HIC-5 Is a Novel Repressor of Lymphoid Enhancer Factor/T-cell Factor-driven Transcription
2005; Elsevier BV; Volume: 281; Issue: 3 Linguagem: Inglês
10.1074/jbc.m505869200
ISSN1083-351X
AutoresStephen Mbigha Ghogomu, Stephanie van Venrooy, Martin Ritthaler, Doris Wedlich, Dietmar Gradl,
Tópico(s)RNA Research and Splicing
ResumoActivation of Wnt/β-catenin target genes is regulated by a heterodimer of β-catenin and the high mobility group box transcription factors of the lymphoid enhancer factor (LEF)/T-cell factor (TCF) family. In vertebrates, four LEF/TCF family members have been identified. They all contain a conserved β-catenin-binding motif at the N terminus and a highly conserved high mobility group box for DNA binding. The core sequence between these motifs is less conserved and contributes to the specific properties of the individual family members. To identify interacting proteins that allocate specific functions to the individual LEF/TCF transcription factors, we performed a yeast two-hybrid screen using the less conserved core sequence as bait. We isolated the murine LIM protein HIC-5 (hydrogen peroxide-induced clone 5; also termed ARA-55 (androgen receptor activator of 55 kDa)) and cloned the highly conserved Xenopus homolog. In addition, we report that the LIM domain-containing C-terminal half of HIC-5 binds to a conserved alternatively spliced exon in LEF/TCF transcription factors. Our functional analyses revealed that HIC-5 acts as negative regulator of a subset of LEF/TCF family members, which have been characterized as activators in reporter gene analyses and in the Xenopus axis induction assay. In addition, we observed a repressive interference of LEF/TCF family members with HIC-5-mediated activation of glucocorticoid-driven transcription, which again could be allocated to specific LEF/TCF subtypes. With the characterization of HIC-5 as a binding partner of the alternatively spliced exon in LEF/TCF transcription factors, we identified a novel molecular mechanism in the dialog of steroid and canonical Wnt signaling that is LEF/TCF subtype-dependent. Activation of Wnt/β-catenin target genes is regulated by a heterodimer of β-catenin and the high mobility group box transcription factors of the lymphoid enhancer factor (LEF)/T-cell factor (TCF) family. In vertebrates, four LEF/TCF family members have been identified. They all contain a conserved β-catenin-binding motif at the N terminus and a highly conserved high mobility group box for DNA binding. The core sequence between these motifs is less conserved and contributes to the specific properties of the individual family members. To identify interacting proteins that allocate specific functions to the individual LEF/TCF transcription factors, we performed a yeast two-hybrid screen using the less conserved core sequence as bait. We isolated the murine LIM protein HIC-5 (hydrogen peroxide-induced clone 5; also termed ARA-55 (androgen receptor activator of 55 kDa)) and cloned the highly conserved Xenopus homolog. In addition, we report that the LIM domain-containing C-terminal half of HIC-5 binds to a conserved alternatively spliced exon in LEF/TCF transcription factors. Our functional analyses revealed that HIC-5 acts as negative regulator of a subset of LEF/TCF family members, which have been characterized as activators in reporter gene analyses and in the Xenopus axis induction assay. In addition, we observed a repressive interference of LEF/TCF family members with HIC-5-mediated activation of glucocorticoid-driven transcription, which again could be allocated to specific LEF/TCF subtypes. With the characterization of HIC-5 as a binding partner of the alternatively spliced exon in LEF/TCF transcription factors, we identified a novel molecular mechanism in the dialog of steroid and canonical Wnt signaling that is LEF/TCF subtype-dependent. The four vertebrate lymphoid enhancer factor (LEF) 2The abbreviations used are: LEFlymphoid enhancer factorTCFT-cell factorHMGhigh mobility groupPPARγperoxisome proliferator-activated receptor γXXenopusmmurinehhumanGSTglutathione S-transferaseMMTVmouse mammary tumor virusHEKhuman embryonic kidneyCMVcytomegalovirus. 