T396I Mutation of Mouse <i>Sufu</i> 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 (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.刺猬信号通路（Hedgehog signaling）主要通过两种转录因子进行转导：Gli2主要作为全长激活形式发挥功能，而Gli3则通常会从全长形式（Gli3FL）经蛋白水解加工成为N端抑制形式（Gli3REP）。此前借助Sufu基因敲除小鼠开展的研究表明，Sufu可在信号级联反应的多个步骤中调控Gli2与Gli3的激活及抑制活性，但针对二者的特异性调控机制仍有待阐明。本研究构建了ENU（N-乙基-N-亚硝基脲）诱变获得的小鼠等位基因系列品系。对其中一个错义等位基因SufuT396I的分析显示，Sufu的苏氨酸396残基在Gli3活性调控中发挥关键作用。SufuT396I/T396I纯合突变胚胎表现出严重的多指畸形，该表型提示Gli3活性受损。与此同时，在体内及体外实验中均观察到未加工的Gli3（Gli3FL）与经加工的Gli3（Gli3REP）的水平均出现显著的定量降低。遗传学实验表明，组成型Gli3REP等位基因（Gli3∆699）可挽救SufuT396I/T396I小鼠肢芽的模式发育缺陷，这强烈提示SufuT396I突变会降低截短型Gli3抑制子的水平。与之相反，SufuT396I突变在定性层面未对Gli2的调控产生任何影响。综上，本研究结果证实，Sufu的苏氨酸396残基仅特异性参与Gli3的调控，而非Gli2。这一发现提示了一种全新的Sufu介导的调控机制，即Gli2激活子与Gli3抑制子受到差异化调控。