Ecdysone Response Element Research Articles

Drosophila ecdysone receptor functions as a constitutive activator in yeast

Transcriptional activation of the Drosophila ecdysone receptor (EcR) was studied in yeast cells, which carry a reporter plasmid containing the ecdysone response element in the absence or presence of its heterodimeric partners, ultraspiracle protein (USP) or human retinoid X receptor (RXRα). High constitutive transcriptional activation was detected in the yeast strain expressing EcR, but not USP or RXRα in the absence of ponasterone or muristerone A. Incubation of these ligands with yeast cells coexpressing EcR and USP or RXRα did not enhance the constitutive transcriptional activity. However, specific ligand binding using [ 3H]ponasterone A as a radioactive ligand was detected only in yeast extracts prepared from the yeast strain coexpressing EcR and USP, but not from yeast strains expressing only EcR or USP. The ligand binding characteristics of the EcR/USP complexes were similar to those reported in an insect cell line with a K d value of 1.8 nM for [ 3H]ponasterone A. These data are in contrast to mammalian cell transfection studies, and indicate that the EcR is the only member of the nuclear receptor superfamily of ligand-activated transcription factors which functions as a constitutive transcriptional activator in yeast, although the EcR/USP complexes exhibit normal ligand binding properties.

Expression of EcR and USP in Escherichia coli: purification and functional studies.

The functional ecdysteroid receptor complex consists of a nuclear receptor heterodimer of ecdysteroid receptor (EcR) and ultraspiracle (USP). EcR and USP of both Chironomus tentans and Drosophila melanogaster were expressed in Escherichia coli as fusion proteins with glutathione S-transferase (GST). Cell lysis and protein solubilization with the anionic detergent sarkosyl yielded preparations of EcR and USP with properties similar to those of the endogenous receptors in various respects. The heterodimer of the expressed proteins specifically bound the labeled ecdysteroid (Ec) [3H]ponasterone A. Furthermore, it preferentially recognized the palindromic ecdysone response element (EcRE) PALI. Interestingly, binding to the PAL1 element was also observed for EcR homodimers. USP homodimers, in turn, preferentially bound to the direct repeat element DR1. When incubated with native polytene chromosomes of Chironomus, EcR/USP specifically accumulated at the early Ec-inducible puff site IV-2B.

Expression of EcR and USP in Escherichia coli: Purification and functional studies

The functional ecdysteroid receptor complex consists of a nuclear receptor heterodimer of ecdysteroid receptor (EcR) and ultraspiracle (USP). EcR and USP of both Chironomus tentans and Drosophila melanogaster were expressed in Escherichia coli as fusion proteins with glutathione S-transferase (GST). Cell lysis and protein solubilization with the anionic detergent sarkosyl yielded preparations of EcR and USP with properties similar to those of the endogenous receptors in various respects. The heterodimer of the expressed proteins specifically bound the labeled ecdysteroid (Ec) [3H]ponasterone A. Furthermore, it preferentially recognized the palindromic ecdysone response element (EcRE) PAL1. Interestingly, binding to the PAL1 element was also observed for EcR homodimers. USP homodimers, in turn, preferentially bound to the direct repeat element DR1. When incubated with native polytene chromosomes of Chironomus, EcR/USP specifically accumulated at the early Ec-inducible puff site IV-2B. Arch. Insect Biochem. Physiol. 35:59–69, 1997. © 1997 Wiley-Liss, Inc.

The role of ecdysone in the induction and maintenance of hsp27 transcripts during larval and prepupal development of Drosophila

The Drosophila melanogaster small heat-shock gene hsp27 carries the canonic ecdysone response element (EcRE) at -537. This EcRE has been used extensively both in cultured cells and in vitro for studies of the ecdysone response. We have characterised the developmental expression of hsp27 in wild-type larval and prepupal salivary glands in parallel with that of the E74B and E74A primary ecdysone response transcripts, which are induced in the mid and late third larval instar by a minor and major increase in ecdysone titre respectively. The induction of hsp27 occurs between these two events in larvae and in parallel with that of E74A in prepupae. Transcript levels are severely reduced in ecd1 and dor (22) (deep orange) mutant larvae but are only moderately reduced in larvae mutant for the Broad-Complex allele br (t435) . By culturing salivary glands of different ages with low (1.8×10(-8) M) or high (1.8×10(-6) M) concentrations of hormone, we show that the response of an EcRE varies during development and that the timing of the response cannot be predicted solely from its apparent strength in cell line analyses.