2The abbreviations used are: LEFlymphoid enhancer factorTCFT-cell factorHMGhigh mobility groupPPARγperoxisome proliferator-activated receptor γXXenopusmmurinehhumanGSTglutathione S-transferaseMMTVmouse mammary tumor virusHEKhuman embryonic kidneyCMVcytomegalovirus./T-cell factor (TCF) transcription factors TCF-1, TCF-3, TCF-4, and LEF-1 are the nuclear transducers of an activated Wnt/β-catenin pathway. They all contain a highly conserved β-catenin-binding domain and an even more conserved DNA-binding site, the high mobility group (HMG) box. In general, they are activated by recruiting the coactivator β-catenin, which is thought to replace the repressor Groucho (available at www.stanford.edu/~rnusse/wntwindow.html) (1Logan C.Y. Nusse R. Annu. Rev. Cell Dev. Biol. 2004; 20: 781-810Crossref PubMed Scopus (4137) Google Scholar, 2Daniels D.L. Weis W.I. Nat. Struct. Mol. Biol. 2005; 4: 364-371Crossref Scopus (419) Google Scholar). Thereby, LEF/TCF target genes that are repressed in the absence of Wnt/β-catenin signaling become activated. Apart from a complex regulatory network in the cytoplasm that controls β-catenin stability and binding behavior, modulator proteins in the nucleus further decide the cell competence to respond to canonical Wnt signaling. In addition, the Wnt/β-catenin pathway is influenced by cross-talk with other signaling cascades, including transforming growth factor-β/SMAD (3Nishita M. Hashimoto M.K. Ogata S. Laurent M.N. Ueno N. Shibuya H. Cho K.W. Nature. 2000; 403: 781-785Crossref PubMed Scopus (394) Google Scholar), transforming growth factor-β/Nemo-like kinase (4Ishitani T. Ninomiya-Tsuji J. Matsumoto K. Mol. Cell. Biol. 2003; 23: 1379-1389Crossref PubMed Scopus (181) Google Scholar), and Delta/Notch (5Galceran J. Sustmann C. Hsu S.-C. Folberth S. Grosschedl R. Genes Dev. 2004; 18: 2718-2723Crossref PubMed Scopus (152) Google Scholar), and by protein modulators such as ALY (6Hsu S.-C. Galceran J. Grosschedl R. Mol. Cell. Biol. 1998; 18: 4807-4818Crossref PubMed Scopus (335) Google Scholar) and PIAS (7Sachdev S. Bruhn L. Sieber H. Pichler A. Melchior F. Grosschedl R. Genes Dev. 2001; 15: 3088-3103Crossref PubMed Scopus (462) Google Scholar). lymphoid enhancer factor T-cell factor high mobility group peroxisome proliferator-activated receptor γ Xenopus murine human glutathione S-transferase mouse mammary tumor virus human embryonic kidney cytomegalovirus. lymphoid enhancer factor T-cell factor high mobility group peroxisome proliferator-activated receptor γ Xenopus murine human glutathione S-transferase mouse mammary tumor virus human embryonic kidney cytomegalovirus. Although the general view of LEF/TCF action is well understood, the functional specificity of individual LEF/TCF proteins are often ignored. Most of the LEF/TCF-binding proteins mentioned above do not discriminate between the different family members or splice variants. Furthermore, proteins such as CtBP and ALY that bind selectively to individual LEF/TCF proteins cannot completely explain their functional differences. For example, CtBP binds to PLSL(T/V) motifs in the C termini of TCF-3 and TCF-4E, resulting in repression of target genes (8Valenta T. Lukas J. Korinek V. Nucleic Acids Res. 2003; 31: 2369-2380Crossref PubMed Scopus (100) Google Scholar, 9Brannon M. Brown J.D. Bates R. Kimelman D. Moon R.T. Development (Camb.). 