Regulation of Wnt Signaling by Sox Proteins

Molecular Cell, Vol. 4, 487–498, October, 1999, Copyright 1999 by Cell Press

Regulation of Wnt Signaling by Sox Proteins: XSox17a/b and XSox3 Physically Interact with b-catenin Aaron M. Zorn,*k Grant D. Barish,‡ Bart O. Williams,‡ Paul Lavender,*† Michael W. Klymkowsky,§# and Harold E. Varmus‡# * Wellcome Trust/Cancer Research Campaign Institute of Cancer and Developmental Biology Tennis Court Road Cambridge CB2 1QR † Department of Respiratory Medicine and Allergy King’s College London, Guy’s Campus London, SE1 9RT United Kingdom ‡ Division of Basic Sciences National Cancer Institute National Institutes of Health 49 Convent Drive Bethesda, Maryland 20892 § Molecular, Cellular, and Developmental Biology University of Colorado, Boulder Boulder, Colorado 80309

Summary Using a functional screen in Xenopus embryos, we identified a novel function for the HMG box protein XSox17b. Ectopic expression of XSox17b ventralizes embryos by inhibiting the Wnt pathway downstream of b-catenin but upstream of the Wnt-responsive gene Siamois. XSox17b also represses transactivation of a TCF/LEF-dependent reporter construct by Wnt and b-catenin. In animal cap experiments, it both activates transcription of endodermal genes and represses b-catenin-stimulated expression of dorsal genes. The inhibition activity of XSox17b maps to a region C-terminal to the HMG box; this region of XSox17b physically interacts with the Armadillo repeats of b-catenin. Two additional Sox proteins, XSox17a and XSox3, likewise bind to b-catenin and inhibit its TCF-mediated signaling activity. These results reveal an unexpected mechanism by which Sox proteins can modulate Wnt signaling pathways. Introduction Secreted proteins including FGFs, TGFb/BMPs, hedgehogs, and Wnts initiate signaling cascades, and their interplay drives cellular differentiation during embryogenesis. An outstanding challenge is to understand how signaling inputs are integrated to generate cell type– specific patterns of gene expression and behavior. How does a cell control the nature of its response to a signal? How can the same signaling pathway elicit a different, yet appropriate, transcriptional response in different cells? k To whom correspondence should be addressed (e-mail: amz@

mole.bio.cam.ac.uk). # These authors contributed equally to this work.

The Wnt signaling pathway is used to modulate cell fate and proliferation in many different adult and embryonic cells (reviewed in Cadigan and Nusse, 1997; Moon et al., 1997). Secreted Wnt ligands interact with Wntreceptors of the Frizzled family (Bhanot et al., 1996), resulting in the inhibition of the constitutively active serine/threonine kinase glycogen synthase kinase-3b (GSK-3b) (Yanagawa et al., 1995; Cook et al., 1996). In the absence of a Wnt signal, GSK-3b phosphorylates the cytoplasmic protein b-catenin, causing it to be rapidly degraded (Aberle et al., 1997). In the presence of a Wnt signal, GSK-3b activity is inhibited, and cytoplasmic b-catenin is stabilized (He et al., 1995; Yost et al., 1996; Wilbert and Nusse, 1998). As a result, more b-catenin is available to interact with the nuclear effectors of Wnt signaling, members of the TCF family of DNA-binding proteins, TCF1, LEF1, TCF3, and TCF4 (see Cadigan and Nusse, 1997; Clevers and van de Wetering, 1997). TCFs are sequence-specific DNA-binding proteins that contain a single high-mobility group domain (HMG box). TCFs can regulate target gene expression through their interactions with additional factors (Giese and Grosscheld, 1993), such as the coactivators ALY (Bruhn et al., 1997) and b-catenin (Behrens et al., 1996; Huber et al., 1996; Molenaar et al., 1996; van de Wetering et al., 1997) or corepressors such as Groucho-like proteins (Levanon et al., 1998; Roose et al., 1998) or CBP (Waltzer and Bienz, 1998). During early Xenopus development, b-catenin and XTCF3 interact to modulate expression of the dorsalizing genes Siamois, Twin, and Xnr3 (Brannon et al., 1997; Laurent et al., 1997; Klymkowsky, 1997; McKendry et al., 1997). In mammals, there are at least 16 different Wnt ligands and eight Frizzled receptors that regulate many different cellular responses (Moon et al., 1997). In contrast to the large number of Wnt ligands and receptors, only four TCFs have been identified in humans (Clevers and van de Wetering, 1997). Studies indicate that all TCFs bind to a common DNA consensus motif (Clevers and van de Wetering, 1997). Therefore, it is unclear how appropriate Wnt target genes are selected. One emerging idea is that a particular cell’s response to a signal is determined by the combination of transcriptional regulatory proteins within that cell. In this model, the set of regulatory factors present in the cell and their availability and activity determine which genes are expressed in response to a specific signaling event. In a functional screen to identify genes that regulate Xenopus embryonic axis formation, we discovered that an HMG box protein belonging to the Sox family, XSox17b, can regulate Wnt-responsive gene expression. The Sox proteins are related to TCF/LEFs and bind specific AT-rich DNA sequences (Gubbay et al., 1990; Pevny and Lovell-Badge, 1997; Wegner, 1999). Some can function as classical transcription factors, but most Sox proteins appear to work as context-dependent transcriptional regulators that require other proteins for activity (Kamachi et al., 1995, 1998; Wegner, 1999). More than 20 Sox proteins have been identified in mammals. They are expressed in a wide variety of tissues during

