Interaction between Hhex and SOX13 Modulates Wnt/TCF Activity
2009; Elsevier BV; Volume: 285; Issue: 8 Linguagem: Inglês
10.1074/jbc.m109.046649
ISSN1083-351X
AutoresVanessa Marfil, Marta Moya, Christophe E. Pierreux, José V. Castell, Frédéric P. Lemaigre, Francisco X. Real, Roque Bort,
Tópico(s)Epigenetics and DNA Methylation
ResumoFine-tuning of the Wnt/TCF pathway is crucial for multiple embryological processes, including liver development. Here we describe how the interaction between Hhex (hematopoietically expressed homeobox) and SOX13 (SRY-related high mobility group box transcription factor 13), modulates Wnt/TCF pathway activity. Hhex is a homeodomain factor expressed in multiple endoderm-derived tissues, like the liver, where it is essential for proper development. The pleiotropic expression of Hhex during embryonic development and its dual role as a transcriptional repressor and activator suggest the presence of different tissue-specific partners capable of modulating its activity and function. While searching for developmentally regulated Hhex partners, we set up a yeast two-hybrid screening using an E9.5–10.5 mouse embryo library and the N-terminal domain of Hhex as bait. Among the putative protein interactors, we selected SOX13 for further characterization. We found that SOX13 interacts directly with full-length Hhex, and we delineated the interaction domains within the two proteins. SOX13 is known to repress Wnt/TCF signaling by interacting with TCF1. We show that Hhex is able to block the SOX13-dependent repression of Wnt/TCF activity by displacing SOX13 from the SOX13·TCF1 complex. Moreover, Hhex de-repressed the Wnt/TCF pathway in the ventral foregut endoderm of cultured mouse embryos electroporated with a SOX13-expressing plasmid. We conclude that the interaction between Hhex and SOX13 may contribute to control Wnt/TCF signaling in the early embryo. Fine-tuning of the Wnt/TCF pathway is crucial for multiple embryological processes, including liver development. Here we describe how the interaction between Hhex (hematopoietically expressed homeobox) and SOX13 (SRY-related high mobility group box transcription factor 13), modulates Wnt/TCF pathway activity. Hhex is a homeodomain factor expressed in multiple endoderm-derived tissues, like the liver, where it is essential for proper development. The pleiotropic expression of Hhex during embryonic development and its dual role as a transcriptional repressor and activator suggest the presence of different tissue-specific partners capable of modulating its activity and function. While searching for developmentally regulated Hhex partners, we set up a yeast two-hybrid screening using an E9.5–10.5 mouse embryo library and the N-terminal domain of Hhex as bait. Among the putative protein interactors, we selected SOX13 for further characterization. We found that SOX13 interacts directly with full-length Hhex, and we delineated the interaction domains within the two proteins. SOX13 is known to repress Wnt/TCF signaling by interacting with TCF1. We show that Hhex is able to block the SOX13-dependent repression of Wnt/TCF activity by displacing SOX13 from the SOX13·TCF1 complex. Moreover, Hhex de-repressed the Wnt/TCF pathway in the ventral foregut endoderm of cultured mouse embryos electroporated with a SOX13-expressing plasmid. We conclude that the interaction between Hhex and SOX13 may contribute to control Wnt/TCF signaling in the early embryo. IntroductionWnt signaling is a conserved signaling pathway that plays crucial roles in animal life by controlling the genetic programs of embryonic development and adult homeostasis. Recent reports have particularly highlighted the essential role of Wnt during liver and pancreas development. Wnt repression in the anterior endoderm is required for liver and pancreas specification, whereas active Wnt signaling in the posterior endoderm suppresses these fates (1.McLin V.A. Rankin S.A. Zorn A.M. Development. 2007; 134: 2207-2217Crossref PubMed Scopus (267) Google Scholar). However, immediately after the induction of the hepatic program in the endoderm, Wnt signaling is apparently required for the further outgrowth of the endoderm into a liver bud. In fact, the expression of Wnt2b in the lateral plate mesoderm appears essential for liver specification in the endoderm and bud induction (2.Ober E.A. Verkade H. Field H.A. Stainier D.Y. Nature. 