Novel negative regulation of LIN-12/Notch in Caenorhabditis elegans

Proper cell fate specification is crucial for development, and dysregulation in cellular signaling pathways can lead to deleterious effects like cancer. The conserved LIN- 12/Notch signaling pathway mediates fate specification in many cellular contexts, and multiple regulatory mechanisms ensures appropriate LIN-12/Notch activity in each context. Here, I have identified several cis-regulatory domains and trans-acting factors that contribute to the negative regulation of LIN-12/Notch in Caenorhabditis elegans. In this thesis, I find that LIN-12/Notch requires binding to LAG-1/CSL and association with the nuclear complex for protein turnover in the C. elegans vulval precursor cells (VPCs). I also identify two layers of negative regulation in the VPCs and their descendants. The E3 ubiquitin ligase SEL-10/Fbw7 mediates degradation of LIN-12/Notch via the PEST domain in the VPCs, while a novel structural conformation in the C-terminal end of the LIN-12/Notch intracellular domain is required for downregulation in the descendants. Through an RNAi screen for negative regulators, I isolated 13 conserved kinases that downregulate LIN-12/Notch activity. Of these 13 kinases, CDK-8 had been previously implicated in Notch turnover, while the other 12 are novel negative regulators. I provide evidence that 5 of the kinases regulate LIN-12/Notch through modulation of the intracellular domain. Furthermore, I conduct a deeper investigation into CDK-8, which is the kinase component of the Mediator complex. I determine that CDK-8 acts with the rest of the Cdk8 Kinase Module and independent of the Mediator core to negatively regulate LIN-12/Notch, and that CDK-8 kinase activity is required for this process. Lastly, I find that sur-2/MED23 and lin-25/MED24 are required for LIN-12/Notch ligand transcription, independent of the Cdk8 Kinase Module and Mediator Head and Tail components.