1999; 126: 3159-3170Crossref PubMed Google Scholar). But even after depletion of the CtBP-binding site, XTCF-3 still neither activates target genes nor induces a secondary axis in Xenopus embryos (10Gradl D. König A. Wedlich D. J. Biol. Chem. 2002; 277: 14159-14171Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Recently, we have shown that the core region between the β-catenin-binding domain and the HMG box confers specific properties to individual LEF/TCF transcriptions factors (10Gradl D. König A. Wedlich D. J. Biol. Chem. 2002; 277: 14159-14171Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). This region has formerly been described as the interaction domain for Groucho (11Levanon D. Goldstein R.E. Bernstein Y. Tang H. Goldenberg D. Stifani S. Paroush Z. Groner Y. Proc. Nat. Acad. Sci. U. S. A. 1998; 95: 11590-11595Crossref PubMed Scopus (407) Google Scholar, 12Brantjes H. Roose J. van de Werering M. Clevers H. Nucleic Acids Res. 2001; 29: 1410-1419Crossref PubMed Scopus (308) Google Scholar) and ALY (6Hsu S.-C. Galceran J. Grosschedl R. Mol. Cell. Biol. 1998; 18: 4807-4818Crossref PubMed Scopus (335) Google Scholar). It contains a highly conserved alternatively spliced exon, which is termed exon IVa in TCF-1, exon VI in LEF-1, and exon VIII in TCF-4. Interestingly, a Xenopus LEF-1 RNA containing this exon has not been reported so far. Two small repressive peptide motifs adjacent to this exon are alternatively expressed in TCF-4 (10Gradl D. König A. Wedlich D. J. Biol. Chem. 2002; 277: 14159-14171Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 13Pukrop T. Gradl D. Henningfeld K. Knöchel W. Wedlich D. Kühl M. J. Biol. Chem. 2001; 276: 8968-8978Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). With the exception of this conserved exon, the sequence between the β-catenin-binding domain and the HMG box is less conserved and only poorly characterized. In addition to the modulators of LEF/TCF activity mentioned above, steroid receptors not only bind β-catenin (14Yang F. Li X. Sharma M. Sasaki C.Y. Longo D.L. Lim B. Sun Z. J. Biol. Chem. 2002; 277: 11336-11344Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar, 15Song L.-N. Herrell R. Byers S. Shah S. Wilson E.M. Gelmann E.P. Mol. Cell. Biol. 2003; 23: 1674-1687Crossref PubMed Scopus (142) Google Scholar), but also interact directly with TCF proteins (16Amir A.L. Barua M. McKnight N.C. Cheng S. Yuan X. Balk S.P. J. Biol. Chem. 2003; 278: 30828-30834Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 17El-Tanani M. Fernig D.G. Barraclough R. Green C. Rudland P. J. Biol. Chem. 2001; 276: 41675-41682Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 18Smith E. Frenkel B. J. Biol. Chem. 2005; 280: 2388-2394Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar). Steroid receptors belong to the family of ligand-activated zinc finger transcription factors and consist of an N-terminal transactivation domain, two zinc fingers, and a hormone-binding site. Upon ligand binding, the steroid receptor changes its conformation, which reduces the binding affinity of inhibitors (e.g. hsp90). After dimerization, it enters the nucleus and activates target genes. Interestingly, the cross-talk between Wnt/β-catenin signaling and steroid response is bidirectional: glucocorticoids inhibit the transcriptional activity of LEF/TCF proteins, whereas TCF proteins modulate estrogen receptor activity. Depending on individual TCF family members, the latter can result in enhancement (TCF-1) or suppression (TCF-4) of the estrogen-driven promoter response (16Amir A.L. Barua M. McKnight N.C. Cheng S. Yuan X. Balk S.P. J. Biol. Chem. 2003; 278: 30828-30834Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 