2006; 442: 688-691Crossref PubMed Scopus (277) Google Scholar). In short, Wnt activity is extremely dynamic in the course of liver specification and morphogenesis, suggesting that a tight and fast control of Wnt signaling is essential for the proper development of the organ.Hhex is a homeobox transcription factor of the Antennapedia/Ftz class. Hhex is expressed in the anterior definitive endoderm (3.Thomas P.Q. Brown A. Beddington R.S. Development. 1998; 125: 85-94PubMed Google Scholar) that gives rise to the liver and pancreas. After gastrulation, Hhex is also expressed in mesoderm- and endoderm-derived tissues such as hematopoietic and vascular progenitors, endocardium of the heart, liver, thyroid, lung, thymus, gallbladder, and pancreas (4.Bogue C.W. Zhang P.X. McGrath J. Jacobs H.C. Fuleihan R.L. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 556-561Crossref PubMed Scopus (37) Google Scholar, 5.Bort R. Martinez-Barbera J.P. Beddington R.S. Zaret K.S. Development. 2004; 131: 797-806Crossref PubMed Scopus (206) Google Scholar, 6.Crompton M.R. Bartlett T.J. MacGregor A.D. Manfioletti G. Buratti E. Giancotti V. Goodwin G.H. Nucleic Acids Res. 1992; 20: 5661-5667Crossref PubMed Scopus (156) Google Scholar, 7.Martinez Barbera J.P. Clements M. Thomas P. Rodriguez T. Meloy D. Kioussis D. Beddington R.S. Development. 2000; 127: 2433-2445PubMed Google Scholar). Hhex plays a role in cell proliferation and morphogenesis during organogenesis (5.Bort R. Martinez-Barbera J.P. Beddington R.S. Zaret K.S. Development. 2004; 131: 797-806Crossref PubMed Scopus (206) Google Scholar, 8.Kubo A. Chen V. Kennedy M. Zahradka E. Daley G.Q. Keller G. Blood. 2005; 105: 4590-4597Crossref PubMed Scopus (52) Google Scholar, 9.Obinata A. Akimoto Y. Omoto Y. Hirano H. Dev. Growth Differ. 2002; 44: 281-292Crossref PubMed Scopus (21) Google Scholar). Loss-of-function of Hhex in mice results in embryonic lethality at E10.5 and shows different degrees of defects in organs derived from the three embryonic germ layers (4.Bogue C.W. Zhang P.X. McGrath J. Jacobs H.C. Fuleihan R.L. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 556-561Crossref PubMed Scopus (37) Google Scholar, 7.Martinez Barbera J.P. Clements M. Thomas P. Rodriguez T. Meloy D. Kioussis D. Beddington R.S. Development. 2000; 127: 2433-2445PubMed Google Scholar, 10.Keng V.W. Yagi H. Ikawa M. Nagano T. Myint Z. Yamada K. Tanaka T. Sato A. Muramatsu I. Okabe M. Sato M. Noguchi T. Biochem. Biophys. Res. Commun. 2000; 276: 1155-1161Crossref PubMed Scopus (150) Google Scholar, 11.Hallaq H. Pinter E. Enciso J. McGrath J. Zeiss C. Brueckner M. Madri J. Jacobs H.C. Wilson C.M. Vasavada H. Jiang X. Bogue C.W. Development. 2004; 131: 5197-5209Crossref PubMed Scopus (85) Google Scholar). The defects observed in endoderm-derived organs, such as thyroid, liver, and pancreas, are associated with alterations in cell proliferation and cell migration in embryonic progenitors (5.Bort R. Martinez-Barbera J.P. Beddington R.S. Zaret K.S. Development. 2004; 131: 797-806Crossref PubMed Scopus (206) Google Scholar, 7.Martinez Barbera J.P. Clements M. Thomas P. Rodriguez T. Meloy D. Kioussis D. Beddington R.S. Development. 2000; 127: 2433-2445PubMed Google Scholar, 12.Bort R. Signore M. Tremblay K. Martinez Barbera J.P. Zaret K.S. Dev. Biol. 2006; 290: 44-56Crossref PubMed Scopus (212) Google Scholar, 13.Hunter M.P. Wilson C.M. Jiang X. Cong R. Vasavada H. Kaestner K.H. Bogue C.W. Dev. Biol. 2007; 308: 355-367Crossref PubMed Scopus (111) Google Scholar). Recently, Hhex locus has been robustly associated in genome-wide association studies with type 2 diabetes (14.Sladek R. Rocheleau G. Rung J. Dina C. Shen L. Serre D. Boutin P. Vincent D. Belisle A. Hadjadj S. Balkau B. Heude B. Charpentier G. Hudson T.J. Montpetit A. Pshezhetsky A.V. Prentki M. Posner B.I. Balding D.J. Meyre D. Polychronakos C. Froguel P. Nature. 2007; 445: 881-885Crossref PubMed Scopus (2352) Google Scholar). Human and mouse Hhex proteins share 95% homology throughout the full sequence and the N-terminal domain.Hhex is a transcriptional repressor in Xenopus embryos and cultured cells (15.Brickman J.M. Jones C.M. Clements M. Smith J.C. Beddington R.S. Development. 2000; 127: 2303-2315PubMed Google Scholar, 16.Cong R. Jiang X. Wilson C.M. Hunter M.P. Vasavada H. Bogue C.W. Biochem. Biophys. Res. Commun. 