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

Makino S, Zhulyn O, Mo R, Puviindran V, Zhang X, Murata T, Fukumura R, Ishitsuka Y, Kotaki H, Matsumaru D, Ishii S, Hui CC, Gondo Y - PLoS ONE (2015)

Bottom Line: Concomitantly, significant quantitative reductions of unprocessed Gli3 (Gli3FL) and processed Gli3 (Gli3REP) were observed in vivo as well as in vitro.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.

View Article: PubMed Central - PubMed

Affiliation: Mutagenesis and Genomics Team, RIKEN BioResource Center, Tsukuba, Ibaraki, Japan.

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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Gli3∆699 suppresses the A-P polarity defects in the SufuT396I/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), SufuT396I/T396I (B, E, H, and K), and SufuT396I/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.
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pone.0119455.g003: Gli3∆699 suppresses the A-P polarity defects in the SufuT396I/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), SufuT396I/T396I (B, E, H, and K), and SufuT396I/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.

Mentions: It is known that mutual antagonistic interactions between Gli3 and Hand2 are required to establish early AP patterning in the limb and correct expression of downstream genes including Alx4 and Pax9 in the anterior mesenchyme [28,29] and Hand2 and Hoxd12 in the posterior mesenchyme [30]. Analysis of SufuT396I/T396I limbs revealed decreased expression of Gli3 target genes Alx4 and Pax9 in the anterior of the limb (Fig. 3A, B, D, E) and ectopic expression of posterior genes Hoxd12 and Hand2 throughout the limb bud mesenchyme (Fig. 3G, H, J, K). These phenotypes were consistent with compromised Gli3REP function [31–33]. To determine whether digit defects in SufuT396I/T396I are due to impaired Gli3REP activity, we forced expression of a constitutive Gli3REP allele (Gli3∆699), which produces a truncated protein due to a premature termination codon [34], in the SufuT396I/T396I background. We showed that expression of Gli3∆699 was sufficient to rescue the expression of Alx4 and Pax9 (Fig. 3C, F) and restore polarized expression of Hand2 and Hoxd12 (Fig. 3I, L). These findings indicate that the patterning defects in the SufuT396I/T396I limb may be attributed to compromised Gli3REP activity.


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

Makino S, Zhulyn O, Mo R, Puviindran V, Zhang X, Murata T, Fukumura R, Ishitsuka Y, Kotaki H, Matsumaru D, Ishii S, Hui CC, Gondo Y - PLoS ONE (2015)

Gli3∆699 suppresses the A-P polarity defects in the SufuT396I/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), SufuT396I/T396I (B, E, H, and K), and SufuT396I/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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4356511&req=5

pone.0119455.g003: Gli3∆699 suppresses the A-P polarity defects in the SufuT396I/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), SufuT396I/T396I (B, E, H, and K), and SufuT396I/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.
Mentions: It is known that mutual antagonistic interactions between Gli3 and Hand2 are required to establish early AP patterning in the limb and correct expression of downstream genes including Alx4 and Pax9 in the anterior mesenchyme [28,29] and Hand2 and Hoxd12 in the posterior mesenchyme [30]. Analysis of SufuT396I/T396I limbs revealed decreased expression of Gli3 target genes Alx4 and Pax9 in the anterior of the limb (Fig. 3A, B, D, E) and ectopic expression of posterior genes Hoxd12 and Hand2 throughout the limb bud mesenchyme (Fig. 3G, H, J, K). These phenotypes were consistent with compromised Gli3REP function [31–33]. To determine whether digit defects in SufuT396I/T396I are due to impaired Gli3REP activity, we forced expression of a constitutive Gli3REP allele (Gli3∆699), which produces a truncated protein due to a premature termination codon [34], in the SufuT396I/T396I background. We showed that expression of Gli3∆699 was sufficient to rescue the expression of Alx4 and Pax9 (Fig. 3C, F) and restore polarized expression of Hand2 and Hoxd12 (Fig. 3I, L). These findings indicate that the patterning defects in the SufuT396I/T396I limb may be attributed to compromised Gli3REP activity.

Bottom Line: Concomitantly, significant quantitative reductions of unprocessed Gli3 (Gli3FL) and processed Gli3 (Gli3REP) were observed in vivo as well as in vitro.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.

View Article: PubMed Central - PubMed

Affiliation: Mutagenesis and Genomics Team, RIKEN BioResource Center, Tsukuba, Ibaraki, Japan.

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.

Show MeSH
Related in: MedlinePlus