Repression mechanisms of the Notch-signalling pathway in Drosophila

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Notch signalling governs intercellular communication in higher eukaryotes. Since several human diseases are linked to defects in Notch signalling, understanding of its regulation is of fundamental importance. Activation of the name giving receptor Notch results in the release of the intracellular Notch domain (ICN), which in the nucleus acts as a coactivator within a large activation complex to drive expression of Notch target genes and to guide the cellular response. The crystal structure of the activator complex has been resolved. According to this, the central component is the transcription factor CSL which recruits several coactivators apart from ICN. In the absence of Notch signals, CSL together with several corepressors assembles a repressor complex that silences Notch target genes. The aim of this project is a structural characterization of the Notch repressor complex in the model system Drosophila. Here, the CSL-homologue Su(H) assembles a repressor complex containing the protein Hairless (H) plus additional general corepressors. With its C-terminal domain (CTD), Su(H) binds the just 16 amino acids spanning NT-box of H. In H-NT we identified a single amino acid essential for Su(H) binding. The affinity of H for Su(H) binding matches that of ICN. Nevertheless, ICN is able to displace H from the repressor complex in vitro, presumably provoking a conformational change of Su(H). The structure of the H-Su(H) repressor complex shall be analysed in collaboration with Rhett Kovall (University of Cincinnati). First raw data indicate a hydrophobic contact between the H-NT domain and the CTD of Su(H). Based on these data, Su(H) shall be changed by in vitro mutagenesis to affect H binding. First results have been achieved and are very promising. The binding properties of the new Su(H) mutants will be tested biochemically and by in vitro assays, and their biological activity investigated by in vivo approaches. The focus of to the proposal is 'gene-engineering' allowing the in vivo studies of the H and Su(H) mutants, respectively, generated in vitro. In a first step, the wild type H and Su(H) locus is each replaced by an attP target sequence. The attP site permits position specific integration of any sequence - here the mutant gene copy -, which can be subsequently studied directly in the organism within the normal context. Hence the typical negative effects resulting from the overexpression of the given gene can be avoided. Via homologous recombination, we have already created the attP-fly line at the H locus and established the proof-of-principle. Now we can start to generate and study the first engineered H mutants. Engineering at the Su(H) locus shall follow. Our goal is to eventually unravel the details of Notch signal repression.