The ecdysone response enhancer of the Fbp1 gene of Drosophila melanogaster is a direct target for the EcR/USP nuclear receptor.

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102

The steroid hormone 20-hydroxyecdysone plays a key role in the induction and modulation of morphogenetic events throughout Drosophila development. Previous studies have shown that a heterodimeric nuclear receptor composed of the EcR and USP proteins mediates the action of the hormone at the transcriptional level through binding to palindromic ecdysteroid response elements (EcREs) such as those present in the promoter of the hsp27 gene or the fat body-specific enhancer of the Fbp1 gene. We show that in addition to palindromic EcREs, the EcR/USP heterodimer can bind in vitro with various affinities to direct repetitions of the motif AGGTCA separated by 1 to 5 nucleotides (DR1 to DR5), which are known to be target sites for vertebrate nuclear receptors. At variance with these receptors, EcR/USP was also found to bind to a DR0 direct repeat with no intervening nucleotide. In cell transfection assays, direct repeats DR0 to DR5 alone can render the minimum viral tk or Drosophila Fbp1 promoter responsive to 20-hydroxyecdysone, as does the palindromic hsp27 EcRE. In a transgenic assay, however, neither the palindromic hsp27 element nor direct repeat DR3 alone can make the Fbp1 minimal promoter responsive to premetamorphic ecdysteroid peaks. In contrast, DR0 and DR3 elements, when substituted for the natural palindromic EcRE in the context of the Fbp1 enhancer, can drive a strong fat body-specific ecdysteroid response in transgenic animals. These results demonstrate that directly repeated EcR/USP binding sites are as effective as palindromic EcREs in vivo. They also provide evidence that additional flanking regulatory sequences are crucially required to potentiate the hormonal response mediated by both types of elements and specify its spatial and temporal pattern.

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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confidence: 99%

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Direct Repeats Bind the EcR/USP Receptor and Mediate Ecdysteroid Responses in Drosophila melanogaster

Antoniewski

Mugat

Delbac

et al. 1996

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102

“…The mosquito and tobacco hornworm USP isoforms appear to be functionally distinct, displaying differential response to activation by 20E and thus contributing to the tissue and stage specificity of 20E action (36,69). Differential ecdysteroid response may also be effected through the differential recognition of a variety of EcREs, including inverted and direct repeats with various spacers (1,2,14,49,70).…”

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Molecular Determinants of Differential Ligand Sensitivities of Insect Ecdysteroid Receptors

Wang

Ayer

Segraves

et al. 2000

The functional receptor for insect ecdysteroid hormones is a heterodimer consisting of two nuclear hormone receptors, ecdysteroid receptor (EcR) and the retinoid X receptor homologue Ultraspiracle (USP). Although ecdysone is commonly thought to be a hormone precursor and 20-hydroxyecdysone (20E), the physiologically active steroid, little is known about the relative activity of ecdysteroids in various arthropods. As a step toward characterization of potential differential ligand recognition, we have analyzed the activities of various ecdysteroids using gel mobility shift assays and transfection assays in Schneider-2 (S2) cells. Ecdysone showed little activation of the Drosophila melanogaster receptor complex (DmEcR-USP). In contrast, this steroid functioned as a potent ligand for the mosquito Aedes aegypti receptor complex (AaEcR-USP), significantly enhancing DNA binding and transactivating a reporter gene in S2 cells. The mosquito receptor also displayed higher hormoneindependent DNA binding activity than the Drosophila receptor. Subunit-swapping experiments indicated that the EcR protein, not the USP protein, was responsible for ligand specificity. Using domain-swapping techniques, we made a series of Aedes and Drosophila EcR chimeric constructs. Differential ligand responsiveness was mapped near the C terminus of the ligand binding domain, within the identity box previously implicated in the dimerization specificity of nuclear receptors. This region includes helices 9 and 10, as determined by comparison with available crystal structures obtained from other nuclear receptors. Site-directed mutagenesis revealed that Phe529 in Aedes EcR, corresponding to Tyr611 in Drosophila EcR, was most critical for ligand specificity and hormone-independent DNA binding activity. These results demonstrated that ecdysone could function as a bona fide ligand in a species-specific manner.

“…Germ line transformation analysis of its promoter has pinpointed a 70-bp enhancer (Ϫ69 to Ϫ138) sufficient to specify the spatially and temporally ecdysteroid-controlled pattern of Fbp1 expression (24). The EcR/ USP heterodimer binds to a pseudopalindromic site in the 70-bp Fbp1 enhancer (4). Mutations of this site completely abolished the ability of the Fbp1 enhancer to confer a fat body-specific ecdysteroid response onto a minimal promoterlacZ reporter transgene, indicating that activation by the EcR/ USP heterodimer is a strict requirement for the activity of the Fbp1 enhancer (5).…”

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Dual Requirement for the EcR/USP Nuclear Receptor and the dGATAb Factor in an Ecdysone Response in Drosophila melanogaster

Brodu

Mugat

Roignant

et al. 1999

The EcR/USP nuclear receptor controls Drosophila metamorphosis by activating complex cascades of gene transcription in response to pulses of the steroid hormone ecdysone at the end of larval development. Ecdysone release provides a ubiquitous signal for the activation of the receptor, but a number of its target genes are induced in a tissue-and stage-specific manner. Little is known about the molecular mechanisms involved in this developmental modulation of the EcR/USP-mediated pathway. Fbp1 is a good model of primary ecdysone response gene expressed in the fat body for addressing this question. We show here that the dGATAb factor binds to three target sites flanking an EcR/USP binding site in a 70-bp enhancer that controls the tissue and stage specificity of Fbp1 transcription. We demonstrate that one of these sites and proper expression of dGATAb are required for specific activation of the enhancer in the fat body. In addition, we provide further evidence that EcR/USP plays an essential role as a hormonal timer. Our study provides a striking example of the integration of molecular pathways at the level of a tissue-specific hormone response unit.