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

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 (Gli3^FL) to an N-terminal repressor (Gli3^REP). 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, Sufu^T396I, showed that Thr³⁹⁶residue of Sufu played a key role in regulation of Gli3 activity. Sufu^T396I/T396I embryos exhibited severe polydactyly, which is indicative of compromised Gli3 activity. Concomitantly, significant quantitative reductions of unprocessed Gli3 (Gli3^FL) and processed Gli3 (Gli3^REP) were observed in vivo as well as in vitro. Genetic experiments showed that patterning defects in the limb buds of Sufu^T396I/T396I were rescued by a constitutive Gli3^REP allele (Gli3^Δ699), strongly suggesting that Sufu^T396I reduced the truncated Gli3 repressor. In contrast, Sufu^T396I qualitatively exhibited no mutational effects on Gli2 regulation. Taken together, the results of this study show that the Thr³⁹⁶residue 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.