A UAS site substitution approach to study the regulation of an Ecdysone Response Unit : interplay between EcR/USP, GATAb and AEF-1. C. Antoniewski 1, V. Brodu 2, B. Mugat 1, P. Fichelson 3, J.-A. Lepesant 1. 1) Dept Biol du Developpement, Inst Jacques Monod, Paris, France; 2) M. R. C., London, UK; 3) UMR 7622, Université Paris VI, Paris, France.
A complex ecdysone response unit (EcRU) directs the expression of the Fat Body Protein 1 (Fbp1) gene in the larval fat body in response to the late third instar ecdysone peak. We had shown previously that dGATAb binds to three GATA binding sites GBS1, GBS2 and GBS3 in the Fbp1 EcRU and is strictly required, in addition to the EcR/USP ecdysone receptor, for the activity of this regulatory element in the fat body. We have undertaken to establish more precisely the functional role of GATAb in the Fbp1 EcRU activity. To this aim, we have substituted the various GBS sites with UAS sites for the yeast GAL4 activator and tested the activity of the mutagenized Fbp1 EcRUs in transgenic lines, in the presence or absence of GAL4. Unexpectedly, we found that ubiquitously expressed GAL4 can replace GATAb at GBS1 without change in the tissue-specificity of the Fbp1 EcRU. We showed that this is due to a redundant tissue-specific role of GATAb binding to GBS3. Combinations of GBS substitutions with UAS revealed that GATAb exerts two distinguishable functions at the Fbp1 EcRU, both of them contributing to the highly tissue-specific activity of this regulatory element. On the one hand, GATAb mediates a fat body specific transcriptional activation. On the other hand, it antagonizes specifically in the fat body an ubiquitous repressor which maintains the Fbp1 EcRU in an inactive state, refractory to activation by GAL4. Using gel shift assays and mutation analysis, we identified this repressor as AEF-1, a factor previously shown to be involved in the regulation of Adh and yp1-yp2 genes. Our results demonstrate that substitution of specific regulatory target sites by UAS GAL4 target sites provides a powerful alternative to the widely used disruption approach for a functional dissection of complex promoter-dependent regulatory pathways.