The Notch repressor complex in Drosophila: in vivo analysis of Hairless mutants using overexpression experiments
The Notch repressor complex in Drosophila: in vivo analysis of Hairless mutants using overexpression experiments
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DOI：
10.1007/s00427-018-00624-2
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发表时间：
2019-01
影响因子：
2.4
通讯作者：
Thomas K. Smylla;Markus Meier;A. Preiss;D. Maier
中科院分区：
文献类型：
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作者：
Thomas K. Smylla;Markus Meier;A. Preiss;D. Maier
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特化。在没有信号传导的情况下，Notch靶基因被阻遏物复合物沉默。在模式生物黑腹果蝇中，阻遏物复合物包括无毛转录因子Suppressor of Hairless [Su（H）]和无毛转录因子Suppressor of Hairless（H）加上一般的辅阻遏物。最近对果蝇Notch阻遏物的晶体结构分析揭示了Su（H）-H复合物的细节。它们通过任一蛋白质的突变分析得到证实;然而，仅对Su（H）突变体进行了体内进一步研究。在这里，我们分析了三个H变体预测影响苏（H）结合。为此，将苯丙氨酸237、亮氨酸245和247以及色氨酸258替换为丙氨酸引入H蛋白。基于细胞的报告基因测定表明Su（H）与相应突变蛋白HFA、HLLA A和HWA的结合基本丧失。对于体内分析，产生UAS-系HFA、HLLAA和HWA以允许空间限制的过表达。在这些试验中，所有三个突变体类似于HLD对照，之前显示缺乏Su（H）结合，表明H活性强烈降低。例如，H变异体在翅缘形成中受损，但意外地诱导了异位翅脉序。然而，与Su（H）同时过表达表明所有突变H蛋白同种型在体内仍然能够结合Su（H）。我们的结论是，在H-Su（H）阻遏复合物的凝聚力的削弱是足以破坏其在体内的功能。
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 organismDrosophila 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 theDrosophilaNotch repressor revealed details of the Su(H)-H complex. They were confirmed by mutational analyses of either protein; however, onlySu(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 HFA, HLLAAand HWA. For in vivo analysis, UAS-linesHFA,HLLAAandHWAwere generated to allow spatially restricted overexpression. In these assays, all three mutants resembled theHLDcontrol, shown before to lack Su(H) binding, indicating a strong reduction ofHactivity. For example, theHvariants were impaired in wing margin formation, but unexpectedly induced ectopic wing venation. Concurrent overexpression withSu(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.