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

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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).