17El-Tanani M. Fernig D.G. Barraclough R. Green C. Rudland P. J. Biol. Chem. 2001; 276: 41675-41682Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Apart from the internal transactivation domain, several adaptor proteins that bind to steroid receptors and activate expression of target genes have been identified. Among them, ARA-55 (androgen receptor activator of 55 kDa; also named HIC-5 (hydrogen peroxide-induced clone 5)) was initially described as a component of the focal adhesion complex, in which it binds to the focal adhesion kinase (19Fujita H. Kamiguchi K. Cho D. Shibanuma M. Morimoto C. Tachibana K. J. Biol. Chem. 1998; 273: 26516-26521Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 20Nishiya N. Tachibana K. Shibanuma M. Mashimo J.I. Nose K. Mol. Cell. Biol. 2001; 21: 5332-5345Crossref PubMed Scopus (86) Google Scholar). HIC-5/ARA-5 (referred to below only as HIC-5) belongs to the paxillin family of LIM proteins because it shares common protein-protein interaction motifs with paxillin: HIC-5 possesses three LD domains in the N-terminal half and four LIM domains in the C-terminal half. Recent studies confirmed a nuclear role of HIC-5 as coactivator of steroid receptors (21Yang L. Guerrero J. Hong H. DeFranco D.B. Stallcup M.R. Mol. Biol. Cell. 2000; 11: 2007-2018Crossref PubMed Scopus (117) Google Scholar, 22Guerrero-Santoro J. Yang L. Stallcup M.R. DeFranco D.B. J. Cell. Biochem. 2004; 92: 810-819Crossref PubMed Scopus (23) Google Scholar) and peroxisome proliferator-activated receptor γ (PPARγ) (23Drori S. Girnun G.D. Tou L. Szwaya J.D. Mueller E. Kia X. Shivdasani R.A. Spiegelman B.M. Genes Dev. 2005; 19: 362-375Crossref PubMed Scopus (83) Google Scholar) and as co-regulator of transcription factors SMAD3 and Sp1 (24Shibanuma M. Kim-Kaneyama J.-I. Sato R. Nose K. J. Cell. Biochem. 2004; 91: 633-645Crossref PubMed Scopus (55) Google Scholar, 25Wang H. Song K. Sponseller T.L. Danielpour D. J. Biol. Chem. 2005; 280: 5154-5162Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). The pleiotropic functions of HIC-5 imply an important role as a regulatory protein, allowing the cell to integrate the input of different signaling cascades. In this study, we identify HIC-5 as a novel binding partner of LEF/TCF proteins. The HIC-5 C terminus containing the LIM domains binds to a conserved exon in LEF/TCF proteins. This interaction of HIC-5 and LEF/TCF proteins is conserved in vertebrates and results in a complex that represses both LEF/TCF target gene activation and HIC-5-induced steroid receptor activation. However, this does not present a general regulatory mechanism of LEF/TCF target gene activation because only those family members that contain the conserved exon and that act as activators (Xenopus (X) TCF-4C, murine (m) LEF-1, human (h) TCF-1, and hTCF-4) are repressed by HIC-5. Other family members that activate target genes but do not contain the exon (e.g. XLEF-1) are not regulated by HIC-5. However, transcription factors that do not activate target gene promoters (TCF-3 and XTCF-4A) are not regulated by HIC-5. Instead, they repress HIC-5-induced steroid receptor activation. Thus, HIC-5 is a novel LEF/TCF binding partner that mediates the TCF subtype-specific cross-talk between Wnt/β-catenin signaling and steroid receptor activation. Constructs—The coding regions of XLEF-1, XTCF-3, and XTCF-4 corresponding to amino acids 63–274, 63–328, and 63–353, respectively, were fused to the LexA DNA-binding domain in BTM116. Additionally, the same constructs and XTCF-3 amino acids 193–249 and XTCF-4 amino acids 220–316 were fused to glutathione S-transferase (GST) and His tags in pET-M30. hTCF-1, mTCF-3, and hTCF-4 in pcDNA3.1 were kindly provided by Hans Clevers, whereas