2006; 346: 535-545Crossref PubMed Scopus (19) Google Scholar, 17.Guo Y. Chan R. Ramsey H. Li W. Xie X. Shelley W.C. Martinez-Barbera J.P. Bort B. Zaret K. Yoder M. Hromas R. Blood. 2003; 102: 2428-2435Crossref PubMed Scopus (71) Google Scholar). The N-terminal proline-rich domain (aa 2The abbreviations used are: aaamino acidsGSTglutathione S-transferaseEGFPenhanced green fluorescence proteinHAhemagglutininshRNAshort hairpin RNACMVcytomegalovirusPBSphosphate-buffered salineRTreverse transcriptionqRTquantitative real-timeLZleucine zipperHMGhigh mobility groupX-gal5-bromo-4-chloro-3-indolyl-β-d-galactopyranosideChIPchromatin immunoprecipitationTCFT cell factor. 1–137) is highly conserved among species and exerts a repressing activity (18.Tanaka T. Inazu T. Yamada K. Myint Z. Keng V.W. Inoue Y. Taniguchi N. Noguchi T. Biochem. J. 1999; 339: 111-117Crossref PubMed Scopus (72) Google Scholar). Goosecoid, vascular endothelial growth factor, and endothelial cell-specific molecule 1 are among the best characterized targets transcriptionally repressed by Hhex. But Hhex can also function as a transcriptional activator of the Na+/taurocholate cotransporting polypeptide and sodium iodide symporter promoters (19.Denson L.A. Karpen S.J. Bogue C.W. Jacobs H.C. Am. J. Physiol. Gastrointest. Liver Physiol. 2000; 279: G347-G355Crossref PubMed Google Scholar, 20.Puppin C. Puglisi F. Pellizzari L. Manfioletti G. Pestrin M. Pandolfi M. Piga A. Di Loreto C. Damante G. BMC Cancer. 2006; 6: 192Crossref PubMed Scopus (29) Google Scholar). The acidic C-terminal domain (aa 197–271) is responsible for the transactivating properties of Hhex (21.Kasamatsu S. Sato A. Yamamoto T. Keng V.W. Yoshida H. Yamazaki Y. Shimoda M. Miyazaki J. Noguchi T. J. Biochem. 2004; 135: 217-223Crossref PubMed Scopus (15) Google Scholar). Finally, the homeodomain (aa 138–196) is responsible for sequence-specific binding to DNA.Given the wide expression of Hhex and the complexity of its functions, we speculated that an elevated number of interactors in different tissues contributes to its differential specificity and activity. Therefore, we searched an E9.5-E10.5 mouse embryo library for developmentally regulated proteins that interact with Hhex. Using a yeast two-hybrid screening approach we identified SOX13 as a Hhex interactor. Two SOX13 isoforms of 768 bp (255 aa) and 1815 bp (604 aa) were isolated from human cDNA, but only the longer isoform interacted with Hhex. We mapped the interaction domains in both proteins and showed that Hhex and SOX13 are colocalized in the nucleus. Finally, we addressed the function of the Hhex/SOX13 interaction and show in cultured cells and mouse embryos that it de-represses Wnt activity by disrupting the SOX13·TCF1 complex.DISCUSSIONWe have shown that binary interactions between Hhex, SOX13, and TCF regulate Wnt activity in vitro and in vivo. The three genes are co-expressed in several embryonic tissues, like the liver and pancreas (44.Bogue C.W. Ganea G.R. Sturm E. Ianucci R. Jacobs H.C. Dev. Dyn. 2000; 219: 84-89Crossref PubMed Scopus (79) Google Scholar, 45.Wang Y. Ristevski S. Harley V.R. J. Histochem. Cytochem. 2006; 54: 1327-1333Crossref PubMed Scopus (25) Google Scholar). The β-catenin-TCF complex is a downstream effector of the Wnt signaling pathway (Fig. 10). SOX13 binds the transcription factor, TCF1, possibly through the LZ-Q domain to disrupt the TCF1-β-catenin complex and inhibit Wnt activity (40.Melichar H.J. Narayan K. Der S.D. Hiraoka Y. Gardiol N. Jeannet G. Held W. Chambers C.A. Kang J. Science. 2007; 315: 230-233Crossref PubMed Scopus (130) Google Scholar). The addition of Hhex results in the formation of the Hhex·SOX13 complex (Fig. 4) to dislodge SOX13 from the SOX13·TCF1 complex (Fig. 7) and results in a restoration/elevation of the Wnt levels (Fig. 6A, 8, and 9). In short, Hhex and SOX13 act as a pair of antagonists of the Wnt activity to achieve the right level in different biological contexts.Through the screening for Hhex-binding proteins with an E9.5-E10.5 mouse embryo library (24.Hollenberg S.M. Sternglanz R. Cheng P.F. Weintraub H. Mol. Cell. Biol. 1995; 15: 3813-3822Crossref PubMed Scopus (582) Google Scholar), we isolated cDNA for a HMG box protein, SOX13. In the only previous yeast two-hybrid screening using Hhex as bait, two interactors were isolated; that is, the HC8 subunit of the proteasome (46.Bess K.L. Swingler T.E. Rivett A.J. Gaston K. Jayaraman P.S. Biochem. J. 2003; 374: 667-675Crossref PubMed Google Scholar) and