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Figure 1. Uch37 positively regulates Wnt signalling downstream of β-catenin stabilization.(a) TOPflash assay using whole embryos (stage 10.5, 10 embryos were used for each sample). Four-cell stage embryos were animally injected with indicated reagents (150 pg TOPflash reporter; 50 pg pRL-TK; 40 ng Co MO; 40 ng Uch37 MO; 20 pg Wnt8 mRNA; 1 ng Re.Uch37 mRNA). (b) Expression levels of Wnt target genes (siamois and nodal3.1) were examined by RT-PCR analysis using Xenopus animal cap tissues. Two-cell stage embryos were animally injected with indicated reagents (5 pg Wnt8 mRNA; 20 ng Co MO; 20 ng Uch37 MO; 1 ng Re.Uch37 mRNA). Animal cap explants were isolated at stage 9 and cultured until stage 11. WE, Whole embryos; (-), -RT; ODC, ornithine decarboxylase loading control. Full images are presented in Supplementary information (Fig. S12) (c) Axis duplication assay. Four-cell stage embryos were injected in one ventrovegetal blastomere with indicated reagents (5 pg Wnt8 mRNA; 1 ng Uch37 mRNA; 20 ng Uch37 MO). See also Table S2. (d) Quantified result of c. (e) TOPflash assay in HepG2 cells. After transfection of reporter constructs into stable cells (sh Control or sh Uch37) for 48 h, luciferase activity was measured. Knockdown of β-catenin indicates a positive control for downregulation of Wnt activity. (f) Quantitative real-time PCR (qPCR) analysis for the expression of Wnt-target genes (cyclinD1 and c-myc) in stable HepG2 cells. Knockdown of β-catenin indicates a positive control for downregulation of Wnt activity. The quantities of indicated mRNA were normalized by β-actin. Data represent average values from three independent experiments performed. Error bars indicate standard deviations of triplicate. *p < 0.002; **p < 0.001 (two-tailed Student’s ttest). (g) Axis duplication assay. Four-cell stage embryos were injected in one ventrovegetal blastomere with indicated reagents (5 pg Wnt8 mRNA; 200 pg Dvl2 mRNA; 25 pg β-catenin mRNA; 20 ng Co MO; 20 ng Uch37 MO). See also Supplementary Fig. S5a and Table S3).
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Figure 2. Uch37 physically interacts with Tcf7 in nucleus.(a) Co-IP assay in HEK293FT cells. Cells were transfected with indicated plasmids. myc-ADRM1 is known as an interacting partner of Uch37 and used as a positive control11. 48 h after transfection, cell lysates were precipitated with anti-myc antibody. (b) GST pulldown assay using purified proteins. His-Uch37 and GST-Tcf7 proteins were incubated and then subjected to immunoblotting. (c) Co-IP assay using nuclear extract of Xenopus gastrula embryo. One-cell stage embryos were injected with myc-Tcf7 (500 pg) and HA-Uch37 (500 pg) and cultured until stage 11, and then subjected to fractionation. Nuclear extracts were subjected to Co-IP assay using anti-myc or anti-HA antibody. (d) Co-IP assay using HEK293FT cells, HA-Uch37 and indicated myc-tagged truncated mutants of Tcf7 were transfected. Cell lysates were immunoprecipitated with anti-myc antibody. Truncated mutants of Tcf7 are depicted on the left. Tcf7, a full length; Tcf7 1–196, amino acids 1–196; Tcf7 240–365, amino acids 240–365; Tcf7 52–239, amino acids 52–239. Full images of all Fig. 2 are presented in Supplementary information (Fig. S9).
