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Characterization of an EcR/USP heterodimer target site that mediates ecdysone responsiveness of the Drosophila Lsp-2 gene

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Abstract

The Larval serum protein-2 gene (Lsp-2) of Drosophila melanogaster is uniquely expressed in the fat body tissue from the beginning of the third instar to the end of adult life. Accumulation of the larval Lsp-2 transcript is enhanced by 20-hydroxyecdysone. To study the molecular basis for ecdysone regulated Lsp-2 activity, deletion mutants of the Lsp-2 5′-flanking region were constructed by fusion to either the Escherichia coli chloramphenicol acetyltransferase (CAT) gene or to an hsp70-lacZ hybrid gene encoding β-galactosidase. Constructs transfected into Drosophila S2/M3 cells were shown to confer transient ecdysone inducibility on the reporter genes. A single functional ecdysone response element (EIRE) was localized at position — 75 relative to the Lsp-2 transcription initiation site. In gel mobility shift assays using fat body nuclear extracts or nuclear receptors synthesized in vitro, a 27-bp sequence harboring the EcRE bound both the Drosophila ecdysone receptor and the Drosophila retinoid-X homologue, Ultraspiracle, in a cooperative manner. Competition experiments indicate that the affinity of the Lsp-2 EcRE for the ecdysone receptor complex is comparable to that of the canonical EcRE of the hsp27 gene and is at least 4-fold greater than that of Fbp1, another fat body-specific Drosophila gene. Our results suggest that structural features of this EcRE determine its ability to induce ecdysone responsiveness at a lower ligand concentration and may form the basis for differential hormone responsiveness within the fat body.

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Author notes

  1. Michael S. O'Grady

    Present address: Department of Zoology, University of British Columbia, 2354-6270 University Blvd., V6T 1Z4, Vancouver, B.C., Canada

Authors and Affiliations

  1. Institut Jacques Monod, Centre National de la Recherche Scientifique and Université de Paris, 7 - Denis Diderot, 75251, Paris Cedex 05, France

    Christophe Antoniewski

  2. Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Slot 516, 4301 West Markham St., 72205, Little Rock, Arkansas, USA

    Michael S. O'Grady, Ronda G. Edmondson, Suzanne M. Lassieur & Helen Beneš

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  1. Christophe Antoniewski
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  2. Michael S. O'Grady
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  4. Suzanne M. Lassieur
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  5. Helen Beneš
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Communicated by J. A. Campos-Ortega

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Antoniewski, C., O'Grady, M.S., Edmondson, R.G. et al. Characterization of an EcR/USP heterodimer target site that mediates ecdysone responsiveness of the Drosophila Lsp-2 gene. Molec. Gen. Genet. 249, 545–556 (1995). https://doi.org/10.1007/BF00290580

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