During development of higher animals, the Notch signalling pathway governs cell type specification by mediating appropriate gene expression responses. In the absence of signalling, Notch target genes are silenced by repressor complexes. In the model organism Drosophila melanogaster, the repressor complex includes the transcription factor Suppressor of Hairless [Su(H)] and Hairless (H) plus general co-repressors. Recent crystal structure analysis of the Drosophila Notch repressor revealed details of the Su(H)-H complex. They were confirmed by mutational analyses of either protein; however, only Su(H) mutants have been further studied in vivo. Here, we analyse three H variants predicted to affect Su(H) binding. To this end, amino acid replacements Phenylalanine 237, Leucines 245 and 247, as well as Tryptophan 258 to Alanine were introduced into the H protein. A cell-based reporter assay indicates substantial loss of Su(H) binding to the respective mutant proteins H^FA, H^LLAA and H^WA. For in vivo analysis, UAS-lines H^FA, H^LLAA and H^WA were generated to allow spatially restricted overexpression. In these assays, all three mutants resembled the H^LD control, shown before to lack Su(H) binding, indicating a strong reduction of H activity. For example, the H variants were impaired in wing margin formation, but unexpectedly induced ectopic wing venation. Concurrent overexpression with Su(H), however, suggests that all mutant H protein isoforms are still able to bind Su(H) in vivo. We conclude that a weakening of the cohesion in the H-Su(H) repressor complex is sufficient for disrupting its in vivo functionality.

中文翻译：

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果蝇中的Notch阻遏物复合物：使用过表达实验对无毛突变体进行体内分析。

在高等动物的发育过程中，Notch信号通路通过介导适当的基因表达反应来控制细胞类型规范。在没有信号传导的情况下，Notch靶基因被阻遏物复合物沉默。在模型生物果蝇（Drosophila melanogaster）中，阻遏物复合物包括无毛[Su（H）]和无毛（H）的转录因子抑制子以及一般的共抑制子。果蝇Notch阻遏物的最新晶体结构分析揭示了Su（H）-H配合物的详细信息。通过对两种蛋白质的突变分析证实了它们；但是只有Su（H）突变体已在体内进行了进一步研究。在这里，我们分析了三个预计会影响Su（H）绑定的H变体。为此，将氨基酸替代物苯丙氨酸237，亮氨酸245和247，以及色氨酸258替换为丙氨酸到H蛋白中。基于细胞的报告基因检测表明Su（H）与相应的突变蛋白H ^FA，H ^LLAA和H ^WA的结合大量丧失。为了进行体内分析，生成了UAS系H ^FA，H ^LLAA和H ^WA，以实现空间受限的过表达。在这些测定中，所有三个突变体均类似于H ^LD对照，显示缺乏Su（H）结合，表明H活性大大降低。例如，H变体在机翼边缘形成中受损，但是意外地诱发了异位机翼通气。然而，Su（H）的同时过表达表明，所有突变H蛋白同工型仍然能够在体内结合Su（H）。我们得出的结论是，H-Su（H）阻遏物复合物的内聚力减弱足以破坏其体内功能。