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Figure 3. Uch37 deubiquitinates Tcf7 protein, but is not involved in protein stabilization.(a) In vivo ubiquitination assay in HEK293FT cells. Cells were transfected with indicated plasmids (1 μg myc-Tcf7; 3 μg flag-Ub; 2 μg and 4 μg Uch37). After 48 h, samples were prepared as described in materials and methods and then precipitated with anti-myc antibody. (b) In vitro ubiquitination assay. HEK293FT cells were co-transfected with both myc-Tcf7 and HA-Ub plasmids to express polyubiquitinated Tcf7. After 48 h, cell lysates were immunoprecipitated using anti-myc antibody to prepare Polyubiquitinated Tcf7. Precipitated polyubiqutinated Tcf7 was then incubated with indicated amount of His-Uch37 protein. (c) Signal intensity of polyubiquitinated Tcf7 in b was quantified using image J software. (d) In vivo ubiquitination assay in HEK293FT cells. Cells were transfected with indicated plasmids and siRNAs. After 72 h, total proteins were precipitated with anti-myc antibody. Cell lysis and detailed procedures are described in materials and methods. In HA-K63, all lysines except Lys-63 were mutated to arginines. In HA-K48, all lysines except Lys-48 were mutated to arginines. (e) Pulse-chase test using HEK293FT cells. 48 hours after the transfection as indicated, 100 μg/ml cycloheximide (CHX, sigma) was treated for 0, 12, and 24 h. myc-Tcf7, Uch37, and HA-Uch37 were monitored by western blot analysis, and Actin levels were used as a loading control. Full images of all Fig. 2 are presented in Supplementary information (Figs S10 and 11).
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Figure 4. Deubiquitinating activity of Uch37 is required for Tcf7-mediated gene transcription by mediating DNA-binding of Tcf7.(a) Expression levels of Wnt target genes (siamois and nodal3.1) were examined by RT-PCR analysis using Xenopus animal cap tissues. Embryos were animally injected at four-cell stage. Animal caps were isolated at stage 9, grown to stage 11. Injected reagents are as follows, 25 pg myc-Tcf7 mRNA; 20 ng Co MO; 20 ng Uch37 MO; 1 ng wild type Uch37 mRNA (WT); 1 ng catalytically inactive Uch37 mRNA (IN). Full images are presented in Supplementary information (Fig. S12). (b) TOPflash assay using whole embryos (stage 10.5, 10 embryos). Embryos were animally injected at four-cell stage. 150 pg TOPflash reporter; 50 pg pRL-TK; 40 ng Co MO; 40 ng Uch37 MO; 50 pg Tcf7 mRNA; 1 ng wild type of Uch37 mRNA (WT); 1 ng catalytically inactive Uch37 mRNA (IN). (c) qPCR analysis for the expression of Wnt-target genes (c-myc, cyclinD1 and c-jun) in stable HepG2 cells. Tcf7 was transiently transfected alone or co-transfected with wild type of Uch37 or catalytically inactive Uch37. Error bars indicate standard deviations of triplicate. *p < 0.05; **p < 0.02; ***p < 0.003 (two-tailed Student’s t test). (d) Co-IP assay using Xenopus embryos (stage 11). Two-cell stage embryos were animally injected with indicated reagents (1 ng myc-Tcf7 mRNA; 20 pg Wnt8 mRNA; 40 ng Co MO; 40 ng Uch37 MO). Total proteins were precipitated with anti-myc antibody. Active state of Wnt signalling is indicated with enhanced β-catenin level in input panel. Full images are presented in Supplementary information (Fig. S11). (e) ChIP assay using Xenopus embryos (stage 11). 70 embryos were injected at two-cell stage as indicated (25 pg myc-Tcf7 mRNA; 1 ng wild type of Uch37 (WT); 1 ng catalytically inactive Uch37 (IN); 40 ng Co MO; 40 ng Uch37 MO). Lysates were precipitated with anti-myc antibody. Precipitated Wnt target DNAs were analysed by PCR. EF1α was used as a control for specificity. Full images are presented in Supplementary information (Fig. S11). (f) ChIP assay using stable HepG2 cells. Cells were transfected with either wild type of Uch37 (WT) or catalytically inactive Uch37 (IN). Lysates were precipitated with normal rabbit IgG or anti-Tcf7 antibody. DNA-binding of Tcf7 was assessed by qPCR. gapdh was used as a negative control. **p < 0.02; ***p < 0.003 (two-tailed Student’s t test).
