XB-ART-53116Sci Rep. March 28, 2017; 7 42590.
Ubiquitin C-terminal hydrolase37 regulates Tcf7 DNA binding for the activation of Wnt signalling.
The Tcf/Lef family of transcription factors mediates the Wnt/β-catenin pathway that is involved in a wide range of biological processes, including vertebrate embryogenesis and diverse pathogenesis. Post-translational modifications, including phosphorylation, sumoylation and acetylation, are known to be important for the regulation of Tcf/Lef proteins. However, the importance of ubiquitination and ubiquitin-mediated regulatory mechanisms for Tcf/Lef activity are still unclear. Here, we newly show that ubiquitin C-terminal hydrolase 37 (Uch37), a deubiquitinase, interacts with Tcf7 (formerly named Tcf1) to activate Wnt signalling. Biochemical analyses demonstrated that deubiquitinating activity of Uch37 is not involved in Tcf7 protein stability but is required for the association of Tcf7 to target gene promoter in both Xenopus embryo and human liver cancer cells. In vivo analyses further revealed that Uch37 functions as a positive regulator of the Wnt/β-catenin pathway downstream of β-catenin stabilization that is required for the expression of ventrolateral mesoderm genes during Xenopus gastrulation. Our study provides a new mechanism for chromatin occupancy of Tcf7 and uncovers the physiological significance of Uch37 during early vertebrate development by regulating the Wnt/β-catenin pathway.
PubMed ID: 28198400
PMC ID: PMC5309806
Article link: Sci Rep.
Genes referenced: gapdh jun myc nodal3.1 odc1 sia1 tcf7 ubc
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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).|
|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).|
|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).|
|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.|