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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 Brodu1,*, Bruno Mugat2, Pierre Fichelson3, Jean-Antoine Lepesant1 and Christophe Antoniewski1,{ddagger}

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



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  		Fig. 1. Analysis of the expression conferred in vivo by the Fbp1 EcRU to the lacZ reporter transgene. The structures and names of the transgenic constructs are indicated on the left-hand side. Positions of the A (A) and enhancer (E) elements in the Fbp1 EcRU (-194 to –69 relative to the Fbp1 transcription start) are indicated. All constructs include the minimal Fbp1 promoter (-69 to +80) fused to the lacZ reporter gene. GATA-binding sites GBS1 (-130/-124), GBS2 (-90/-85) and GBS3 (-75/-70) are indicated with black boxes, EcR/USP-binding site EBS (-91/-103) is indicated by arrows. Late third instar larval tissues from transgenic lines with the indicated genotypes (right-hand side) were dissected and histochemically stained for determination of ß-galactosidase activity.

 


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  		Fig. 2. dGATAb is required for the specific transactivation of the Fbp1 EcRU by GAL4. Late third instar larval tissues from transgenic lines with the indicated genotypes (right-hand side) were dissected and histochemically stained for ß-galactosidase activity. Structures of the constructs in transgenic lines are depicted to the left-hand side, as in Fig. 1. Substituting UAS sites are indicated by hatched boxes. Disruptions of GBS2 and GBS3 are indicated by black crosses. Expression of the AE[UAS1] reporter construct in either the absence (A) or the presence (B) of the GAL4daG32 driver is shown.

 


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  		Fig. 3. GAL4 activation of the Fbp1 EcRU is dependent upon the integrity of at least one of the two GBS1 or GBS3 sites. Late third instar larval tissues from transgenic lines with the indicated genotypes (right-hand side) were histochemically stained for ß-galactosidase activity. Structures of the constructs in transgenic lines (left-hand side) are depicted as in Fig. 2. Expression of the AE[UAS2-3] reporter construct in either the absence (A) or the presence (B) of the GAL4daG32 driver is shown.

 


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  		Fig. 4. AEF-1 silences the activation of the Fbp1 EcRU by GAL4. Late third instar larval tissues from transgenic lines with the indicated genotypes (left-hand side) were histochemically stained for ß-galactosidase activity. Structures of the constructs in transgenic lines (left-hand side) are depicted as in Fig. 2. Disruptions of the binding sites for EcR/USP and AEF-1 are indicated with black crosses.

 


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  		Fig. 5. AEF-1 in third instar fat body nuclear extract binds to element A. Binding of proteins in a nuclear extract from late third instar fat body was analyzed in a gel shift assay using the element A (-194 to -138) as a probe in the presence or absence of competitor DNAs, AEF-1 antibody and protein A as indicated. C1, specific AEF-1 retarded complex; S1, AEF-1 supershifted complex; S2, nonspecific complex that formed when rabbit serum was incubated in the presence of fat body nuclear extract. The sequence of the element A probe and positions of the competitor DNAs are depicted at the bottom.

 


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  		Fig. 6. Tentative model for the regulation of the Fbp1 EcRU activity in the fat body during development. Before the third larval instar, AEF-1 bound to element A (red bar) silences the activity of the Fbp1 enhancer (green bar). During the third larval instar, GATAb bound to GBS1 and GBS3 and associated with unidentified co-factors (C?) counteracts the repressing activity of AEF-1 in the fat body. Final activation of the Fbp1 promoter during late third larval instar results from synergistic transactivation by the GATAb multiprotein complex at GBS1 and the EcR/USP ecdysone receptor activated by its 20-hydroxyecdysone (20E) ligand.

 




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