hTCF-3 was from W. Birchmeier. psp64T3-mLEF-1 and pCS2-XLEF/XTCF constructs were as described previously (10Gradl D. König A. Wedlich D. J. Biol. Chem. 2002; 277: 14159-14171Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). Full-length mHIC-5 constructs in pcDNA-3.1 and pGEX were kindly provided by Michael Stall-cup. mHIC-5 fused to GST was separated in the N-terminal half containing the LD domains and in the C-terminal half containing the LIM domains by PCR. 3Primer sequences are available upon request. The mouse mammary tumor virus (MMTV)-luciferase reporter construct was provided by Olivier Kassel. The Xenopus fibronectin reporter and the TOPFlash promoter have been described (26Gradl D. Kühl M. Wedlich D. Mol. Cell. Biol. 1999; 19: 5576-5587Crossref PubMed Google Scholar, 27Korinek V. Barker N. Morin P.J. van Wichen D. de Weger R. Kinzler K.W. Vogelstein B. Clevers B. Science. 1997; 275: 1784-1787Crossref PubMed Scopus (2895) Google Scholar). Yeast Two-hybrid Screen—A mouse embryonic day 10 library cloned in the pVP16 vector was kindly provided by Jürgen Behrens. L40 yeast cells were transformed with BTM116 constructs and used for screening ∼1 × 105 transformants of library-transformed cells. After the interacting clones of the bait vector were cured, the clones were tested for specific interaction with XTCF-3 and XTCF-4 by mating with AMR70 previously transformed with the respective bait construct (28Hollenberg S.M. Sternglanz R. Cheng P.F. Weintraub H. Mol. Cell. Biol. 1995; 7: 3813-3822Crossref Scopus (580) Google Scholar). Positive clones were isolated, sequenced, and analyzed. Bacterial Expression of LEF/TCF Proteins—Transformed BL21(DE3) bacteria were induced at A600 = 0.7 with 1 mm isopropyl 1-thio-β-d-galactopyranoside for 4 h at 30 °C. After centrifugation, bacterial pellets were lysed in phosphate-buffered saline (137 mm NaCl, 2.7 mm KCl, 6.5 mm Na2HPO4, and 1.5 mm KH2PO4, pH 7.5) additionally containing 400 mm NaCl, 1 mg/ml lysozyme, and protease inhibitors. Aliquots of the cleared lysates were stored in liquid nitrogen. Pull-down Assays—Bacterially expressed GST-tagged HIC-5 protein was immobilized on glutathione-Sepharose beads for 2 h at 4°C in buffer A (10 mm Tris-Cl, pH 7.8, 150 mm NaCl, 1 mm MgCl2·6H2O, 0.75 mm CaCl2·2H2O, 2% Nonidet P-40, and protease inhibitors) and incubated with buffer A lysate from transfected human embryonic kidney (HEK) epithelial 293 cells. After binding for 2 h at 4 °C,thesamples were washed three times with buffer A, boiled in SDS sample buffer for 5 min, and subjected to 10% SDS-PAGE. Proteins were transferred onto nitrocellulose, probed with anti-Myc monoclonal antibody 9E10, and revealed by the chemiluminescence reaction (ECL, Amersham Biosciences). Immobilized GST-HIC-5 constructs and in vitro translated 35S-labeled LEF/TCF proteins or immobilized GST-LEF/TCF constructs and in vitro translated 35S-labeled HIC-5 proteins were incubated in buffer A, washed, eluted and separated on a 10% SDS gel. After Coomassie Blue staining, the gel was dried and subjected to PhosphorImager (Raytest) analysis to visualize the bound protein. Injection Experiments—mRNA was synthesized in vitro using the mMESSAGE mMACHINE kit (Ambion, Inc.). 500 pg of LEF-1 or HIC-5 mRNA or 70 pg of XWNT-8 mRNA were injected into both ventral blastomeres of Xenopus four-cell stage embryos. Embryos were kept as described previously (29Kühl M. Sheldahl L.C. Malbon C.C. Moon R.T. J. Biol. Chem. 2000; 275: 12701-12711Abstract Full Text Full Text PDF PubMed Scopus (395) Google Scholar) and analyzed for the appearance of secondary axis, dorso-anteriorization, and target gene expression. Transfection and Reporter Gene Assays—HeLa and HEK293 cells were routinely grown in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. HEK293 cells were transfected by calcium phosphate precipitation according to Gorman (30Gorman C. Glover D.M. DNA Cloning: a Practical Approach. IRL Press, Oxford1985: 143-190Google Scholar), and HeLa cells were transfected with MATra reagent (IBA GmbH, Göttingen, Germany) according to the manufacturer's recommendations. 