Role of Su(H) in the repression of Notch signaling activity in Drosophila

在高等真核生物中，细胞间的通讯是基于Notch通路的，由于其与许多人类疾病有关，其调控引起了人们的极大兴趣。Notch受体激活后，细胞内Notch结构域（ICN）与转录因子CSL一起激活Notch靶基因。在缺乏Notch信号的情况下，CSL使抑制因子复合体中的Notch靶基因沉默。在果蝇中，抑制复合物包含csl同源的Su(H)和Hairless (H)。与R. Kovall （Cincinnati）合作，在体内分析并证实了H-Su(H)配合物的晶体结构：通过疏水接触，H深度侵入Su(H)，引发了剧烈的构象变化，不包括ICN结合。采用基因工程的方法对H和Su(H)位点的特异性突变进行了介绍和分析。我们可以证明，Su(H)结合缺陷H等位基因与零突变体相似，即H的主要功能是抑制Notch。此外，我们还在核进口中产生了特异性的H*NLS等位基因缺陷。此外，我们还发现了三个在H结合上有缺陷的Su(H)突变体，它们是隐性致死的，并显示出Notch功能的增益，这表明Notch抑制对果蝇发育是必要的。最后，我们观察到Su(H)蛋白的核定位及其稳定性不仅取决于其与H或ICN的结合。该项目的目标是更深入地了解Notch信号抑制。我们感兴趣的是从激活到抑制转变的动力学。为此，我们希望研究H和Su(H)的亚细胞定位和稳定性，以及与染色质调控的功能和结构联系，特别是H和组蛋白伴侣Asf1之间的联系。首先，将确定单个H核定位信号的贡献。利用H*NLS等位基因和DNA结合缺陷的Su(H)S5等位基因，我们可以确定Su(H)蛋白的稳定性依赖于核定位或DNA结合。用热诱导的Su(H)构建体在S2细胞培养和体内测量Su(H)的半衰期，以及蛋白酶体的贡献。可能泛素化的赖氨酸残基应该在Su(H)和RBP-J中突变，以揭示它们对Su(H)稳定性的贡献。利用酵母双杂交和报告基因分析，将分析H和Asf1之间的分子相互作用。为了研究Asf1在h介导的Notch靶基因抑制中的作用，研究人员在体外将Asf1- h相互作用区域引入突变，并在细胞培养和过表达实验中进行研究。最终产生特异性的H和Asf1等位基因，前者通过基因组工程，后者通过Crispr/Cas9。其中部分实验将与R. Kovall（辛辛那提）、S. Bray（剑桥）和F. Oswald（乌尔姆）合作进行。

Cell-cell communication in higher eukaryotes is based on the Notch pathway, whose regulation is of great interest due to its involvement in many human diseases. Upon activation of the Notch receptor, the intracellular Notch domain (ICN) together with the transcription factor CSL activates Notch target genes. In the absence of Notch signals, CSL silences Notch target genes in a repressor complex. In Drosophila, the repressor complex contains the CSL-homologue Su(H) and Hairless (H). In collaboration with R. Kovall (Cincinnati) the crystal structure of the H-Su(H) complex was analysed and confirmed in vivo: via hydrophobic contacts, H deeply intrudes into Su(H), provoking a dramatic conformational change which excludes ICN binding. Using the method of gene-engineering specific mutations in the H and Su(H) loci were introduced and analyzed. We could show that Su(H)-binding deficient H alleles were similar to a null mutant, i.e. that the primary function of H is Notch repression. Moreover, we generated specific H*NLS alleles defective in nuclear import. In addition, three Su(H) mutants defective in H binding were established that are recessive lethal and show a Notch gain of function, demonstrating the requirement of Notch repression for fly development. Finally we observed that not only the nuclear localisation of Su(H) protein but also its stability depends on the binding to H or ICN. The goal of this project is a deeper understanding of Notch signal repression. We are interested in the dynamics of the transition from activation to repression. To this end we want to investigate the subcellular localisation and stability of H and Su(H), as well as the functional and structural links to chromatin regulation, notably between H and the histone chaperon Asf1. At first the contribution of individual H nuclear localisation signals will be determined. Using H*NLS alleles and the DNA-binding defective Su(H)S5 allele, we can determine the dependency of Su(H) protein stability on nuclear localization or DNA binding. The half life of Su(H) is being measured with heat inducible Su(H) constructs in S2 cell culture and in vivo, as well as the contribution of the proteasome. Potentially ubiquitylated lysine residues shall be mutated in Su(H) and RBP-J to reveal their contribution to Su(H) stability. Using yeast two-hybrid and reporter assays, the molecular interactions between H and Asf1 shall be analysed. To investigate the role of Asf1 in H-mediated repression of Notch target genes, mutations are being introduced in vitro in the Asf1-H interaction domain and studied in cell culture and overexpression experiments. Eventually, specific H and Asf1 alleles are generated, the former by genome engineering, the latter by Crispr/Cas9. Part of these experiments will be performed in a collaboration with R. Kovall (Cincinnati), S. Bray (Cambridge) and F. Oswald (Ulm).