the CK2β (47.Soufi A. Noy P. Buckle M. Sawasdichai A. Gaston K. Jayaraman P.S. Nucleic Acids Res. 2009; 37: 3288-3300Crossref PubMed Scopus (30) Google Scholar). In our study we have used an embryo-derived library instead of a chronic myelogenous leukemia cell line (K562)-derived cDNA library, which lacks any developmental context. Other studies using different bait have identified Hhex as an interacting partner of Jun (48.Schaefer L.K. Wang S. Schaefer T.S. J. Biol. Chem. 2001; 276: 43074-43082Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar), GATA2 (49.Minami T. Murakami T. Horiuchi K. Miura M. Noguchi T. Miyazaki J. Hamakubo T. Aird W.C. Kodama T. J. Biol. Chem. 2004; 279: 20626-20635Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar), Sox10 (50.Wissmüller S. Kosian T. Wolf M. Finzsch M. Wegner M. Nucleic Acids Res. 2006; 34: 1735-1744Crossref PubMed Scopus (117) Google Scholar), TLE1 (34.Swingler T.E. Bess K.L. Yao J. Stifani S. Jayaraman P.S. J. Biol. Chem. 2004; 279: 34938-34947Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar), or PML (51.Topcu Z. Mack D.L. Hromas R.A. Borden K.L. Oncogene. 1999; 18: 7091-7100Crossref PubMed Scopus (57) Google Scholar). Noteworthy, our screening confirmed the interaction of Hhex with TLE1. Besides Sox13, we obtained other 68 candidates, 24 of them with 2 repetitions or more. Sox13 was isolated 8 times with a common spanning region of ∼100 aa (Fig. 2).Among the different SOX13 mRNAs described in the literature, we identify SOX13 (1815 bp) and S-SOX13 (768 bp) as the isoforms present in human tissues. Our identification is in conflict with Roose et al. (37.Roose J. Korver W. de Boer R. Kuipers J. Hurenkamp J. Clevers H. Genomics. 1999; 57: 301-305Crossref PubMed Scopus (15) Google Scholar) who described a longer open reading frame of 2673 bp isolated from embryonic thymic cDNA. We did not find a significant amount of this longer mRNA in any of the 18 tissues tested, including adult thymus, which is also in agreement with a later report from Harley and co-workers (36.Kasimiotis H. Myers M.A. Argentaro A. Mertin S. Fida S. Ferraro T. Olsson J. Rowley M.J. Harley V.R. Diabetes. 2000; 49: 555-561Crossref PubMed Scopus (59) Google Scholar).SOX transcription factors are involved in the differentiation of multiple cell types and developmental processes from the endoderm. Sox17 plays an essential role in definitive endoderm formation in the mouse (52.Kanai-Azuma M. Kanai Y. Gad J.M. Tajima Y. Taya C. Kurohmaru M. Sanai Y. Yonekawa H. Yazaki K. Tam P.P. Hayashi Y. Development. 2002; 129: 2367-2379Crossref PubMed Google Scholar). Sox9 is expressed in the pancreas where it plays a role in the maintenance of a progenitor cell pool (53.Seymour P.A. Freude K.K. Tran M.N. Mayes E.E. Jensen J. Kist R. Scherer G. Sander M. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 1865-1870Crossref PubMed Scopus (435) Google Scholar). It is also expressed asymmetrically in the ductal plate, and it seems to control the timing of biliary tubulogenesis (54.Antoniou A. Raynaud P. Cordi S. Zong Y. Tronche F. Stanger B.Z. Jacquemin P. Pierreux C.E. Clotman F. Lemaigre F.P. Gastroenterology. 2009; 136: 2325-2333Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar). Sox4 is essential for the normal formation of endocrine pancreas (55.Wilson M.E. Yang K.Y. Kalousova A. Lau J. Kosaka Y. Lynn F.C. Wang J. Mrejen C. Episkopou V. Clevers H.C. German M.S. Diabetes. 2005; 54: 3402-3409Crossref PubMed Scopus (87) Google Scholar). SOX13 is expressed in the pancreas and liver, in the visceral mesoderm of the extraembryonic yolk sac, and also in the spongiotrophoblast layer of the placenta (45.Wang Y. Ristevski S. Harley V.R. J. Histochem. Cytochem. 2006; 54: 1327-1333Crossref PubMed Scopus (25) Google Scholar, 56.Lioubinski O. Müller M. Wegner M. Sander M. Dev. Dyn. 2003; 227: 402-408Crossref PubMed Scopus (105) Google Scholar). At the biochemical level, SOX13 contains a leucine zipper and glutamine-rich domain at the C terminus and the HMG box, an 80-amino acid sequence that mediates DNA binding (37.Roose J. Korver W. de Boer R. Kuipers J. Hurenkamp J. Clevers H. Genomics. 