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Figure 1. Isolation of XSox17b (A) Functional screen to identify genes regulating axis formation in Xenopus. Synthetic mRNA was prepared from pools of a dorsal lip cDNA library, and 2–5 ng was injected into 2–4 cell stage Xenopus embryos. Embryos were assayed for changes in morphology at the tail bud stage. A pool with ventralizing activity was serially subdivided and retested until a single active sequence was isolated. (B) Uninjected control embryos. (C) Dorsal injection of the active RNA (1 ng) resulted in ventralized embryos, while (D) ventral injection (1 ng) of the same RNA resulted in relatively normal development. Histological section of an uninjected control embryo (E) shows typical dorsal structures such as neural tube (nt), notochord (n), and somites (s), which are absent in the ventralized embryos (F). (G) The active clone encodes the HMG box protein, XSox17b.

development and have been implicated in a number of cell fate decisions (Pevny and Lovell-Badge, 1997). However, there is no previous evidence to implicate Sox proteins in Wnt signaling or any other known signaling pathways. XSox17b is a regulator of endodermal differentiation that is expressed throughout the presumptive endoderm in early Xenopus embryos (Hudson et al., 1997). We found that XSox17b can inhibit Wnt signals by repressing b-catenin’s transcriptional activity. This repression involves a physical interaction between the carboxyl terminal domain of XSox17b and Armadillo repeats of b-catenin. Two additional Sox proteins, XSox17a (Hudson et al., 1997) and XSox3 (Penzel et al, 1997), likewise bind to b-catenin and inhibit its TCF-mediated signaling activity. Our findings indicate an unexpected interaction between Sox family proteins and b-catenin, suggesting that Sox proteins may have a role in modulating the cell type–specific transcriptional response to a Wnt signal. Results Isolation of XSox17b We performed a functional screen (Smith and Harland, 1991) to identify genes that regulate axis formation in Xenopus. Synthetic mRNA was prepared from pools of a Xenopus gastrula dorsal lip library and microinjected into early Xenopus embryos. Injected embryos were assayed for changes in axial development (Figure 1A). One pool of RNA ventralized embryos when injected in dorsal blastomeres (Figures 1B and 1C). Histological analysis

of these embryos showed that they lacked typical dorsal structures such as the notochord, neural tube, and somites (Figures 1E and 1F). Ventral injection of this same mRNA had little effect on embryonic development (Figures 1B and 1D). The positive pool was serially subdivided and retested until the cDNA responsible for the ventralizing activity was isolated, sequenced, and found to encode the Sox family HMG box protein, XSox17b (Figure 1G). XSox17b Can Inhibit the Dorsal Wnt-like Pathway in Xenopus Embryos TGF-b, BMP, and Wnt-like signaling systems regulate early dorsal/ventral patterning in Xenopus embryos (see Heasman, 1997; Moon and Kimelman, 1998). TGF-bs, such as the nodal-related proteins Xnr1–4 or Vg1, promote dorsal development. BMP signals promote the development of ventrolateral cell types, while BMP antagonists promote dorsal development. A maternal Wnt pathway is critical for specifying dorsal identity. Using the Xenopus axis duplication assay, we tested whether XSox17b interacted functionally with any of these three signaling pathways. A second dorsal axis can be induced in Xenopus embryos by ventral injection of mRNA encoding an active form of Vg1 (bVg1; Thomsen and Melton, 1993), a dominant-negative BMP receptor (dnBMPR; Graff et al., 1994), or Wnts and activating components of the Wnt pathway (McMahon and Moon, 1989; see Moon et al., 1997). Coinjection of XSox17b mRNA inhibited the second axis induced by Xwnt8 mRNA injection but did not affect the second axis induced by either bVg1 or dnBMPR RNAs (Figure 2A),

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Figure 2. XSox17b Can Inhibit Wnt Signaling Downstream of b-catenin (A) The left panels show representative tail bud stage embryos with a second axis induced by the injection of one ventral blastomere at the 4 cell stage with mRNA encoding dnBMPR (250 pg), bVg1 (100 pg), Xwnt8 (5 pg). Panels on the right show representative embryos similarly injected with the addition of XSox17b (500 pg). XSox17b only inhibits the second axis induced by Xwnt8 (lower right). (B) Summary of second axis assays from three to five separate experiments. Injection of mRNA encoding dnBMPR (250 pg), bVg1 (100 pg), Xwnt8 (5 pg), b-catenin (250 pg), or Siamois (10 pg), resulting in a high frequency of embryos with second axes (gray bars). Embryos similarly injected with the same RNAs 1 XSox17b mRNA (500 pg) were scored for percent second axis (black bars). (C) Ventralization assay. RNA encoding XSox17b (1 ng) or XSox17b (1 ng) 1 Xwnt8 (10 pg); XSox17b (1 ng) 1 b-catenin (250 pg); XSox17b (1 ng) 1 XTcf-3 (2 ng); XSox17b (1 ng) 1 Siamois (20 pg) was injected into both dorsal blastomeres at the 4 cell stage (black bars). Control injections (gray bars) without XSox17b were Xwnt8 (10 pg), b-catenin (250 pg), XTcf3 (2 ng), DN-XTcf3 (500 pg), DN-XTcf3 (500 pg) 1 XTcf3 (2 ng), and Siamois (20 pg). The dorsal development of the resulting embryos were scored by a dorsal anterior index (DAI; Kao and Elinson, 1988). A score of 5 indicates normal development, and a score of 0 indicates severely ventralized embryos. Only Siamois rescued the ventralized phenotype caused by dorsal XSox17b injection. (D) XSox17b operates downstream of b-catenin but upstream from Siamois.

indicating that XSox17b specifically inhibits the dorsal Wnt pathway. XSox17b Acts Downstream of b-catenin and Upstream of Siamois To learn how XSox17b blocks signaling, we tested the epistatic relationship of XSox17b to various components of the Wnt pathway. First, we examined the ability of XSox17b RNA to inhibit the induction of second axis by RNAs encoding various Wnt pathway components. An ectopic second axis is induced in Xenopus embryos by ventral injection of mRNA encoding Xwnt, b-catenin, or the Wnt target homeobox protein Siamois (Figure 2B; McMahon and Moon, 1989; Funayama et al., 1995; Lemaire et al., 1995). Injection of XSox17b mRNA effectively blocked the induction of a second axis by either Xwnt8 or b-catenin mRNAs, but failed to block second axes induced by the injection of Siamois mRNA (Figure 2B).

