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. 2015 Mar 11;10(3):e0119455.
doi: 10.1371/journal.pone.0119455. eCollection 2015.

T396I mutation of mouse Sufu reduces the stability and activity of Gli3 repressor

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T396I mutation of mouse Sufu reduces the stability and activity of Gli3 repressor

Shigeru Makino et al. PLoS One. .

Abstract

Hedgehog signaling is primarily transduced by two transcription factors: Gli2, which mainly acts as a full-length activator, and Gli3, which tends to be proteolytically processed from a full-length form (Gli3FL) to an N-terminal repressor (Gli3REP). Recent studies using a Sufu knockout mouse have indicated that Sufu is involved in regulating Gli2 and Gli3 activator and repressor activity at multiple steps of the signaling cascade; however, the mechanism of specific Gli2 and Gli3 regulation remains to be elucidated. In this study, we established an allelic series of ENU-induced mouse strains. Analysis of one of the missense alleles, SufuT396I, showed that Thr396 residue of Sufu played a key role in regulation of Gli3 activity. SufuT396I/T396I embryos exhibited severe polydactyly, which is indicative of compromised Gli3 activity. Concomitantly, significant quantitative reductions of unprocessed Gli3 (Gli3FL) and processed Gli3 (Gli3REP) were observed in vivo as well as in vitro. Genetic experiments showed that patterning defects in the limb buds of SufuT396I/T396I were rescued by a constitutive Gli3REP allele (Gli3∆699), strongly suggesting that SufuT396I reduced the truncated Gli3 repressor. In contrast, SufuT396I qualitatively exhibited no mutational effects on Gli2 regulation. Taken together, the results of this study show that the Thr396 residue of Sufu is specifically required for regulation of Gli3 but not Gli2. This implies a novel Sufu-mediated mechanism in which Gli2 activator and Gli3 repressor are differentially regulated.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Identification of novel Sufu mutants.
(A) Schematic diagrams of the mouse Sufu protein. The numbers refer to amino acid residues. Filled boxes indicate binding domains with GSK3β [21] and the C- and N-terminal regions of Gli1 [26]. An asterisk marks the position of a 1-base substitution that results in a change of Thr396 to Ile (T396I). Sufu R146X is a 1-base pair deletion in Sufu, leading to premature termination of the protein product after addition of an aberrant 33 amino acid stretch at the C terminal when translated. (B, C) Wild-type and Sufu T396I/T396I embryos at E13.5. Scale bar, 4 mm. (D) Limb phenotype of Sufu T396I/T396I at E15. Scale bar, 2 mm. (E, F) Lungs of wild-type and Sufu T396I/T396I at E15.5. Scale bars, 2 mm. (G, H) Wild-type and Sufu R146X/R146X embryos at E9.5. Scale bars, 1 mm. Homozygous embryos died at approximately E9.5 and exhibited an open brain and failure to undergo embryonic turning, characteristics identical to those reported in Sufu knockout embryos [22,23,51].
Fig 2
Fig 2. SufuT396I does not stabilize Gli3FL protein and reduces the processing of Gli3FL.
(A) Western blotting of lysates prepared from Sufu R146X/R146X at E9.5 and Sufu T396I/T396I, Sufu T396I/+, and wild-type embryos at E10.5 with anti-Gli3, anti-Sufu, and anti-actin antibodies. Each image presented in the Fig. is a representative of independent triplicated experiments. The full gel images are shown in S6A Fig. (B, C) Relative expression of Gli3FL (B) and Gli3REP (B and C). Western blotting was performed two times using lysates prepared from five wild-type and five Sufu T396I/T396I embryos at E10.5 (S2A Fig.). Expression levels were quantified from the band intensity shown in S2A Fig. as relative values of the Gli3FL/actin and Gli3REP/actin expression ratios (B) and the direct ratio of Gli3REP/Gli3FL (C). (**) p < 0.01, two-tailed Student’s t-test. Error bars indicate the