Characterization of Drosophila nuclear receptor interactions and activation patterns

Nuclear receptors (NRs) are transcription factors whose activities are modulated through the binding of small lipophilic ligands. In general, ligand binding induces a conformational change in the NR that drives the recruitment of coactivator protein complexes. In Drosophila, there are 18 NRs. At the beginning of my work, only one of these, the ecdysone receptor (EcR), had a known ligand. The other 17 NRs are orphans whose activity has not yet been demonstrated to be modulated by the binding of ligands. Fushi tarazu-Factor 1 (FTZ-F 1) is an orphan NR that interacts with the homeodomain protein Fushi tarazu (FTZ) in early embryogenesis. I have shown that this interaction is mediated primarily through the ligand binding domain (LBD) of FTZ-F1 and the LXXLL motif, or NR box, of FTZ. I have further demonstrated that the FTZ-F1 LBD is alpha-helical in solution consistent with the known structures of other NR LBDs.;To identify ligands and cofactors for the Drosophila NRs, I have utilized a biochemical approach and an in vivo ligand sensor assay in embryos. I developed a three-step purification procedure using a modified tandem affinity purification (TAP) tag that generates highly purified protein complexes and co-purifies heme as an endogenous ligand for the NR E75. Using a ligand sensor assay, which consists of the yeast GAL4 DNA binding domain fused to the NR LBD and a UASGAL4-responsive reporter, I characterized the temporal and spatial patterns of activity for the 18 NRs in transgenic embryos. Only 9 NRs show activation patterns during embryogenesis, and many of these are activated in the extra-embryonic tissues. By modulating the levels of the EcR ligand 20-hydroxyecdysone (20E), I demonstrate that the ligand sensor transgenic lines respond specifically to alterations in the levels of their cognate ligands. Namely, only the EcR and USP ligand sensor lines are affected in a genetic background deficient for 20E production. Conversely, when ligand sensor embryos are cultured in the presence of exogenous 20E, only the EcR, USP and DHR38 ligand sensor lines show ectopic activation.