Secondly, we determined whether coinjcetion of various Wnt pathway components could suppress the ventralization induced by dorsal injection of XSox17b RNA. The extent of dorsal development was scored by a dorsal anterior index (DAI) (Kao and Elinson, 1988); a score of 5 indicates normal dorsal development, and a score of 0 indicates a totally ventralized embryo. Embryos injected dorsally with XSox17b mRNA alone were ventralized with an average DAI score of 1.0, whereas embryos injected dorsally with Xwnt8, b-catenin, XTcf-3, or Siamois RNAs resulted in average DAI scores of 5.5, 5.5, 2.9, and 6.7, respectively. Coinjection of Xwnt8, b-catenin, or XTcf-3 RNAs did not rescue the ventralized phenotype of XSox17b RNA-injected embryos (average DAI scores of 1.6, 0.4, and 1.6, respectively) (Figure 2C). In contrast, coinjection of Siamois mRNA efficiently rescued dorsal development (DAI 4.9) (Figure 2C). These results indicate that XSox17b inhibits the Wnt

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Figure 3. XSox17b Represses Wnt/b-catenin Stimulated Transcription and Activates Endodermal Genes (A and B) Human 293T cells were cotransfected with OT reporter construct containing TCF consensus binding sites (Korinek et al., 1997) and expression plasmids encoding (A) S37A-b-catenin (0.2 mg), S37A-b-catenin (0.2 mg) 1 XSox17b (2 mg and 4 mg) and (B) Wnt1 (2 mg), Wnt1 (2 mg) 1 XSox17b (2 mg and 4 mg). XSox17b inhibits b-catenin and Wnt1 activation of the OT reporter. (C) Schematic of XSox17b:GAL4 DNA-binding domain fusion constructs and the 5x GAL DNA binding site–luciferase construct. (D) Human 293T cells cotransfected with 5x GAL reporter (0.5 mg) and 1 mg of the indicated fusion construct, with (gray bars) or without (black bars) 1 mg of S37A-b-catenin. The C-terminal region of XSox17b has transactivation capacity. Transfection experiments were repeated two to four times, and a representative experiment is shown. (E) Embryos were injected with mRNA encoding XSox17b (125 pg, 250 pg, 500 pg, 1000 pg), pt-b-catenin (30 pg), and pt-b-catenin (30 pg), 1 XSox17b (125 pg, 250 pg, 500 pg, 1000 pg). Animal caps dissected at blastula stage were cultured until gastrula stage and (F) analyzed by RTPCR for expression of the Wnt/b-catenin responsive genes Siamois and Xnr3, the endodermal gene Endodermin, and Ef-1a as a loading control.

pathway downstream of b-catenin and upstream of the Wnt target gene Siamois (Figure 2D). XSox17b Represses Wnt/b-catenin-Stimulated Transcription The epistasis experiments suggested that XSox17b represses Wnt signaling by inhibiting transcription of TCFregulated genes, such as Siamois. We therefore examined the effects of XSox17b on Wnt/b-catenin-induced

transcription of a Wnt-responsive reporter construct in human 293T cells. The OT luciferase reporter contains three TCF consensus binding sites upstream of a minimal promoter and is strongly activated by positive components of the Wnt signaling pathway (Korinek et al., 1997). Transfection with a stabilized, mutant form of b-catenin (S37A-b-catenin) or Wnt-1 activated transcription of the OT reporter construct. Transfection with XSox17b plasmid alone did not stimulate transcription