The Broad-Complex gene is a tissue-specific modulator of the ecdysone response of the Drosophila hsp23 gene.

The steroid hormone ecdysone causes dramatic changes in the genetic programs leading to the pupariation of Drosophila melanogaster, and the Broad-Complex (BR-C) gene is known to play a key role in this process. Previously we showed that BR-C regulates developmental changes in transcription and chromatin structure of the 67B heat shock gene cluster, which contains four small hsp genes. Importantly, the downregulation of the hsp23 gene in the BR-C mutants correlates with the absence of a DNase I-hypersensitive site (DHS) at position -1400. To study the functional importance of the DHS-1400, we have introduced genomic fragments containing a modified hsp23 gene into the Drosophila germ line. Our analysis shows that the ecdysone response element is necessary but not sufficient for full developmental expression of hsp23 in the late third instar and that there is, indeed, another regulatory element, in the vicinity of DHS-1400. We also show that hsp23 developmental expression is not tissue specific. A construct lacking the ecdysone response element is unable to direct normal hsp23 expression in all tissues except the brain. Similarly, brain-specific expression is BR-C independent, although in the other tissues we find different requirements for BR-C genetic functions. The effect of the br mutations is restricted to wing imaginal discs and midgut tissue, while that of 2Bc is restricted to the fat body and Malpighian tubules, and mutations in the rbp group have no effect in any of the tissues studied. Thus, BR-C regulatory action is mediated through different genetic functions in a tissue-specific manner.

Molecular Analysis of theDrosophilaCatalase Gene

The main objective of this study was to isolate and characterize the catalase gene and accompanyingcis-regulatory regions inDrosophila melanogaster.Genomic clones were obtained on the basis of cross-hybridization to catalase cDNA and a 7-kbSalI–KpnI fragment encompassing the catalase gene was introduced intoDrosophilaby P element-mediated transformation. A single transgene, when placed in a catalase null background, was sufficient to restore resistance to H2O2as well as reduce susceptibility to early death. DNA sequence of the catalase gene domain was obtained. This included 1365 bp of sequence upstream of the transcription initiation site and 1423 bp downstream of the termination codon. TheDrosophilacatalase gene is composed of 3 exons, encoding 19, 307, and 180 amino acids, which are separated by 3520- and 96-bp introns. Sequence analysis of the promoter domain is presented, revealing multiple sequence similarities between catalase and Cu,Zn superoxide dismutase promoter domains. Developmental RNA gel analysis shows that peaks of catalase mRNA accumulation correspond roughly with major peaks of ecdysone titer during third instar and pupal stages. Candidate ecdysone response element sequences are noted downstream of the catalase polyadenylation site.

Bombyx EcR (BmEcR) and Bombyx USP (BmCF1) combine to form a functional ecdysone receptor

The Drosophila ecdysone receptor (DmEcR) is a member of the nuclear receptor superfamily; it functions as an obligate heterodimer with another nuclear receptor, DmUSP. EcR homologs have now been cloned from several other insects. We report here that one such homolog, BmEcR from the commercial silkmoth, Bombyx mori, is a functional ecdysone receptor. Upon dimerization with BmCF1, the silkmoth homolog of DmUSP, BmEcR binds the radiolabeled steroid ligand 125I-iodoponasterone A with K d = 1.1 nM, indistinguishable from that exhibited by DmEcR/DmUSP. BmEcR/BmCF1 forms a specific complex with an ecdysone response element (EcRE) derived from the heat shock protein 27 ( hsp27) gene promoter of Drosophila; and, as with DmEcR/DmUSP, formation of this complex is stimulated by the presence of 20-hydroxyecdysone. Finally, BmEcR can substitute for DmEcR in an EcR-deficient Drosophila tissue culture line, stimulating trans-activation of an ecdysone-inducible reporter gene construct. Thus, BmEcR and BmCF1 are the functional counterparts of DmEcR and DmUSP, respectively and, despite considerable sequence divergence between the Drosophila and Bombyx proteins, the counterparts are—at least qualitatively-functionally equivalent.