1999; 57: 301-305Crossref PubMed Scopus (15) Google Scholar). SOX13 spans 14 exons and belongs to the group D of the SOX family together with SOX5 and SOX6. They are characterized by the absence of the transactivation domain (57.Kiefer J.C. Dev. Dyn. 2007; 236: 2356-2366Crossref PubMed Scopus (4) Google Scholar). SOX13 is able to bind the consensus sequence (A/T)(A/T)CAA(A/T)G as a monomer or homodimer, probably through the LZ domain (58.Roose J. Korver W. Oving E. Wilson A. Wagenaa r G. Markman M. Lamers W. Clevers H. Nucleic Acids Res. 1998; 26: 469-476Crossref PubMed Scopus (40) Google Scholar). Which domains are responsible for Hhex/SOX13 interaction? The N terminus of Hhex (aa 1–137) is apparently necessary and sufficient for SOX13 interaction. When used as bait, it interacts with the LZ-Q domain of SOX13 to transactivate MEL1 and LacZ reporter genes under the control of a heterologous GAL4-responsive upstream activation sequence and promoter elements in yeast (Fig. 2). In a GST pulldown assay, only Hhex itself and its deletion mutants containing the N-terminal domain, i.e. Hhex-(1–137) and Hhex-(1–196), achieve a significant amount of SOX13 pulldown (Fig. 4). On the other hand, the LZ-Q domain in SOX13 is necessary, but not sufficient, for interaction in cells. S-SOX13 contains the LZ-Q domain but does not interact with any fragment of Hhex. ΔSOX13, which lacks only the LZ-Q domain, is not able to interact either. If Hhex interacts through the LZ-Q, then we may expect its effects to be mediated by a disruption of the putative SOX13 homodimer. However, SOX13 might act as a monomer, as suggested by the fact that co-transfection with S-SOX13 (containing the LZ-Q domain but not the HMG domain) did not block the inhibitory effect of SOX13 on Wnt activity (supplemental Fig. S1B). In short, the N-terminal domain of Hhex mediates the binding to SOX13 in mammalian cells, whereas the LZ-Q domain in SOX13 is necessary, but not sufficient, for the interaction. The interaction between Hhex and SOX13 seems to be specific as SOX9 did not interact with Hhex in an immunoprecipitation assay. Finally, we showed that SOX13 and Hhex interact in a cell system and can do so directly without a bridging protein (Fig. 5).Our work also uncovered the independent structural requirements of Hhex and SOX13 to effectively modulate Wnt activity. The Wnt signaling pathway describes a complex network of proteins most well known for their roles in embryogenesis and cancer but also involved in normal physiological processes in adult animals. Hhex has been recently described as an inducer of Wnt by repressing the expression of TLE4 (42.Zamparini A.L. Watts T. Gardner C.E. Tomlinson S.R. Johnston G.I. Brickman J.M. Development. 2006; 133: 3709-3722Crossref PubMed Scopus (42) Google Scholar). However, Hhex-(1–196), which retains the repressor domain but lacks the transactivator domain, did not induce Wnt activity (Fig. 6A). In parallel, Hhex-(138–271), which lacks the repressor domain, induced Wnt activity by almost 4-fold, suggesting that Wnt activation depends on the C terminus of Hhex. It is feasible that the assigned transcriptional function of specific Hhex domains might be more plastic than initially thought. Alternatively, TLE4 might be a secondary target of Hhex. Fragments of Hhex lacking the homeodomain, i.e. Hhex-(197–271) and Hhex-(1–137), did not alter Wnt activity (data not shown). This is possibly linked to their extranuclear localization (supplemental Fig. S3) and the inability to bind DNA. On the other hand, SOX13 integrity seems to be crucial for Wnt repression. A specific deletion of the LZ-Q domain in SOX13 (ΔSOX13) completely abolished Wnt repression. S-SOX13, which lacks the C-terminal domain of SOX13, was also unable to inhibit Wnt activity (supplemental Fig. S1). An inspection of the S-SOX13 structure suggests that it might function as a SOX13 inhibitor. However, it did not interfere with the SOX13-dependent regulation of Wnt. Full-length Hhex is able to restore Wnt inhibition after SOX13 co-transfection in 293T cells. Hhex domains have a statistically significant difference to restore SOX13-dependent Wnt repression. Hhex-(1–196) was as efficient as the full-length protein, whereas Hhex-(138–271) only caused a certain degree of Wnt restoration at the highest dose, which was significantly less pronounced (Fig. 6A). Given that Hhex-(138–271) lacks the N terminus involved in SOX13 interaction, this result suggests that Wnt activity restoration is caused by direct SOX13/Hhex interaction. In agreement with this hypothesis, Hhex-VP2, a modified form of Hhex containing the N terminus, behaves as a transcriptional activator