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of the reporter, but cotransfection of b-catenin or Wnt1 with XSox17b significantly repressed transactivation of the OT reporter in a dose-dependent manner (Figures 3A and 3B). This suggested that XSox17b can block Wnt signaling by inhibiting the transcriptional activity of b-catenin. Since XSox17b repressed Wnt- and b-catenin-stimulated transcription from a TCF-dependent reporter, we asked whether XSox17b had any intrinsic transcriptional repression activity. The N-terminal (amino acids 1–56) and the C-terminal (amino acids 135–373) regions of XSox17b were each fused to a GAL4 DNA-binding domain. These fusion constructs were cotransfected into human 293T cells with a luciferase reporter plasmid containing five GAL4 DNA binding sites (Figure 3C), and the transcriptional activity of the reporter was assayed. Surprisingly, we detected no repressive activity, but the GAL4:Sox17(135–373) fusion potently activated transcription (Figure 3D). Cotransfection of b-catenin with the GAL4:Sox17 fusions did not alter their transcriptional activity (Figure 3D). XSox17b Can Simultaneously Repress Wnt-Responsive Genes and Activate Endodermal Genes Our results with OT and Gal4 reporter constructs in 293T cells suggested that XSox17b represses b-catenin-stimulated transcription, yet harbors a transcriptional activation domain. Using Xenopus animal caps, we tested whether XSox17b could both activate and inhibit the expression of endogenous genes (Figure 3E). When XSox17b is ectopically expressed in Xenopus animal cap cells, the endodermal gene Endodermin is transcribed (Figure 3F; Hudson et al., 1997). Overexpression of b-catenin in animal cap cells induces the transcription of the Wnt-responsive genes Siamois and Xnr3 (Figure 3F; Smith et al., 1995; Carnac et al., 1996). Coinjected of b-catenin and XSox17b RNAs resulted in a simultaneous activation of Endodermin and a repression of Siamois and Xnr3 expression (Figure 3F). Therefore, XSox17b can promote transcription of endodermal genes while it represses Wnt-responsive gene expression, suggesting that XSox17b may normally perform both functions in the embryo. XSox17b Does Not Bind to a TCF Consensus DNA Binding Site Members of the TCF and Sox protein families bind to similar consensus sequences of 59-[A/T][A/T]CAA[A/T] GG239 (Clevers and van de Wetering, 1997). To determine if the repressive effects of XSox17b on Wnt signaling were mediated through a competition between XSox17b and TCF proteins for binding to TCF DNA binding sites, we examined the binding specificity of XSox17b using an electrophoretic mobility shift assay. Epitope-tagged forms of hTCF4, mLEF1, and XTCF3 all bound to a DNA probe containing a TCF consensus sequence, and antibodies directed against the tagging epitope supershifted the TCF/LEF-probe complexes (Figure 4A). In contrast, very little if any XSox17b bound to the TCF probe (Figure 4A). Epitope-tagged XSox17b bound to a consensus sequence for murine Sox17, ATTGTT (Kanai et al., 1996), and addition of antibodies

Figure 4. XSox17b Does Not Bind to a TCF/LEF Consensus DNA Binding Site (A) Electrophoretic mobility shift assays (EMSAs) performed using in vitro translated TCF4-V5, XSox17b-V5, LEF1-V5, and XTCF3-HA epitope–tagged proteins, without and with anti-epitope tag antibody and TCF DNA consensus sequence probe. (B) EMSAs performed with TCF4-V5, XSox17b-V5, and LEF1-V5, without and with anti-epitope tag antibody and a murine Sox17 DNA consensus sequence probe.

against the epitope-tagged XSox17b protein supershifted the complex (Figure 4B). TCF4 failed to bind to the Sox probe, as did XTCF3 (data not shown), but LEF1 appeared to bind modestly (Figure 4B). While it may be possible that some endogenous promoters have sites that can be bound by either TCF or XSox17b, our data suggest that the ability of XSox17b to suppress Wnt/ b-catenin signaling is unlikely to be due to occupation of TCF binding sites. The C-Terminal Region of XSox17b Is Critical for Repression of b-catenin Activity To better understand the mechanism by which XSox17b represses b-catenin signaling, we performed a structure–function analysis of the XSox17b protein. Various XSox17b deletion mutants (Figure 5A) were tested for their ability to inhibit b-catenin activity in Xenopus second axis (Figure 5B) and 293T cell transcription assays

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Figure 5. The C Terminus of XSox17b Is Essential for Repressing Wnt Signaling (A) Schematic of the XSox17b deletion mutants. (B) Secondary axis assay. mRNA encoding mutant stabilized pt-b-catenin (Yost et al., 1996) (0.1 ng), XSox17b (1 ng), or pt-b-catenin (0.1 ng) 1 each of the dXSox17b mutants (1 ng) was injected into one ventral blastomere at the 4 cell stage. Tailbud stage embryos were scored for the presence of second axes, and the compiled results of four separate injection experiments are shown. (C) OT transcription assay. 293T cells were cotransfected with the OT reporter construct (1 mg) and expression plasmids encoding S37Ab-catenin (0.3 mg), wtXSox17b-V5 (2 mg and 4 mg), S37A-b-catenin (0.3 mg) 1 each dXSox17b-V5 deletion construct (2 mg and 4 mg; black triangles indicate increasing dose). Experiments were repeated two to four times, and a representative experiment is shown. The C-terminal region of XSox17b is essential for repression of b-catenin transactivation.

(Figure 5C). Ventral injection of mRNA encoding a stabilized b-catenin mutant (pt-b-catenin; Yost et al., 1996) induced secondary axis formation at high frequency (80%). Coinjection of pt-b-catenin and wild-type XSox17b RNAs completely repressed second axis (0%) induction (Figure 5B), whereas coinjection of RNAs encoding b-catenin and the unrelated HMG box protein HBP had no effect on secondary axis induction by b-catenin (78%, n 5 35; data not shown). Removal of amino acids 315–373 (d1–315) completely eliminated the ability of XSox17b to repress the second axis induced by b-catenin. In contrast, the N-terminal deletion mutants d56–373 and d119–373 were as effective as wild-type XSox17b in repressing b-catenin-induced second axes. N-terminal deletions beyond amino acid 135, however, failed to inhibit b-catenin activity significantly. Thus, the C-terminal region of XSox17b, without a DNA-binding HMG box, is sufficient to inhibit b-catenin’s activity in this assay. XSox17b deletion mutants were also tested for their ability to repress b-catenin activation of the OT reporter construct in human 293T cells. Cells were transfected with stabilized S37A-b-catenin or with S37A-b-catenin plus increasing doses of the XSox17b deletion mutants (Figure 5C). Consistent with the second axis assay, the C-terminal region of XSox17b was required to repress b-catenin transactivation of the reporter. Cotransfection