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Figure 5. Uch37 is required for the expression of ventrolateral mesoderm genes by promoting zygotic Wnt signalling during Xenopus gastrulation.(a) Expressions of ventrolateral mesoderm genes including MyoD, Xpo, and Vent1. Vegetal-view of stage 10.5 embryos, MOs and mRNAs were unilaterally injected at four-cell stage embryos. β-galactosidase (LacZ) mRNA was co-injected to trace the injected site (Inj., injected site). (20 ng Co MO; 20 ng Uch37 MO; 100 pg Lef1; 2 ng Re.Uch37; 300 pg β-galactosidase). See also Table S4. (b) Expressions of Vent1 and Xpo. Vegetal-view of stage 10.5 embryo, Four-cell stage embryos were injected in VMZ and cultured until stage 10.5. Left panel, Co MO (40 ng); middle and right panels, injection of pCSKA-Wnt8 plasmids (500 pg) along with either Co MO (40 ng) or Uch37 MO (40 ng). See also Table S5. (c) RT-PCR analysis using animal cap explants. Four-cell stage embryos were animally injected with indicated reagents (20 ng Co MO; 20 ng Uch37 MO; 300 pg pCSKA-Wnt8; 1 ng Uch37 mRNA (WT); 1 ng a catalytically inactive Uch37 mRNA (IN)). Animal cap explants were dissected at stage 9, and then cultured in activin (5 ng/ml)–treated 1xMR until stage 11. Gsc, a dorsal mesoderm marker; Xpo, Vent1, and Vent2, ventrolateral mesoderm markers. Full images are presented in Supplementary information (Fig. S12).
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Figure 6. Proposed model depicting how Uch37 regulates Tcf7 protein.Uch37 interacts with Tcf7 and removes polyubiquitin chain from Tcf7 protein. As a result, Tcf7 stably binds target DNA for gene transcription. However, absence of Uch37 promotes polyubiquitination on Tcf7 protein, causing transcriptional silence.
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Fig.S.2 Embryonic expression of Uch37. (a) Expression of Uch37 in Xenopus embryo was analyzed by RT-PCR. ODC, ornithine decarboxylase loading control; (-), -RT; egg, unfertilized; St, Nieuwkoop and Faber (NF) developmental stage. (b) Spatial expression of Uch37 from cleavage to gastrula stage. Red box indicates the magnified mesoderm region. (c) Expression of Uch37 in various dissected tissues at stage 10.5. WE, whole embryo; AC, animal cap; DMZ, dorsal marginal zone; VMZ, ventral marginal zone; Chordin, dorsal marker; Msx1, ventral marker; Xbra, pan-mesodermal marker.
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Fig.S.3 Phenotype of Uch37 morphant. (a) Uch37 MO specifically inhibits the translation of C-terminally HA-tagged Uch37 mRNA (1ng), but not MO-resistant Uch37 (Re.Uch37) mRNA (1ng) and human Uch37 mRNA (1ng). Four-cell stage embryos were injected in animal blastomeres with indicated reagents. Embryos were cultured to stage 10.5 and then subjected to western blotting. (b) Phenotypes of Uch37- depleted embryos. Embryos were injected at the four-cell stage with the indicated reagents (40 ng Co MO; 40 ng Uch37 MO; 2ng Re.Uch37) ventrally (b, left) or dorsally (b, right) and cultured to tadpole stage. (c, d) Morphant phenotypes were tabulated.
