kit (Promega, USA).