of wild-type XSox17b with b-catenin inhibited transactivation by b-catenin, while d1–56, d1–150, d1–270, and d1–315 were largely ineffective in inhibiting activation of the reporter construct. In contrast to our results in secondary axis assays, the HMG box was also required, as d119–373 failed to inhibit activation of a Wnt-responsive reporter. Western blot analysis indicated that all of the mutant polypeptides were expressed efficiently in 293T cells (data not shown). The basis for the difference between the OT reporter and embryo assay systems is unclear, but, importantly, both indicate that the C-terminal region of XSox17b is essential for inhibiting b-catenin signaling activity. The C Terminus of XSox17b Binds b-catenin To determine how XSox17b suppresses b-catenin’s signaling activity, we asked if XSox17b and b-catenin could physically interact. Using a GST-b-catenin fusion protein immobilized on agarose beads, we found that 35 S-labeled XSox17b protein bound to b-catenin to a similar extent as the positive control, XTcf-3, while DNXTcf-3, which lacks the b-catenin-binding domain (Molenaar et al., 1996) exhibited negligible binding to GST-b-catenin (Figure 6A). Based on sequence comparisons with the established b-catenin binding site in TCFs (Behrens et al., 1996;

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Figure 6. The C Terminus of XSox17b Binds to b-catenin (A) XSox17b binds b-catenin in vitro. 35S-labeled XTcf-3, DNXTcf-3, and XSox17b proteins were incubated with 1 mg of GST-b-catenin fusion protein (b-cat) or 1 mg of GST alone, immobilized to agarose beads. Ten percent of the input proteins (i) and bound proteins were resolved on SDS-PAGE and visualized by fluorography. (B) b-catenin binds to the C terminus of XSox17b. The indicated 35 S-labeled XSox17b deletion proteins were incubated with GSTb-catenin agarose. Ten percent of the input proteins (i) and the GST-b-catenin bound proteins (b) were resolved on SDS-PAGE and visualized by fluorography. A representative binding experiment is shown. Relative binding of each mutant, summarized from three to four experiments is shown; 111, 60%–100% of wild-type binding; 11, 30%–60%; 1, 10%–30%; 6, z10%; and 2, ,10%. (C) XSox17b associates with b-catenin in vivo. 293T cells were transfected with (1) or without (2) S37A-b-catenin-HA (2 mg) and expression plasmids (8 mg) encoding mLEF1-V5, hTCF4-V5, DN-hTCF4V5, XSox17b-V5, or dSox17b 1–150. The top panel shows an anti-V5 Western of the resulting input lysates. Cell lysates were subjected to immunoprecipitation with anti-HA antibody, followed by anti-HA Western to show levels of b-catenin-HA (middle panel) and an antiV5 epitope Western (bottom panel) to visualize proteins coprecipitating with b-catenin-HA.

Huber et al., 1996; Molenaar et al., 1996; Hsu et al., 1998), no obvious b-catenin binding region is apparent in XSox17b. We therefore mapped the b-catenin binding site on XSox17b in vitro using GST-b-catenin and the XSox17b deletion mutants. Figure 6B shows an example of a binding assay and the relative binding of each mutant summarized from three to four different binding experiments. Progressive C-terminal deletions of XSox17b dramatically reduced GST-b-catenin binding. The mutant protein d1–315 bound about 30% as well as wildtype XSox17b, d1–270 showed about 10% binding, and d-1–150 failed to interact with GST-b-catenin. In contrast, N-terminal deletions up to and including the HMG box had no effect on b-catenin binding; mutant proteins d56–373, d119–373, and d135–373 bound to GSTb-catenin almost as well as full-length XSox17b. Further deletions of amino acids 135–200 significantly impaired b-catenin binding. These results suggest that at least two regions of XSox17b, one just carboxyl to the HMG box and the other at the extreme C terminus, are important for b-catenin binding. To confirm that the interaction between XSox17b and b-catenin occurs in vivo, coimmunoprecipitation experiments were performed with HA-tagged b-catenin and V5 epitope–tagged versions of the wild-type XSox17bV5 or the d1–150-V5 deletion mutant. 293T cells were transfected with mLEF1-V5, hTCF4-V5 (positive controls), DN-hTCF4-V5 (negative control lacking the b-catenin binding site), XSox17b-V5, and d1–150-V5 with or without HA-S37A-b-catenin. b-catenin was precipitated from the resulting cell lysates with anti-HA antibodies, and the immunoprecipitates were then subjected to anti-V5 epitope Western blotting. XSox17b-V5 coprecipitated with HA-b-catenin, as did the positive control proteins, mLEF1-V5 and hTCF4-V5 (Figure 6C). The XSox17b deletion, d1–150-V5, which lacks the b-catenin binding site, failed to coprecipitate with HA-b-catenin, as did the negative control, DN-hTCF4-V5. Therefore, XSox17b and b-catenin can form a physical complex in vivo via the C-terminal amino acids 135–373 of XSox17b, the same regions required for inhibition of b-catenin activity. We conclude that the repression of Wnt/b-catenin signaling by XSox17b is due largely to the physical interaction between b-catenin and XSox17b. XSox17b and XTCF3 Bind to Overlapping Sites on b-catenin To better understand the mechanism by which XSox17b inhibits b-catenin/TCF activity, we examined the possibility that XSox17b and TCF compete for a limited pool of b-catenin. If such a mechanism functions in vivo, then a low dose of XSox17b should be sufficient to inhibit b-catenin activity in embryos. Using the Xenopus dorsal axis induction assay, we found that as little as 50 pg of injected XSox17b RNA could inhibit the second axis induced by 100 pg of pt-b-catenin RNA (Figure 7A). This amount of injected XSox17b RNA is similar to the levels that induce endodermal genes (Figure 3F; Hudson et al., 1997) and is lower than the RNA doses typically used in inhibition experiments with Wnt signaling antagonists such as DNTCF, Groucho, and GSK-3 (Molenaar et al., 1996; Yost et al., 1996; Roose et al., 1998). Furthermore, in both the second axis assay (Figure 7A) and the OT

