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

Su(H) 在抑制果蝇 Notch 信号活性中的作用

• 批准号: 357709120
• 负责人: Dr. Dieter Maier
• 金额: --
• 依托单位: Institut für Genetik (240) (aufgelöst)
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2017
• 资助国家: 德国
• 起止时间: 2016-12-31 至 2020-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/357709120?language=en
• 关键词: Role; Su; repression; Notch; signaling

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).

高等真核生物中的细胞间通讯是基于Notch通路，由于其参与许多人类疾病，其调节引起了极大的兴趣。在Notch受体活化后，胞内Notch结构域（ICN）与转录因子CSL一起活化Notch靶基因。在Notch信号不存在的情况下，CSL使阻遏物复合物中的Notch靶基因沉默。在果蝇中，阻遏复合物包含CSL同源物Su（H）和Hairless（H）。与R. Kovall（辛辛那提）在体内分析并证实了H-Su（H）复合物的晶体结构：通过疏水接触，H深深侵入Su（H），引起显著的构象变化，排除了ICN结合。采用基因工程的方法，对H和Su（H）基因座的特异性突变进行了介绍和分析。我们可以证明Su（H）结合缺陷型H等位基因类似于无效突变体，即H的主要功能是Notch阻遏。此外，我们产生了核输入缺陷的特定H*NLS等位基因。此外，建立了三个H结合缺陷的Su（H）突变体，它们是隐性致死的，并显示出Notch功能的获得，证明了果蝇发育需要Notch阻遏。最后，我们观察到，不仅苏（H）蛋白的核定位，而且其稳定性取决于结合到H或ICN。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的转变过程。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Asf 1之间的联系。首先，将确定单个H核定位信号的贡献。使用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）蛋白稳定性对核定位或DNA结合的依赖性。Su（H）的半衰期用S2细胞培养物和体内的热诱导Su（H）构建体测量，以及蛋白酶体的贡献。在Su（H）和RBP-J中，潜在的泛素化赖氨酸残基将被突变，以揭示它们对Su（H）稳定性的贡献。应使用酵母双杂交和报告基因试验，分析H和Asf 1之间的分子相互作用。为了研究Asf 1在H介导的Notch靶基因阻遏中的作用，在体外将突变引入Asf 1-H相互作用结构域，并在细胞培养和过表达实验中进行研究。最终，产生了特定的H和Asf 1等位基因，前者通过基因组工程，后者通过Crispr/Cas9。这些实验的一部分将与R。Kovall（辛辛那提）、S. Bray（剑桥）和F.奥斯瓦尔德（乌尔姆）。

项目成果

Nucleo-cytoplasmic shuttling of Drosophila Hairless/Su(H) heterodimer as a means of regulating Notch dependent transcription.

• DOI: 10.1016/j.bbamcr.2019.07.008
• 发表时间: 2019-10
• 期刊: Biochimica et biophysica acta. Molecular cell research
• 作者: Dorina B. Wolf;Thomas K. Smylla;Jan Reichmuth;Philipp Hoffmeister;L. Kober;M. Zimmermann;A. Turkiewicz;T. Borggrefe;A. Nagel;F. Oswald;A. Preiss;D. Maier

The Notch repressor complex in Drosophila: in vivo analysis of Hairless mutants using overexpression experiments

• DOI: 10.1007/s00427-018-00624-2
• 发表时间: 2019-01
• 期刊: Development Genes and Evolution
• 影响因子: 2.4
• 作者: Thomas K. Smylla;Markus Meier;A. Preiss;D. Maier

Conservation of the Notch antagonist Hairless in arthropods: functional analysis of the crustacean Daphnia pulex Hairless gene

• DOI: 10.1007/s00427-017-0593-4
• 发表时间: 2017-08
• 期刊: Development Genes and Evolution
• 影响因子: 2.4
• 作者: A. Zehender;Melanie Bayer;Milena Bauer;B. Zeis;A. Preiss;D. Maier

The evolution of transcriptional repressors in the Notch signaling pathway: a computational analysis

• DOI: 10.1186/s41065-019-0081-0
• 发表时间: 2019-01-17
• 期刊: HEREDITAS
• 影响因子: 2.7
• 作者: Maier, Dieter