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Fig.S.4 Uch37 specifically regulates Wnt signaling. (a-d) RT-PCR analysis using animal cap explants. Co MO (20ng) or Uch37 MO (20ng) were respectably or co-injected with indicated mRNAs encoding Wnt8 (10pg and 20pg), BMP4 (50pg and 100pg), Xnr1 (50pg and 100pg), or eFGF (100pg and 200pg) in animal blastomeres of four-cell stage embryo. Animal cap explants were dissected at stage 9 and cultured until stage 11. siamois, Wnt-target gene (a); Sizzled, Bmp4-target gene (b); Xbra, target gene of Xnr1 (c) and eFGF (d). (e) Short hairpin RNA (shRNA) of Uch37 efficiently reduced levels of Uch37 protein in HepG2 cells. (f) TOPflash assay in HepG2 cells. Reporter constructs were transfected alone or co-transfected with constitutively active LRP6 (LRP6∆N) into stable cells (sh Control and sh Uch37). 48h after transfection, luciferase activity was measured. (g) qPCR analysis for the expression of Wnt-target genes (c-myc and cyclinD1) in stable HepG2 cells (sh Control or sh Uch37) treated with or without LiCl (25mM). The quantities of indicated mRNA were normalized by β-actin. Data represent average values from three independent experiments performed. Error bars indicate standard deviations of triplicate. *, p <0.005; **, p< 0.001 (two-tailed Student’s ttest).
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Fig. S.5 Uch37 promotes Wnt signaling downstream of ß-catenin destruction complex. (a) Result of axis duplication assay in Fig 1g. (b, c) RT-PCR analysis using animal cap explants. Embryos were animally injected at four-cell stage. Animal cap explants were isolated at stage 9 and cultured until stage 11. Injected reagents are as follows, Co MO (20ng), Uch37 MO (20ng), myc-Dvl2 mRNA (200pg), ß-catenin mRNA (25pg), Re.Uch37 (1ng). (d) Western blot analysis using animal cap explants. Embryos were animally injected at two-cell stage. Animal cap explants were dissected at stage 9 and cultured until stage 11. Injected reagents are as follows, Co MO (40ng), Uch37 MO (40ng), Wnt8 mRNA (20pg), myc-Uch37 mRNA (1ng).
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Fig.S.6 Uch37 interacts with Tcf7 in nucleus of Xenopus gastrula embryo.
(a) Fractionated lysates from Xenopus gastrula embryos were used as expression input for Co-IP analysis in Fig 1A. (b) Uch37 is co-localized with Tcf7 in nucleus. Four- cell stage embryos were animally co-injected with HA-Uch37 (1 ng) and myc-Tcf7 (50pg). Animal cap explants were isolated at stage 10 and fixed with 4% paraformaldehyde for 2h. HA-Uch37 and myc-Tcf7 were immunostained with anti-HA (mouse, santacruz; 2nd antibody, Alexa-488, invitrogen) and anti-myc antibodies (rabbit santacruz; 2nd antibody, Alexa-594, invitrogen). Scale bar (white line) indicates 50μm. (c) Co-IP assay using HEK293FT cells, HA-Uch37 and indicated myc-tagged truncated mutants of Tcf7 were transfected. Cell lysates were immunoprecipitated with anti-myc antibody. Truncated mutants of Tcf7 are depicted on the left.
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Fig.S.8 Uch37 MO specifically blocks Tcf7-mediated Wnt activity and Uch37 is required for Tcf7 binding to promoter of Vent2. (a) Axis duplication assay. Four- cell stage embryos were injected in one ventrovegetal blastomere with indicated
reagents (400pg Tcf7 mRNA; 400pg Lef1; 40ng Co MO; 40ng Uch37 MO). (b) Quantified result of a. (c) ChIP assay using Xenopus embryos (stage 11). 70 embryos were injected at two-cell stage as indicated (25pg myc-Tcf7 mRNA; 1ng wild type of Uch37 (WT); 1ng catalytically inactive Uch37 (IN); 40ng Co MO; 40ng Uch37 MO). Lysates were precipitated with anti-myc antibody. Precipitated Wnt target DNAs were analyzed by PCR. EF1α was used as a control for specificity.
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