and is still able to reinstate Wnt activity (Fig. 6B). Thus, Hhex influences Wnt signaling in the presence of SOX13 and does not reside in Hhex transcriptional activity. Reporter assay analysis in HepG2 cells confirmed that Hhex is able to block the inhibitory effect of SOX13 either by endogenous expression in HepG2 cells (Fig. 6C) or by overexpression in 293T cells (Fig. 6A, group 6, black bar). Moreover, knockdown of endogenous HHEX in HepG2 restored the inhibitory activity of SOX13 (Fig. 6C, group 3). In summary, HepG2 cells behave as 293T cells in terms of SOX13-dependent Wnt inhibition, when endogenous Hhex is knocked down.We validated in cultured mouse embryos the results obtained in cell cultures (Fig. 8). It is somewhat striking that ectopic expression of Hhex cDNA in 293T cells induces Wnt activity, whereas in the embryo it inhibits Wnt activity. We can best explain this discrepancy by suggesting that Wnt induction is a cell-autonomous effect of Hhex. In the embryo, non-cell autonomous effects, such as interaction between Hhex expressing endoderm and mesoderm during patterning, may induce expression of the Wnt antagonist that ultimately accounts for a general inhibition of Wnt activity. In fact, phenotypes observed in response to overexpression of Hhex resemble those obtained after expression of Wnt antagonists (15.Brickman J.M. Jones C.M. Clements M. Smith J.C. Beddington R.S. Development. 2000; 127: 2303-2315PubMed Google Scholar). Loss-of-function of Hhex in the mouse causes a dorsalization of the embryo, where most of the ventral structures are missing, i.e. forehead, thyroid, liver, or ventral pancreas (5.Bort R. Martinez-Barbera J.P. Beddington R.S. Zaret K.S. Development. 2004; 131: 797-806Crossref PubMed Scopus (206) Google Scholar, 7.Martinez Barbera J.P. Clements M. Thomas P. Rodriguez T. Meloy D. Kioussis D. Beddington R.S. Development. 2000; 127: 2433-2445PubMed Google Scholar), which is compatible with extended Wnt activity (59.Fagotto F. Guger K. Gumbiner B.M. Development. 1997; 124: 453-460PubMed Google Scholar).Keeping in mind that our reporter assay is based on an heterologous promoter, we wanted to confirm our results in an endogenous promoter. We chose DKK1 because it is a relevant developmentally regulated gene and it has been validated extensively as a β-catenin-TCF1 target (28.Niida A. Hiroko T. Kasai M. Furukawa Y. Nakamura Y. Suzuki Y. Sugano S. Akiyama T. Oncogene. 2004; 23: 8520-8526Crossref PubMed Scopus (429) Google Scholar). The mRNA levels of DKK1 reproduced the expression pattern obtained with the TOPflash reporter plasmid. Moreover, DKK1 mRNA expression profile was paralleled by TCF1 occupancy of the DKK1 promoter. In summary, the results obtained in reporter assay were validated with endogenous TCF1-regulated promoter by qRT-PCR and a chromatin immunoprecipitation assay (Fig. 9).There seems to be an intense tissue-specific cross-regulation between Hhex and Wnt during development. Activation of Wnt/β-catenin signaling in zebrafish (61.Bischof J. Driever W. Dev. Biol. 2004; 276: 552-562Crossref PubMed Scopus (13) Google Scholar) induces Hhex expression in the dorsal yolk syncytial layer. In agreement, β-catenin-deficient mouse embryos do not express Hhex in the anterior-posterior axis on embryonic day 5.5 (62.Huelsken J. Vogel R. Brinkmann V. Erdmann B. Birchmeier C. Birchmeier W. J. Cell Biol. 2000; 148: 567-578Crossref PubMed Scopus (514) Google Scholar). However, inhibition of the canonical Wnt pathway in Xenopus induces Hhex in the underlying endoderm of the heart field (63.Foley A.C. Mercola M. Genes Dev. 2005; 19: 387-396Crossref PubMed Scopus (163) Google Scholar). Do modulators like SOX13 determine the output of Hhex and Wnt interrelationship in different tissues? Wnt regulation is particularly essential for the specification of the liver and pancreas from the ventral foregut endoderm. Wnt down-regulation in the anterior endoderm is shown to be crucial for liver and pancreas specification in the ventral foregut endoderm. But immediately after inducing the hepatic program in the endoderm, Wnt signaling is apparently required for the endoderm to outgrow further into a liver bud (1.McLin V.A. Rankin S.A. Zorn A.M. Development. 2007; 134: 2207-2217Crossref PubMed Scopus (267) Google Scholar). In zebrafish, the expression of Wnt2b in the lateral plate mesoderm, acting through the β-catenin canonical pathway, appears essential for liver specification in the endoderm and bud induction (2.Ober E.A. Verkade H. Field H.A. Stainier D.Y. Nature. 