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Figure 7. Functional and Structural Analysis of the Interaction between XSox17b and b-catenin (A) Second axis assay. Embryos were injected with the indicated doses of XSox17b and pt-b-catenin RNA, and the frequency of secondary axis was scored. (B) OT reporter assay. Human 293T cells were cotransfected with OT reporter construct containing TCF consensus binding sites (Korinek et al., 1997) and the indicated amounts of expression plasmids encoding S37Ab-catenin and XSox17b. (C and D) The indicated 35S-labeled Xb-catenin deletion proteins were incubated with nickelagarose resin or nickel-resin coupled with 1 mg of His-XSox17b protein (C). The relative binding of each deletion protein to HisXSox17b (based on three separate experiments) is indicated (D), and a representative experiment is shown. The minimal XSox17b binding site overlaps with the established TCF binding site.

reporter assay (Figure 7B), increasing the amount of b-catenin alleviated the repression by XSox17b suggesting that the repression involves competing for a limited pool of b-catenin. To investigate how the b-catenin/XSox17b interaction could inhibit b-catenin/TCF activity, we mapped the XSox17b binding site on b-catenin. Truncated 35 S-labeled Xb-catenin proteins were incubated with nickel-agarose alone or nickel-agarose coupled with His-XSox17b fusion protein. The relative binding of truncated b-catenin proteins to His-XSox17b and a representative experiment are shown in Figures 7C and 7D. We found that a minimum XSox17b binding site resided between the Armadillo repeats 3–6 of b-catenin, which overlaps with the established TCF binding site between Armadillo repeats 4–9 (Behrens et al., 1996; van de Wetering et al., 1997; Wilbert and Nusse, 1998). These results suggest that XSox17b and TCF bind overlapping sites on b-catenin. Other Sox Proteins Can Bind to b-catenin and Inhibit Its Signaling Activity To assess whether the interaction with b-catenin is a more general feature of Sox proteins, we examined the activities of XSox17a (Hudson et al., 1997), a related protein, and XSox3 (Penzel et al., 1997), a protein from a different Sox subfamily. In Xenopus second axis assays

(Figure 8A) and OT reporter assays (Figure 8B), both XSox17a and XSox3 repressed b-catenin signaling, while an unrelated HMG box protein XHBP did not (Figure 8A). Furthermore, XSox17a and XSox3 bound to GST-b-catenin in vitro (Figure 8C) and coimmunoprecipitated with b-catenin in extracts from 293T cells (Figure 8D). These results suggest that the ability of Sox proteins to interact with b-catenin and repress its signaling activity may be a common feature of many Sox proteins. Discussion In a functional screen to identify inhibitors of axis formation, we have discovered that a Sox protein, XSox17b, as well as XSox17a and XSox3, can repress Wnt/ b-catenin-responsive gene expression through physical association with b-catenin. Over 20 Sox proteins are known, showing highly restricted patterns of expression during development. There is compelling evidence from mutations in humans (Wagner et al., 1994), mice (LovellBadge and Robertson, 1990; Schillam et al., 1996), and flies (Nambu and Nambu, 1996; Russell et al., 1996) that Sox genes are functionally important for development. Our findings with XSox17a/b and XSox3 suggest that Sox proteins can modulate the response to Wnt signals during development. Specifically, cells expressing relatively high levels of XSox17a/b or XSox3 would not transcribe b-catenin/TCF target genes in response to a Wnt

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Figure 8. XSox17a and XSox3 Bind to b-catenin and Interfere with Its Signaling (A) Secondary axis assay. mRNA encoding mutant stabilized pt-b-catenin (Yost et al., 1996) (0.1 ng), or pt-b-catenin (0.1 ng) 1 XSox17b, XSox17a, and XSox3 (0.5 ng) was injected into one ventral blastomere at the 4 cell stage. Tail bud stage embryos were scored for second axes. (B) OT transcription assay. 293T cells were cotransfected with the OT reporter construct (1 mg) and expression plasmids encoding S37A-b-catenin (0.3 mg), XSox17b-V5 (4 mg), XSox17a-V5 (4 mg), XSox3-V5 (4 mg), or S37Ab-catenin (0.3 mg) 1 each XSox17b-V5, XSox17a-v5, XSox3-V5 (2 mg and 4 mg; black triangles indicate increasing dose). (C) XSox17a and XSox3 bind b-catenin in vitro. 35S-labeled XSox17b, dXSox17b 1–150, XSox17a, and XSox3 proteins were incubated with 1 mg of GST-b-catenin fusion protein immobilized to agarose beads. Ten percent of the input proteins (i) and bound proteins (b). (D) XSox17a and XSox3 associate with b-catenin in vivo. 293T cells were cotransfected with S37A-b-catenin (2 mg) and expression plasmids (8 mg) encoding DN-hTCF4-V5 (lane 1), mLEF1-V5 (lane 2), hTCF4-V5 (lane 3), XSox17b-V5 (lane 4), XSox3-V5 (lane 5), or XSox17a-V5 (lane 6). The top panel shows an anti-V5 Western of the resulting extracts. Cell lysates were divided and subjected to immunoprecipitation with anti-b-catenin (1) or a negative control anti-mER antibody (2), followed by an anti-b-catenin Western to show levels of b-catenin (middle panel) and an anti-V5 epitope Western (bottom panel) to visualize proteins coprecipitating with b-catenin.