48 h after transfection, cells were harvested. Reporter gene assays were performed as described (26Gradl D. Kühl M. Wedlich D. Mol. Cell. Biol. 1999; 19: 5576-5587Crossref PubMed Google Scholar). To analyze the glucocorticoid response, transfected cells were treated with 10 nm dexamethasone. Reverse Transcription-PCR—1 μg of HeLa cell total RNA was reverse-transcribed using MMTV reverse transcriptase (Promega). cDNA corresponding to 20 ng of RNA was amplified for 28 (glyceraldehyde-3-phosphate dehydrogenase) or 34 (p21 and p27) cycles with the glyceraldehyde-3-phosphate dehydrogenase forward primer (5′-GTGGATATTGTTGCCATCAAT-3′) and reverse primer (5′-CGCTGTTGAAGTCAGAGGAG-3′), the p21 forward primer (5-ATGTCCGTCAGAACCCATG-3′) and reverse primer (5′-TTAGGGCTTCCTCTTGGAGA-3′, or the p27 forward primer (5′-GTCTAACGGGAGCCCTAGCC-3′) and reverse primer (5′-CTAACCCCGTCTGGCTGTCC-3′). 1 μgof Xenopus stage 10.5 total RNA was reverse-transcribed using MMTV reverse transcriptase. cDNA corresponding to 20 ng of RNA was amplified for 26 (histone H4) or 34 (siamois and Xnr-3) cycles with the histone H4 forward primer (5′-CGGGATAACATTCAGGGTATCACT-3′) and reverse primer (5′-ATCCATGGCGGTAACTGTCTTCCT-3), the siamois forward primer (5′-CTCCAGCCACCAGTACCAGAT-3′) and reverse primer (5′-GGGGAGAGTGGAAAGTGGTTG-3′, and the Xnr-3 forward primer (5′-TCCACTTGTGCAGTTCCACAG-3′) and reverse primer (5′-ATCTCTTCATGGTGCCTCAGG-3′). Isolation of Full-length XHIC-5—Using degenerated primers, we amplified a 500-bp fragment of the 5′-region of XHic-5 from stage 18 cDNA. 3Primer sequences are available upon request. To obtain the entire open reading frame of XHic-5, we screened a Xenopus tailbud λ-ZAP cDNA library with this fragment. The open reading frame of XHic-5 (GenBank™ accession number AY971603) was subcloned into the NcoI/XhoI sites of pCS2-Myc. Immunostaining—HeLa cells were fixed with 3% formaldehyde and permeabilized by incubation for 8 min in phosphate-buffered saline containing 0.1% Triton X-100. Localization of the anti-HIC-5 (polyclonal; Santa Cruz Biotechnology, Inc.) and anti-TCF-3/4 and anti-LEF-1 (monoclonal; Pierce) primary antibodies was visualized with Cy2-conjugated goat anti-rabbit and Cy3-conjugated goat anti-mouse IgG, respectively. XHIC-5 Discriminates between XTCF-3/4 and XLEF-1—To identify new binding partners, we screened a mouse embryonic day 10 library using the core domain (between the β-catenin-binding site and the HMG box) of Xenopus TCF-3 and TCF-4 as bait. We identified HIC-5 as a candidate protein that binds to both XTCF-3 and XTCF-4 (data not shown). The full-length XHic-5 cDNA (GenBank™ accession number AY971603) was isolated by screening a Xenopus tailbud λ-ZAP library. The XHIC-5 protein is 40% identical to its human ortholog. The similarity in the conserved three LD and four LIM domains ranges from 55% (LIM domain 2) to 100% (LD domain 2) (Fig. 1A). The most obvious differences between the Xenopus and mammalian HIC proteins are the absence of a 33-amino acid proline-rich region flanking a highly conserved part and the presence of a 55-amino acid acidic and serine-rich region between LD domains 2 and 3 in the Xenopus protein. To confirm the physical interaction found in the yeast two-hybrid screen, we carried out GST pull-down assays using bacterially expressed GST-LEF/TCF fusion proteins and in vitro translated 35S-labeled