2006; 442: 688-691Crossref PubMed Scopus (277) Google Scholar). Briefly, there must be fast and sharp control of Wnt in the early steps of liver and pancreas specification. Part of this control is exerted by the expression of the Wnt inhibitors (15.Brickman J.M. Jones C.M. Clements M. Smith J.C. Beddington R.S. Development. 2000; 127: 2303-2315PubMed Google Scholar). In this study we propose that the reciprocal interactions between the triad Hhex, SOX13, and TCF1 together with the autoregulatory loop Hhex⇔Wnt contribute to achieve the correct Wnt intensity in the appropriate spatiotemporal dimension. IntroductionWnt signaling is a conserved signaling pathway that plays crucial roles in animal life by controlling the genetic programs of embryonic development and adult homeostasis. Recent reports have particularly highlighted the essential role of Wnt during liver and pancreas development. Wnt repression in the anterior endoderm is required for liver and pancreas specification, whereas active Wnt signaling in the posterior endoderm suppresses these fates (1.McLin V.A. Rankin S.A. Zorn A.M. Development. 2007; 134: 2207-2217Crossref PubMed Scopus (267) Google Scholar). However, immediately after the induction of the hepatic program in the endoderm, Wnt signaling is apparently required for the further outgrowth of the endoderm into a liver bud. In fact, the expression of Wnt2b in the lateral plate mesoderm appears essential for liver specification in the endoderm and bud induction (2.Ober E.A. Verkade H. Field H.A. Stainier D.Y. Nature. 2006; 442: 688-691Crossref PubMed Scopus (277) Google Scholar). In short, Wnt activity is extremely dynamic in the course of liver specification and morphogenesis, suggesting that a tight and fast control of Wnt signaling is essential for the proper development of the organ.Hhex is a homeobox transcription factor of the Antennapedia/Ftz class. Hhex is expressed in the anterior definitive endoderm (3.Thomas P.Q. Brown A. Beddington R.S. Development. 1998; 125: 85-94PubMed Google Scholar) that gives rise to the liver and pancreas. After gastrulation, Hhex is also expressed in mesoderm- and endoderm-derived tissues such as hematopoietic and vascular progenitors, endocardium of the heart, liver, thyroid, lung, thymus, gallbladder, and pancreas (4.Bogue C.W. Zhang P.X. McGrath J. Jacobs H.C. Fuleihan R.L. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 556-561Crossref PubMed Scopus (37) Google Scholar, 5.Bort R. Martinez-Barbera J.P. Beddington R.S. Zaret K.S. Development. 2004; 131: 797-806Crossref PubMed Scopus (206) Google Scholar, 6.Crompton M.R. Bartlett T.J. MacGregor A.D. Manfioletti G. Buratti E. Giancotti V. Goodwin G.H. Nucleic Acids Res. 1992; 20: 5661-5667Crossref PubMed Scopus (156) Google Scholar, 7.Martinez Barbera J.P. Clements M. Thomas P. Rodriguez T. Meloy D. Kioussis D. Beddington R.S. Development. 2000; 127: 2433-2445PubMed Google Scholar). Hhex plays a role in cell proliferation and morphogenesis during organogenesis (5.Bort R. Martinez-Barbera J.P. Beddington R.S. Zaret K.S. Development. 2004; 131: 797-806Crossref PubMed Scopus (206) Google Scholar, 8.Kubo A. Chen V. Kennedy M. Zahradka E. Daley G.Q. Keller G. Blood. 2005; 105: 4590-4597Crossref PubMed Scopus (52) Google Scholar, 9.Obinata A. Akimoto Y. Omoto Y. Hirano H. Dev. Growth Differ. 2002; 44: 281-292Crossref PubMed Scopus (21) Google Scholar). Loss-of-function of Hhex in mice results in embryonic lethality at E10.5 and shows different degrees of defects in organs derived from the three embryonic germ layers (4.Bogue C.W. Zhang P.X. McGrath J. Jacobs H.C. Fuleihan R.L. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 556-561Crossref PubMed Scopus (37) Google Scholar, 7.Martinez Barbera J.P. Clements M. Thomas P. Rodriguez T. Meloy D. Kioussis D. Beddington R.S. Development. 2000; 127: 2433-2445PubMed Google Scholar, 10.Keng V.W. Yagi H. Ikawa M. Nagano T. Myint Z. Yamada K. Tanaka T. Sato A. Muramatsu I. Okabe M. Sato M. Noguchi T. Biochem. Biophys. Res. Commun. 2000; 276: 1155-1161Crossref PubMed Scopus (150) Google Scholar, 11.Hallaq H. Pinter E. Enciso J. McGrath J. Zeiss C. Brueckner M. Madri J. Jacobs H.C. Wilson C.M. Vasavada H. Jiang X. Bogue C.W. Development. 