Overlapping Lsp‐2 gene sequences target expression to both the larval and adult Drosophila fat body

Larval serum protein-2 gene (Lsp-2) of Drosophila melanogaster encodes one of the major hexameric haemolymph proteins of third-instar larvae and a major component of adult serum. Regulated transcription of Lsp-2 results in high-level, ecdysone-stimulated expression throughout the larval fat body and low-level, spatially restricted expression in the adult fat cells. To localize cis-acting regulatory sequences responsible for the stage- and tissue-specific activity at Lsp-2, the expression of Lsp-2-lacZ fusion genes was studied by P element-mediated germline transformation of Drosophila. A 230 base pair larval enhancer, which includes an ecdysone response element (EcRE), specifically targets gene activity to the larval fat body. Although the adult mode of Lsp-2 expression depends on the larval enhancer, additional negative regulatory elements dictate both tissue-specificity and unique spatial restriction within the adult fat body. Implications of these findings for the identification of fat body-specific gene regulatory units in other insects are discussed.

20-Hydroxyecdysone, but Not Juvenile Hormone, Regulation ofyolk proteinGene Expression Can Be Mapped tocis-Acting DNA Sequences

The threeyolk proteingenes (yps) ofDrosophila melanogasterare expressed in the ovary and fat body of the adult female. Their levels of expression in the fat body depend upon both juvenile hormone (JH) and 20-hydroxyecdysone (20E). Using transformed lines of flies with various flanking sequences from theypgenes andlacZ, Adh,or nativeypgenes as reporters, the regulation of the threeypgenes by 20E and the JH analogue ZR515 (methoprene) was investigated. For 20E, induction of reporter gene expression in males was assayed and, for JH, upregulation of the genes in nutritionally deprived females, which express yolk proteins (YPs) at very low levels, was followed. We were able to map 20E inducible sites upstream ofyp3and sites located 3′ and within the coding sequence or introns ofyp3which can interact to respond to 20E. There are also sites in the intergenic spacer betweenyp1andyp2.Evidence for repressors was also found upstream of theypgenes, suggesting downstream 20E inducible elements may be importantin vivo.There appears to be a difference in the response to 20E in the fat body of the thorax and abdomen between different constructs in males. It is not clear whether those sequences which respond to 20E are genuine ecdysone response elements (i.e., binding sites for the ecdysone receptor) or if the effect is indirect. Methoprene upregulation of YPs, however, was only ever observed using nativeypgenes as reporters, suggesting that this hormone may act on intron sequences orypcoding sequences, or perhaps by influencing stability of theypmRNA.

Isolation and sequencing of the gene encoding Sp23, a structural protein of spermatophore of the mealworm beetle, Tenebrio molitor

The cDNA for Sp23, a structural protein of the spermatophore of Tenebrio molitor, had been previously cloned and characterized (Paesen, G.C., Schwartz, M.B., Peferoen, M., Weyda, F. and Happ, G.M. (1992a) Amino acid sequence of Sp23, a structure protein of the spermatophore of the mealworm beetle, Tenebrio molitor. J. Biol. Chem. 257, 18852–18857). Using the labeled cDNA for Sp23 as a probe to screen a library of genomic DNA from Tenebrio molitor, we isolated a genomic clone for Sp23. A 5373-base pair (bp) restriction fragment containing the Sp23 gene was sequenced. The coding region is separated by a 55-bp intron which is located close to the translation start site. Three putative ecdysone response elements (EcRE) are identified in the 5' flanking region of the Sp23 gene. Comparison of the flanking regions of the Sp23 gene with those of the D-protein gene expressed in the accessory glands of Tenebrio reveals similar sequences present in the flanking regions of the two genes. The genomic organization of the coding region of the Sp23 gene shares similarities with that of the D-protein gene, three Drosophila accessory gland genes and two Drosophila 20-OH ecdysone-responsive genes.

