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A UAS site substitution approach to the in vivo dissection of promoters: interplay between the GATAb activator and the AEF-1 repressor at a Drosophila ecdysone response unit

Véronique Brodu^1,*, Bruno Mugat², Pierre Fichelson³, Jean-Antoine Lepesant¹ and Christophe Antoniewski^1,

¹ Institut Jacques-Monod, CNRS UMR7592, Université Paris 6 P. et M. Curie, Université Paris 7-Denis-Diderot, 2, place Jussieu, F-75251, Paris cedex 05, France
² Institut de Génétique Humaine, CNRS, 141, rue de la Cardonille, F-34396 Montpellier, cedex 5, France
³ Laboratoire de Biologie du Développement, Université Paris 6-P et M. Curie, 9 quai St Bernard, case 24, F-75005 Paris, France
^* Present address: Medical Research Council, National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 1AA, UK

Author for correspondence (e-mail: antoniewski{at}ijm.jussieu.fr)

Accepted April 17, 2001

An ecdysone response unit (EcRU) directs the expression of the Fat body protein 1 (Fbp1) gene in the third instar larval Drosophila fat body. The tissue-specific activity of this regulatory element necessitates the binding of both the ligand-activated EcR/USP ecdysone receptor and GATAb. To analyze the role played by GATAb in the regulation of the Fbp1 EcRU activity, we have replaced the GATA-binding sites GBS1, GBS2 and GBS3 in the Fbp1 EcRU with UAS sites for the yeast GAL4 activator and tested the activity of the mutagenized Fbp1 EcRUs in transgenic lines, either in the presence or absence of ubiquitously expressed GAL4. Our results reveal that GATAb plays two distinguishable roles at the Fbp1 EcRU that contribute to the 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 a ubiquitous repressor that maintains the Fbp1 EcRU in an inactive state, refractory to activation by GAL4. We identified this repressor as AEF-1, a factor previously shown to be involved in the regulation of the Drosophila Adh and yp1-yp2 genes. These results show that, for a functional dissection of complex promoter-dependent regulatory pathways, the replacement of specific regulatory target sites by UAS GAL4 binding sites is a powerful alternative to the widely used disruption approach.

Key words: Drosophila, Ecdysone, Nuclear receptor, GATAb, serpent, AEF-1, Fbp1

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