Title: Regulation of the LIN-12/Notch Core Nuclear Complex Components in Caenorhabditis elegans Reproductive Development
Description: LIN-12/Notch is a conserved transmembrane receptor that is required during animal development for proper cell-fate decisions and specification. In Caenorhabditis elegans, activation of LIN-12 occurs through binding to ligand expressed by an adjacent cell. This binding event triggers two cleavage steps and results in the release of the LIN-12 intracellular domain [LIN-12(intra)], which translocates to the nucleus to form a ternary complex with two other proteins: LAG-1/Su(H)/Cbf1 and SEL-8/Mastermind/Mastermind-like. This ternary complex will then transcriptionally activate target genes via LAG-1 Binding Sites (LBSs). LAG-1 is the sole DNA-binding component within the complex, and in the absence of LIN-12(intra), can act as a transcriptional repressor. LIN-12 signal transduction can be studied in the C. elegans Vulval Precursor Cells (VPCs), which exhibit precise spatiotemporal patterning regulated by LIN-12 activity. Here, I show that LAG-1 is positively autoregulated by LIN-12 activity in cells where LIN-12 activity is high.

Autoregulation is mediated by an enhancer element that contains a cluster of 18 LBSs that are located within a conserved high occupancy target region, which is a span of DNA that is pulled down promiscuously in ChIP-Seq experiments. Mutation of the LBSs abrogates preferential expression mediated by the enhancer in cells with high LIN-12 signal transduction. When the HOT region is deleted from the endogenous lag-1 locus, expression in the VPCs is strongly reduced and no overt Lag phenotype occurs. Instead, cold-sensitive vulval and egg-laying defects, reminiscent of phenotypes seen in lin-12 hypomorphs, are found. Autoregulation of lag-1, therefore, appears to contribute to the robustness of LIN-12 cell fate specification in response to stochastic environmental and genetic perturbations. Under adverse environmental conditions, C. elegans enter a state of diapause in which they form dauer larvae, which are long-lived and stress-resistant. The VPCs of dauer larvae remain developmentally arrested indefinitely until favorable conditions are reintroduced.

Experimentally, this arrest can be relieved by depletion of the Forkhead transcription factor DAF-16. I show that expression of the components of the LIN-12/SEL-8/LAG-1 ternary complex are downregulated during the L2d-dauer molt (prior to dauer entry) and that this downregulation is not relieved by DAF-16 depletion. Instead, DAF-16 depletion leads to resumption of LIN-12 signaling and expression of ternary complex only in completely formed dauer larvae. These observations suggest that DAF-16 is required for the maintenance but not the initiation of blocking LIN-12 signaling. The components of the ternary complex are required to effect LIN-12 signaling. This work contributes to better understanding how these components are regulated and how their expression can affect LIN-12 -mediated cell fate decisions.