2004; 131: 5197-5209Crossref PubMed Scopus (85) Google Scholar). The defects observed in endoderm-derived organs, such as thyroid, liver, and pancreas, are associated with alterations in cell proliferation and cell migration in embryonic progenitors (5.Bort R. Martinez-Barbera J.P. Beddington R.S. Zaret K.S. Development. 2004; 131: 797-806Crossref PubMed Scopus (206) Google Scholar, 7.Martinez Barbera J.P. Clements M. Thomas P. Rodriguez T. Meloy D. Kioussis D. Beddington R.S. Development. 2000; 127: 2433-2445PubMed Google Scholar, 12.Bort R. Signore M. Tremblay K. Martinez Barbera J.P. Zaret K.S. Dev. Biol. 2006; 290: 44-56Crossref PubMed Scopus (212) Google Scholar, 13.Hunter M.P. Wilson C.M. Jiang X. Cong R. Vasavada H. Kaestner K.H. Bogue C.W. Dev. Biol. 2007; 308: 355-367Crossref PubMed Scopus (111) Google Scholar). Recently, Hhex locus has been robustly associated in genome-wide association studies with type 2 diabetes (14.Sladek R. Rocheleau G. Rung J. Dina C. Shen L. Serre D. Boutin P. Vincent D. Belisle A. Hadjadj S. Balkau B. Heude B. Charpentier G. Hudson T.J. Montpetit A. Pshezhetsky A.V. Prentki M. Posner B.I. Balding D.J. Meyre D. Polychronakos C. Froguel P. Nature. 2007; 445: 881-885Crossref PubMed Scopus (2352) Google Scholar). Human and mouse Hhex proteins share 95% homology throughout the full sequence and the N-terminal domain.Hhex is a transcriptional repressor in Xenopus embryos and cultured cells (15.Brickman J.M. Jones C.M. Clements M. Smith J.C. Beddington R.S. Development. 2000; 127: 2303-2315PubMed Google Scholar, 16.Cong R. Jiang X. Wilson C.M. Hunter M.P. Vasavada H. Bogue C.W. Biochem. Biophys. Res. Commun. 2006; 346: 535-545Crossref PubMed Scopus (19) Google Scholar, 17.Guo Y. Chan R. Ramsey H. Li W. Xie X. Shelley W.C. Martinez-Barbera J.P. Bort B. Zaret K. Yoder M. Hromas R. Blood. 2003; 102: 2428-2435Crossref PubMed Scopus (71) Google Scholar). The N-terminal proline-rich domain (aa 2The abbreviations used are: aaamino acidsGSTglutathione S-transferaseEGFPenhanced green fluorescence proteinHAhemagglutininshRNAshort hairpin RNACMVcytomegalovirusPBSphosphate-buffered salineRTreverse transcriptionqRTquantitative real-timeLZleucine zipperHMGhigh mobility groupX-gal5-bromo-4-chloro-3-indolyl-β-d-galactopyranosideChIPchromatin immunoprecipitationTCFT cell factor. 1–137) is highly conserved among species and exerts a repressing activity (18.Tanaka T. Inazu T. Yamada K. Myint Z. Keng V.W. Inoue Y. Taniguchi N. Noguchi T. Biochem. J. 1999; 339: 111-117Crossref PubMed Scopus (72) Google Scholar). Goosecoid, vascular endothelial growth factor, and endothelial cell-specific molecule 1 are among the best characterized targets transcriptionally repressed by Hhex. But Hhex can also function as a transcriptional activator of the Na+/taurocholate cotransporting polypeptide and sodium iodide symporter promoters (19.Denson L.A. Karpen S.J. Bogue C.W. Jacobs H.C. Am. J. Physiol. Gastrointest. Liver Physiol. 2000; 279: G347-G355Crossref PubMed Google Scholar, 20.Puppin C. Puglisi F. Pellizzari L. Manfioletti G. Pestrin M. Pandolfi M. Piga A. Di Loreto C. Damante G. BMC Cancer. 2006; 6: 192Crossref PubMed Scopus (29) Google Scholar). The acidic C-terminal domain (aa 197–271) is responsible for the transactivating properties of Hhex (21.Kasamatsu S. Sato A. Yamamoto T. Keng V.W. Yoshida H. Yamazaki Y. Shimoda M. Miyazaki J. Noguchi T. J. Biochem. 2004; 135: 217-223Crossref PubMed Scopus (15) Google Scholar). Finally, the homeodomain (aa 138–196) is responsible for sequence-specific binding to DNA.Given the wide expression of Hhex and the complexity of its functions, we speculated that an elevated number of interactors in different tissues contributes to its differential specificity and activity. Therefore, we searched an E9.5-E10.5 mouse embryo library for developmentally regulated proteins that interact with Hhex. Using a yeast two-hybrid screening approach we identified SOX13 as a Hhex interactor. Two SOX13 isoforms of 768 bp (255 aa) and 1815 bp (604 aa) were isolated from human cDNA, but only the longer isoform interacted with Hhex. We mapped the interaction domains in both proteins and showed that Hhex and SOX13 are colocalized in the nucleus. Finally, we addressed the function of the Hhex/SOX13 interaction and show in cultured cells and mouse embryos that it de-represses Wnt activity by disrupting the SOX13·TCF1 complex.
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