standard deviations. (D) Western blotting of cell lysates from wild-type and Sufu T396I/T396I MEFs with indicated antibodies. The Sufu T396I/T396I MEFs were electroporated with 10.0, 1.0, or 0.1 μg of the HA–Sufu construct (lane 3, 4, and 5, respectively), or 10 μg of the HA–SufuT396I construct (lane 6). The mobilities on SDS-PAGE of the wild-type Sufu (lane 3–5) and SufuT396I (lane 6) are identical. The complete gel images are shown in S6B Fig. This image is representative of two independent experiments. (E) Western blotting of cell lysates from Sufu −/− cells with indicated antibodies. The Sufu −/− cells were electroporated with a mixture of the Flag–Gli3 construct (6 μg) and the HA–Sufu construct (4.00, 1.33, 0.44, or 0.15 μg) for the wild-type Sufu cotransfection (lane 2 to 5, respectively), or a mixture of the Flag–Gli3 construct (6 μg) and the HA–SufuT396I construct (4 μg) for mutant Sufu cotransfection (lane 6). The complete gel images are shown in S6C Fig. This image is representative of two independent experiments. (F) Western blotting of immunoprecipitates or lysates from 293T cells transfected with expression constructs as indicated at the top. The complete gel images are shown in S6D Fig. This image is representative of two independent experiments.
Fig 3
Fig 3. Gli3 ∆699 suppresses the A-P polarity defects in the Sufu T396I/T396I limb buds.
(A–L) Expression of Alx4 (A–C), Pax9 (D–F), Hand2 (G–I), and Hoxd12 (J–L) of wild-type (A, D, G and J), Sufu T396I/T396I (B, E, H, and K), and Sufu T396I/T396I; Gli3 ∆699/+ (C, F, I, and L) by RNA in situ hybridization at indicated stages. Genotypes are indicated at the top. Limb buds are oriented with the anterior to the top.
Fig 4
Fig 4. Sufu T396I/T396I shows reduction of Gli3 activity but not Gli2 activity.
(A–H) Immunofluorescence images of transverse sections at thoracic level. Sections from wild-type (A and E), Sufu T396I/T396I (B and F), and Smo G457X/G457X; Sufu T396I/T396I (C and G) embryos at E10.5 and Smo G457X/G457X embryos (D and H) at E9.5 were immunostained with anti-Olig2 (A–D, magenta), anti-Nkx2.2 (A–D, green), and anti-FoxA2 (E–H, green) antibodies. Dashed lines outline the neural tubes. Scale bar, 100 μm. Images with nuclear staining are shown in S7 Fig.
Fig 5
Fig 5. SufuT396I can regulate Gli activator function.
(A) Western blotting of Gli2 from embryo lysates at E9.5. Genotypes and antibodies are indicated at the top and left, respectively. The image presented in the Fig. is representative of independent triplicated experiments. The broad gel images are shown in S5A Fig. and relative expression of Gli2 is shown in S5B Fig. (B, C) Western blotting of immunoprecipitates or lysates from 293T cells transfected with expression constructs as indicated at the top. Antibodies used for immunoprecipitation and western blotting are indicated to the left. The complete gel images are shown in S5C and D Fig. Gli1 protein appears to show multiple bands; the reason for this is unknown [42] [46]. Relative band intensities of lane 5 and 6 (C) are shown. It is note worthy that the expression levels of Gli2 were proportional to the amount of Sufu irrespective of either wild-type or T396I substitutions. (D, E) Luciferase reporter assay in 3T3 transfected with expression constructs as indicated in figures. Error bars indicate standard deviations. In our experimental settings, we observed approximately only 4-fold induction of luciferase reporter activity by Gli1 expression, as had been observed previously [46].

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This work was supported by JSPS (Japan Society for the Promotion of Science) KAKENHI Grant Number 21700454, (SM), 25440096 (SM), 21240043 (YG, SM, TM, and RF), 25241016 (YG), 15200032 (YG) and Incentive Research Grant of RIKEN (SM). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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