signal, while nearby cells expressing lower levels of these Sox proteins would respond. The Mechanism of Sox Interference with Wnt Signaling A number of models could explain how XSox17a/b and XSox3 repress b-catenin/TCF activity. Our data suggests XSox17b can bind to Armadillo repeats that overlap with the TCF binding site on b-catenin. The simplest possibility is that Sox binds to b-catenin and excludes TCFs from interacting with b-catenin. In cells with higher levels of Sox protein, b-catenin would be sequestered and rendered unavailable for interaction with TCF. Thus, the relative levels of TCF and Sox proteins would determine how a cell responds to a Wnt signal. Alternatively, it is possible that Sox proteins bind to a complex of b-catenin and TCF. In this model, Sox proteins would repress TCF/b-catenin activity as part of a ternary or higher order complex. What Are the Endogenous Functions of XSox3 and XSox17a/b in Relation to Wnt Signaling? XSox3 RNA is a maternal transcript that is restricted to the animal hemisphere of the blastula stage embryo (Penzel et al., 1997; Y. Vourgourakis and M. W. K., unpublished data). Given that nuclear b-catenin is present throughout the future dorsal hemisphere of the early Xenopus embyo (see Schneider et al., 1996), we hypothesize that maternal XSox3 acts to restrict the expression of b-catenin-regulated dorsalizing genes, such as Siamois, to the vegetal region of the blastula. Hudson et al. (1997) have shown that both XSox17a and XSox17b are expressed throughout the presumptive endoderm in the gastrula stage embryo, and XSox17a/b

appear to play a critical role in endoderm formation (Figure 3; Hudson et al., 1997). In this study, we have used Xenopus axis formation as an in vivo assay for Wnt signaling, but it is unclear if XSox17a/b are normally involved in axis formation. We speculate that repression of Wnt signals by XSox17a/b may be important for endoderm development. This is an intriguing possibility in light of the fact that, in C. elegans, Wnt signaling is required to repress the TCF-like factor Pop-1 in the presumptive endoderm lineage (Lin et al., 1997; Rocheleau et al., 1997). It is also possible that interaction with b-catenin could be important for transactivation at Sox target gene promoters, and it is tempting to speculate that Sox proteins, like TCFs, could act as Wnt effectors.

Bifunctional Sox Proteins Our results demonstrate that XSox17b is a bifunctional protein that can both activate endodermal genes and repress Wnt signaling. In most cases, Sox proteins have been described as transcriptional activators (van de Wetering et al., 1993; Hosking et al., 1995; Wegner, 1999). This is not the first time that a Sox protein has been shown to have both activator and repressor functions. Sox2 can synergize with Oct-3 to transactivate the FGF-4 enhancer (Yuan et al. 1995) but acts as a corepressor with Oct-4 to inhibit the osteopontin enhancer (Botquin et al., 1998). Recently, another type of transcription factor has been reported to have both DNA-dependent and DNAindependent regulatory functions, similar to those that we have described for XSox17b. The glucocorticoid receptor (GR) acts as a transcriptional activator by binding

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to glucocorticoid response elements in various promoters, but it acts as a transcriptional inhibitor of the collagenase gene by complexing with the Fos and Jun transcription factors, independent of binding to DNA (Reichardt et al., 1998). The duality exhibited by XSox17b and GR may represent a growing paradigm wherein transcription factors have multiple functions. Our results indicate that inhibition of Wnt signaling, as described here, is a feature common to at least three Sox proteins. XSox17a/b and XSox3 represent two different Sox family subgroups, and these proteins have no obvious shared sequences that might form a b-catenin binding site. It is possible, however, that tertiary rather than primary sequences are involved, and preliminary results indicate that a number of Sox proteins from other species and subgroups bind to b-catenin (A. M. Z. and G. D. B., unpublished data). These results raise the intriguing possibility that Sox proteins play a general role in regulating how tissues respond to Wnt signals. Experimental Procedures Xenopus Embryo Manipulations and Expression Cloning Embryos were maintained as previously described (Gurdon, 1977). For expression screening, cDNA was generated from mRNA isolated from the stage 10 dorsal lip region and cloned into the Not1/EcoR1 sites of pRN3 (Lemaire et al., 1995). Plasmid DNA was prepared from plates, each containing 100–200 clones of the unamplified cDNA library. RNA (2–5 ng) prepared from each pool was microinjected into 2–4 cell stage embryos. The positive pool was serially subdivided and retested to isolate the active clone. DNA Constructs and Preparation of Synthetic mRNA XSox17b clones were generated by PCR amplification using Pfu polymerase and cloned into pT7TSHA (Zorn and Krieg, 1997). Plasmids were linearized, and capped synthetic mRNA was transcribed using a MEGA script kit (Ambion), with a cap analog 7mGpppG:GTP ratio of 5:1, as follows: cDNA pools in pRN3 (Sfi1, T3 polymerase); pRN3-XSox17b (Sfi1, T3 polymerase); pT7TSHA-XSox17b and pT7TSHA-XSox17b-deletion constructs (Pst1, T7 polymerase); pT7TSHA-XHBP (Pst1, T7 polymerase); pSPXSox17a (Hudson et al., 1997); pT7TS-XSox3 (a gift from Rob Grainger); p64T-dnBMPR (Graff et al., 1994); pRN3-Sia (Lemaire et al., 1995); pCS2-b-catenin and pCS2-pt-b-catenin (Yost et al., 1996); pcDNA-XTcf-3 and pcDNADNXTcf-3 (Molenaar et al., 1996); p64T-bVg1 (Thomsen and Melton, 1993). Xenopus b-catenin and XSox17b reading frames were amplified using Pfu and cloned into pGEX-3X and pET16 to produce GSTb-catenin and His-XSox71b, respectively. The 5xGal4 luciferase reporter (Li et al., 1998) and OT reporters were generously provided by Jian-Ming Li (NCI, Bethesda, MD) and Bert Vogelstein (Johns Hopkins University, Baltimore, MD), respectively. The pON-mWnt-1 expression plasmid is described in Kitajewski et al. (1992). Fulllength human b-catenin sequence was amplified with Pfu and cloned into pcDNA3.1 (Invitrogen) or an HA epitope–tagged version into pMH (Boehringer Mannheim). A serine 37 to alanine point mutation was generated in human b-catenin using a site-directed mutagenesis kit (Stratagene). To generate V5 epitope–tagged constructs, the appropriate coding regions were amplified with Pfu and cloned into pcDNA6/V5-His expression vectors (Invitrogen). For Sox17:GAL4 fusion experiments, Pfu amplified XSox17b fragments (aa 1–56 and aa 135–373) were cloned in frame with the GAL4 DNA-binding domain into a CMV expression vector (a gift from Tony Kouzarides, Wellcome/CRC Institute). In Vitro Protein Binding Assays GST-b-catenin and His-XSox17b were expressed in bacteria and purified on glutathione or nickel coupled agarose resin, respectively. 35 S-labeled proteins were produced in TNT translation reactions (Promega). Typically, 1–2 ml of a translation reaction was incubated