XHIC-5. Indeed, we found that XHIC-5 bound to XTCF-3 and XTCF-4, but not to the GST control (Fig. 1B). We further confirmed the physical interaction using transfected Myc-tagged XHIC-5 and bacterially expressed GST-LEF/TCF fusion proteins (Fig. 1C). Interestingly, compared with XTCF-3 and XTCF-4, the binding of XHIC-5 to XLEF-1 was very weak (Fig. 1, B and C). HIC-5 Binds to a Conserved Exon—The most obvious difference between XLEF-1 and XTCF-3/4 is a conserved exon that fails in XLEF-1. This exon corresponds to exon IVa in hTCF-1, exon VI in hLEF-1, and exon VIII in hTCF-4, all of which are known to be alternatively spliced. Alternative splicing of this exon has not been reported for the Xenopus homolog of LEF-1; instead, this exon is missing in all XLef-1 cDNAs reported so far (10Gradl D. König A. Wedlich D. J. Biol. Chem. 2002; 277: 14159-14171Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 31Molenaar M. van de Wetering M. Oosterwegel M. Peterson-Maduro J. Godsave S. Korinek V. Roose J. Destree O. Clevers H. Cell. 1996; 86: 391-399Abstract Full Text Full Text PDF PubMed Scopus (1592) Google Scholar). By constructing chimeric proteins, we have recently shown that this exon promotes target gene activation (10Gradl D. König A. Wedlich D. J. Biol. Chem. 2002; 277: 14159-14171Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). To confirm that this conserved exon is the HIC-5-binding domain, we transfected Myc-tagged Xenopus LEF/TCF constructs and analyzed their binding to recombinantly expressed HIC-5 in GST pull-down assays. Indeed, wild-type XLEF-1 hardly bound HIC-5, whereas a chimeric XLEF-1 construct containing the conserved exon of XTCF-3 (XLEF-1+Exon) was precipitated with immobilized GST-HIC-5 (Fig. 2A). Furthermore, in vitro translated HIC-5 bound to the recombinantly expressed exon of XTCF-3 and XTCF-4 (Fig. 2B). Interestingly, HIC-5 did not discriminate among XTCF-4A, -4B, and -4C, which differ in the presence of two small peptide motifs flanking the conserved exon (Fig. 2B). The C Terminus (but Not the N Terminus) of HIC-5 Binds to LEF/TCF Proteins—We next tried to map the binding domain in HIC-5. As HIC-5 contains two putative protein-protein interaction sites, the LD and LIM domains, we fused the LD domain- and LIM domain-containing parts separately to GST. Therefore, we cut the protein into two halves, the N-terminal half containing the three LD domains and the C-terminal half containing the four LIM domains (Fig. 3). GST pull-down assays with in vitro translated LEF/TCF proteins revealed that the LEF/TCF-binding site is the LIM domain-containing C terminus, but not the LD domains (Fig. 3). Thus, the conserved exon of LEF/TCF proteins interacts with the C terminus, most likely with the LIM domains, of HIC-5. Because the binding of HIC-5 to LEF/TCF proteins was observed for the Xenopus and murine proteins and also between the corresponding binding partners of different species, we conclude that the interaction is conserved in vertebrates. HIC-5 Suppresses LEF/TCF-induced Target Gene Activation—Next, we asked whether the physical interaction between LEF/TCF proteins and HIC-5 results in activation or repression of Wnt/β-catenin target genes. Therefore, we cotransfected HEK293 cells with LEF/TCF reporter constructs, HIC-5, and different LEF/TCF expression constructs. Transfected HIC-5 (both the murine and Xenopus orthologs) had only a minor effect on the activity of the TOPFlash promoter by itself. Consistent with previously published data (10Gradl D. König A. Wedlich D. J. Biol. Chem. 2002; 277: 14159-14171Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 13Pukrop T. Gradl D. Henningfeld K. Knöchel W. Wedlich D. Kühl M. J. Biol. Chem. 2001; 276: 