Throughout metazoan development, cell signalling pathways are used to organise gene expression in different cell types at different times. Notch is a highly conserved pathway whose activity controls the expression of many targets in a context dependent manner. Despite the complexity of response, the core pathway is simple. Upon ligand binding, the Notch receptor is cleaved and the Notch intracellular domain (NICD) translocates to the nucleus. In Drosophila melanogaster, NICD forms a complex with Suppressor of Hairless (Su(H)) and Mastermind to activate gene expression. The aim of this PhD was to further our understanding of how NICD is able to initiate target gene activation.

To investigate the recruitment of NICD and its cofactors in the nucleus, it was tagged with a fluorescent protein and deletions were made to the intrinsically disordered C-terminus. Additionally, a mutation in the ankyrin domains of NICD was introduced to prevent dimerization. Confocal fluorescence imaging showed that cleaved NICD was detectably recruited at a target locus, the Enhancer of split-Complex (E(spl)-C), but appeared less robustly enriched over background than Su(H) and Mastermind. When NICD dimerization was perturbed, lower levels of Su(H) were recruited. In contrast, deleting the C-terminus of NICD had little effect on Su(H) recruitment. However, when Mastermind was depleted, there was a more profound effect on Su(H) recruitment with a C-terminal NICD truncation compared to the full-length protein, suggesting Mastermind and the NICD C-terminus may have some overlapping functions. These experiments provide new insights into the role of NICD in promoting a transcription “hub” at target loci to promote transcription.

A second goal was to manipulate the timing of activation and the levels of NICD to determine their impact on the transcriptional response. To achieve this, an optogenetic method was developed to release NICD in a light-dependent manner when a split TEV protease was regenerated. This required the generation of new transgenic flies and when all the components were combined a transcriptional response to light was achieved. Following light induced activation, Su(H) and Mastermind were detectably recruited to the E(spl)-C locus and E(spl)-C mRNA was produced. In addition, an unexpected interaction between Su(H) and NICD in the cytoplasm was detected with the first membrane tethered optogenetic NICD construct generated. This interaction was investigated further as it implied the existence of an additional regulatory mechanism that prevents it occurring with the full-length Notch receptor.