with 1 mg of fusion protein coupled to agarose resin at room temperature for 1 hr in 400 ml of binding buffer. GST-agarose binding buffer contains 25 mM HEPES (pH 7.5), 12.5 mM MgCl, 300 mM KCl, 20% glycerol, 0.1% NP-40, 1 mM DTT, and 1 mg/ml BSA. Nickel-agarose binding buffer contains 20 mM Tris (pH 8), 150 mM NaCl, 40 mM imidazole, 0.5% NP-40, 20% glycerol, 10 mM MgCl2, and 1 mg/ml BSA. GST-agarose pellets were washed five times in NETN (20 mM Tris [pH 8], 800 mM NaCl, 1 mM EDTA, 0.5% NP-40), and nickelagarose pellets were washed five times in 20 mM Tris (pH 8), 300 mM NaCl, 40 mM imidazole, and 0.5% NP-40. Bound proteins were resolved on SDS-PAGE and visualized by fluorography. Cell Culture and Transient Transfections Human 293T cells were transfected by CaPO4 precipitation (Stratagene). For OT reporter assays, 1 mg of OT and 1 mg of pcDNA 3.1-LacZ were cotransfected with the indicated expression plasmids. For Gal4 luciferase reporter studies, 0.5 mg of 5xGal4 luciferase reporter, 1 mg of pcDNA3.1-LacZ, and 1 mg of GAL4 DNA-binding domain fusion constructs were cotransfected, with or without 1 mg of pcDNA3.1 S37A-b-catenin. Transfection was done in duplicate. Cells were harvested, after 36 hr, extracts prepared, and luciferase activity was measured (Promega Luciferase Assay System) and normalized for transfection efficiency by b-galactosidase activity. DNA Binding Assays Probes were generated by annealing the following complementary oligonucleotide pairs: TCF probe, 59-ggtacccCCTTTGATCttacc-39 and 59-aagcttggtaaGATCAAAGGg-39; Sox probe, 59-ggtaccgca gAACAATggcg-39 and 59-aagcttcgccATTGTTctgc-39. Probes were labeled by standard Klenow fill-in reactions. Proteins were made in T7 TNT reactions (Promega), and protein products were confirmed by Western blot analysis (data not shown). Binding reactions and gel shifts were performed as described in Korinek et al. (1997). Immunoprecipitation and Western Blots Cells were lysed on ice with 1 ml of lysis buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 0.5% NP-40, and protease inhibitors) and microcentrifuged at 14,000 rpm for 10 min at 48C. For immunoprecipitations, lysates were normalized for total protein and incubated for 3 hr at 48C, with 1 mg of rabbit anti-HA, anti-b-catenin, or antiestrogen receptor polyclonal antibody (Santa Cruz Biotechnology) and Protein-A agarose beads. Agarose beads were washed three times with 1 ml of 0.23 NP-40 lysis buffer, and bound proteins were eluted in SDS sample buffer. Standard Western immunoblotting procedures were used. AntiV5-HRP antibody (Invitrogen) was diluted 1:2000. Rat anti-HA monoclonal antibody (Boehringer Mannheim) was diluted 1:1000, followed by goat anti-rat IgG-peroxidase (Boehringer Mannheim) diluted 1:10,000. Anti-b-catenin:HRPO antibody (Transduction Laboratories) was diluted 1:1000. Blots were subjected to ECL and exposed to film. Acknowledgments We thank Paul Robbins, Rudolf Grosschedl, Hans Clevers, Bert Vogelstein, Doug Melton, Tony Kouzarides, Hugh Woodland, Rob Grainger, and Randy Moon for generously providing DNA constructs. A. M. Z. thanks J. B. Gurdon for encouragement and support and D. Niranjin for help with the expression screen. We also thank M. Chamorro, J. M. Li, M. Erdos, S. Lipkin, and T. Kouzarides for technical advice and helpful discussions and T. Wynshaw-Boris, G. Fisher, and F. Stennard for critical readings of this manuscript. G. D. B. is a Howard Hughes Medical Institute National Institutes of Health Research Scholar. B. O. W. is a fellow of the Damon Runyon–Walter Winchell Cancer Research Fund. M. W. K. was supported by NIH grant GM54001. This work was supported by grants from the Cancer Research Campaign (UK) and the Wellcome Trust to A. M. Z. Received February 26, 1999; revised July 20, 1999.

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