8968-8978Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar), XTCF-4C and XLEF-1 (but not XTCF-4A and XTCF-3) activated the TOPFlash promoter in HEK293 cells (Fig. 4A). In the presence of HIC-5, however, XTCF-4C did not activate the TOPFlash promoter, and activation by the chimeric XLEF-1+Exon construct was drastically reduced. In the case of XTCF-4C, promoter activation dropped from 2.1 to 1.2-fold (mHIC-5) or 0.8-fold (XHIC-5) and, in the case of XLEF-1+Exon, from 4.1- to 1.9- or 1.5-fold, respectively (Fig. 4A). Consistent with the observation that HIC-5 bound to a conserved exon present in XTCF-3, XTCF-4, and XLEF-1+Exon but missing in XLEF-1, we found no effect of HIC-5 on TOPFlash activation via XLEF-1. The promoter was activated by 1.5–2-fold irrespective of whether HIC-5 was cotransfected or not (Fig. 4A). The specificity of TOPFlash activation and repression is documented by cotransfection of the control promoter FOPFlash, which was neither activated by XLEF-1+Exon nor repressed by HIC-5 (Fig. 4B). This repressive function of HIC-5 is conserved among vertebrates because the results were similar and even more pronounced when we studied mHIC-5 in combination with human or murine LEF/TCF proteins. mLEF-1, hTCF-1, and h-TCF-4 activated the TOPFlash promoter by 3.1–3.5-fold. As shown for the Xenopus LEF/TCF proteins, cotransfection of HIC-5 inhibited mammalian LEF/TCF-induced promoter activation (Fig. 4C). Although TCF-3 contains the conserved exon, it did not activate the TOPFlash promoter and was not regulated by HIC-5. The regulation of Wnt target genes by HIC-5 is not restricted to the artificial TOPFlash promoter. We observed a similar response when we used the Xenopus fibronectin promoter (Fig. 4D). Again, cotransfection of HIC-5 prevented promoter activation by XLEF-1+Exon (1.7-fold versus 3.2-fold) and XTCF-4C (1.6-fold versus 2.3-fold), but not by XLEF-1. Thus, the binding of the conserved exon of LEF/TCF proteins to the C-terminal half of HIC-5 is conserved among vertebrates and prevents activation of Wnt/β-catenin target gene promoters. HIC-5 Suppresses Secondary Axis Formation—Ectopic activation of the Wnt/β-catenin cascade in the ventral hemisphere of Xenopus embryos results in the induction of a secondary Spemann organizer and subsequently in the appearance of a secondary body axis. The only LEF/TCF family member that mimics an activated Wnt/β-catenin cascade and induces a secondary body axis upon ventral injection is LEF-1. We showed recently that the frequency of secondary axis formation is higher following mLEF-1 injection than following XLEF-1 injection and that this difference is due to the presence of the conserved exon (10Gradl D. König A. Wedlich D. J. Biol. Chem. 2002; 277: 14159-14171Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). If HIC-5 is indeed a general repressor that binds to the conserved exon, it should suppress mLEF-1 (but not XLEF-1)-induced secondary axis formation in Xenopus embryos. Therefore, we co-injected 500 pg of HIC-5 mRNA together with 500 pg of mLEF-1 or XLEF mRNA into both ventral blastomeres of Xenopus four-cell stage embryos and scored the appearance of a secondary axis. As expected, co-injected HIC-5 reduced the frequency of secondary axis formation induced by mLEF-1 from 33 to 15%, but had no effect on the frequency of XLEF-1-induced secondary axis formation (Fig. 5, A and B). After injection of 70 pg of XWNT-8 mRNA, most of the embryos (94%, n = 80) showed the most severe canonical Wnt phenotype, a complete dorso-anteriorization. This phenotype is best seen by a ring-shaped cement gland (Fig. 5C). After co-injection of HIC-5, only 3% (n = 104) of the injected embryos showed this complete dorso-anteriorization. Instead, 58% of th
Referência(s)