Animal development unfolds as a succession of life stages, often punctuated by dramatic transitions such as metamorphosis or puberty. The stereotyped course of developmental events suggests that a species life plan is genetically programmed, but the molecular basis for this plan is largely unknown. Our longterm aim is to dissect the temporal programming of development. Studies in a simple genetic organism should yield valuable insights into this process. In C. elegans, the heterochronic loci specify the temporal identity of stage-specific programs. Mutants result in temporal transformations, causing programs to be expressed at earlier (precocious) or later (delayed) larval stages in different tissues. Most identified loci are conserved transcriptional or translational regulators, which have been ordered into a genetic hierarchy, and whose regulatory interactions orchestrate post-embryonic development. Here we describe a new heterochronic gene called dre-1. dre-1 mutants display precocious development of epidermal stem cells and aberrant pathfinding programs in the gonad. dre-1 encodes a novel conserved F-box protein with molecular features that suggest it works in an E3-ubiquitin ligase complex, providing the first hint that heterochronic circuits are also regulated by proteolysis. Preliminary data indicate that DRE-1 works in a cullin-based E3 ligase complex, based on phenotypic congruence and yeast two-hybrid data. Moreover, the initial identification of several dre-1 suppressors reveals potential targets of degradation. The overall goal is to grasp how the E3 ligase complex regulates developmental timing.
The specific aims are to: 1. further characterize the role of dre-1 in the heterochronic circuits, dre-1mutants will be studied for embryonic and post-embryonic phenotypes and cell lineage defects characterized. The dre-1::gfp expression pattern and regulation will be determined and the position of dre-1in the heterochronic pathways revealed by genetic epistasis and synergy. 2. elucidate the E3 complex in which DRE-1 acts. By using RNAi knockdown, candidate E3 components will be discovered through phenotypic congruence. Tests of physical interaction, using yeast two-hybrid and co-immunoprecipitation will be used to characterize the complex. The human ortholog VIT-1 will be similarly analyzed. 3. characterize putative targets of DRE-1, including the trithorax protein LIN- 59/ASH1. lin-59 mutant defects, genetic interactions, expression pattern and regulation will be determined. LIN-59 and other candidate targets will be tested for regulation, binding and ubiquitination by DRE-1. Finally, RNAi screens for dre-1 suppressors will identify other potential targets. VIT1, the human ortholog of DRE-1 is downregulated in vitiligo. Hence, these studies may shed light on pathogenesis of this debilitating disease. Moreover, they may reveal how temporal licensing factors permit cells to shift from stem cell to differentiated states, or motile to non-motile behavior and how such programs may be subverted in cancers.
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