Hairless-binding deficient Suppressor of Hairless alleles reveal Su(H) protein levels are dependent on complex formation with Hairless.
Hairless-binding deficient Suppressor of Hairless alleles reveal Su(H) protein levels are dependent on complex formation with Hairless.
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DOI：
10.1371/journal.pgen.1006774
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发表时间：
2017-05
期刊：
影响因子：
4.5
通讯作者：
Maier D
中科院分区：
文献类型：
--
作者：
Praxenthaler H;Nagel AC;Schulz A;Zimmermann M;Meier M;Schmid H;Preiss A;Maier D
在后生动物发育过程中，细胞命运的选择是由高度保守的Notch信号通路驱动的。Notch受体激活导致Notch胞内结构域（NICD）的释放，该结构域作为dna结合蛋白CSL的转录共激活因子。在缺乏信号的情况下，一个由CSL组成的阻遏子复合物与辅阻遏子结合，使Notch靶基因沉默。果蝇抑制因子复合体包含果蝇CSL同源的无毛抑制因子[Su(H)]和无毛抑制因子(H)。Su(H)-H晶体结构在H结合后显示出Su(H)内部的大构象变化，排除了与NICD的相互作用。根据结构，确定了Su(H)和H中的几个位点专门参与复杂地层。特别是，在Su(H)中发现了三个突变，它们影响与抑制因子H的相互作用，而不影响与激活因子NICD的相互作用。为了分析这些突变体对正常果蝇发育的影响，我们通过基因组工程将这些突变体引入原生的Su(H)位点。我们发现三个H结合缺陷Su(H)等位基因的行为相似。由于这些突变体缺乏形成抑制因子复合物的能力，Notch信号活性在纯合子中强烈增加，相当于H活性的完全丧失。出乎意料的是，我们发现三个突变体Su(H)蛋白变体的丰度发生了变化，野生型Su(H)蛋白在缺乏H蛋白的情况下也发生了变化。然而，在NICD存在的情况下，Su(H)突变蛋白仍然存在。显然，Su(H)蛋白水平取决于与H和NICD的相互作用。基于这些结果，我们提出体内的Su(H)蛋白水平是通过与转录调节复合物的相互作用来稳定的。Notch信号活动在决定细胞命运中起着重要作用。Notch信号通过转录因子CSL和激活的Notch受体胞内结构域（NICD）转导为基因表达变化。CSL也可以作为转录抑制因子，这取决于它的结合辅因子。在果蝇中，Notch靶基因的抑制涉及CSL同源物Hairless Suppressor [Su(H)]和Notch拮抗剂Hairless (H)。H结合Su(H)排除NICD同时结合。基于结构信息，对Su(H)-H相互作用重要的氨基酸发生突变，产生仍然结合NICD但不再结合H的Su(H)分子，从而阻止抑制因子而不是激活因子复合物的形成。将三个这样的突变引入原生的Su(H)基因座，分析它们对果蝇体内发育的影响。这三个等位基因均为纯合致死，证明了Su(H)在果蝇发育过程中作为抑制因子的重要作用。此外，所有三个H结合缺陷Su(H)等位基因都表现出明显的Notch功能增益。出乎意料的是，由于失去与H的相互作用，突变体Su(H)变异体的蛋白质丰度降低。此外，我们发现Su(H)-NICD相互作用增加了突变体Su(H)蛋白水平。综上所述，我们认为Su(H)蛋白通过与转录调节复合物的相互作用在体内稳定下来。
Cell fate choices during metazoan development are driven by the highly conserved Notch signalling pathway. Notch receptor activation results in release of the Notch intracellular domain (NICD) that acts as transcriptional co-activator of the DNA-binding protein CSL. In the absence of signal, a repressor complex consisting of CSL bound to co-repressors silences Notch target genes. The Drosophila repressor complex contains the fly CSL orthologue Suppressor of Hairless [Su(H)] and Hairless (H). The Su(H)-H crystal structure revealed a large conformational change within Su(H) upon H binding, precluding interactions with NICD. Based on the structure, several sites in Su(H) and H were determined to specifically engage in complex formation. In particular, three mutations in Su(H) were identified that affect interactions with the repressor H but not the activator NICD. To analyse the effects these mutants have on normal fly development, we introduced these mutations into the native Su(H) locus by genome engineering. We show that the three H-binding deficient Su(H) alleles behave similarly. As these mutants lack the ability to form the repressor complex, Notch signalling activity is strongly increased in homozygotes, comparable to a complete loss of H activity. Unexpectedly, we find that the abundance of the three mutant Su(H) protein variants is altered, as is that of wild type Su(H) protein in the absence of H protein. In the presence of NICD, however, Su(H) mutant protein persists. Apparently, Su(H) protein levels depend on the interactions with H as well as with NICD. Based on these results, we propose that in vivo levels of Su(H) protein are stabilised by interactions with transcription-regulator complexes. Notch signalling activity plays a major role in determining cell fates. Notch signals are transduced into gene expression changes by the transcription factor CSL and the activated Notch receptor intracellular domain (NICD). CSL can also function as a transcriptional repressor, depending on its bound cofactors. In Drosophila, repression of Notch target genes involves the CSL homologue Suppressor of Hairless [Su(H)] and the Notch antagonist Hairless (H). H binding to Su(H) excludes simultaneous NICD binding. Based on structural information, amino acids important for Su(H)-H interactions were mutated, generating Su(H) molecules that still bind NICD but no longer H, thereby preventing repressor but not activator complex formation. Three such mutations were introduced into the native Su(H) locus to analyse their consequences on fly development in vivo. All three alleles are homozygous lethal, demonstrating the essential role of Su(H) as repressor during fly development. Moreover, all three H-binding deficient Su(H) alleles show marked Notch gain of function. Unexpectedly, protein abundance of mutant Su(H) variants is reduced due to the loss of interactions with H. Moreover, we find that Su(H)-NICD interaction increased mutant Su(H) protein levels. Taken together, we propose that Su(H) protein is stabilised in vivo by interactions with transcription-regulator complexes.