Seven-up Inhibits Ultraspiracle-Based Signaling Pathways In Vitro and In Vivo

Seven-up (Svp), the Drosophila homolog of the chicken ovalbumin upstream transcription factor (COUP-TF); Ultraspiracle (Usp), the Drosophila homolog of the retinoid X receptor; and the ecdysone receptor are all members of the nuclear/steroid receptor superfamily. COUP-TF negatively regulates hormonal signaling involving retinoid X receptor in tissue culture systems. Here we demonstrate that Svp, like COUP-TF, can modulate Ultraspiracle-based hormonal signaling both in vitro and in vivo. Transfection assays in CV-1 cells demonstrate that Seven-up can inhibit ecdysone-dependent transactivation by the ecdysone receptor complex, a heterodimeric complex of Usp and ecdysone receptor. This repression depends on the dose of Svp and occurs with two different Drosophila ecdysone response elements. Ectopic expression of Svp in vivo induces lethality during early metamorphosis, the time of maximal ecdysone responsiveness. Concomitant overexpression of Usp rescues the larvae from the lethal effects of Svp. DNA binding studies show that Svp can bind to various direct repeats of the sequence AGGTCA but cannot bind to one of the ecdysone response elements used in the transient transfection assays. Our results suggest that Svp-mediated repression can occur by both DNA binding competition and protein-protein interactions.

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

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 beta-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 (EcRE) 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.

The moulting hormone ecdysone is able to recognize target elements composed of direct repeats

In Drosophila melanogaster, three temporally distinct ecdysone-responsive puff sets, the so-called intermoult, early and late puffs, have been described on the salivary gland polytene chromosomes. We have analyzed in detail a DNA segment of the 3C polytene region, from which originates one of the most prominent intermoult puffs, with the aim of identifying ecdysone response elements (EcREs). Here we report that two putative EcREs of identical sequence are located at this puff site. Interestingly, these elements display a novel structural feature, being composed of directly repeated half-sites. Our results show that the EcR/USP heterodimer known to constitute the ecdysone functional receptor complex is able to bind to and transactivate through target elements composed of directly repeated half-sites. In addition, we show that these elements are also able to bind efficiently USP alone, suggesting that USP and EcR/USP could compete for their binding to DNA.

Genetically Engineered Insect Virus Pesticides: Present and Future

A computer-assisted analysis identified tentative target sequences for regulatory proteins including ecdysone-inducible factors such as BmFTZ-F1 and Broad-Complex Z4 (BR-C Z4) in the ie1 promoter of BmNPV. A transient expression experiment using BmN cells and a series of truncated ie1 promoter constructs demonstrated that the activity of the ie1 promoter responded to alpha-ecdysone and 20-hydroxyecdysone, which required a tridecameric nucleotide stretch (ie1EcRE, 5′-GTGTTATCGACCT-3′) homologous to the ecdysone response element reported for Drosophila (DmEcRE). RT-PCR demonstrated the expression of BmEcR and BmUSP, which are required as ecdysone-specific activators for EcRE-mediated activation, in BmN cells. Furthermore, the ie1 EcRE-mediated response was confirmed by using a recombinant BmNPV possessing a luciferase gene under the control of the ie1 promoter with or without ie1 EcRE. This is the first report of an ecdysone response element in a baculoviral gene promoter. These results also suggested that the regulation of the ie1 by ecdysone may militate viral replication at least under certain conditions during natural infections in vivo.

Molecular characterization of the gene encoding the precursor protein of diapause hormone and pheromone biosynthesis activating neuropeptide (DH-PBAN) of the silkworm, Bombyx mori and its distribution in some insects

The diapause hormone is a 24 amino acid peptide amide which induces embryonic diapause of the silkworm, Bombyx mori. Diapause hormone, pheromone biosynthesis activating neuropeptide, and other three neuropeptides of FXPRL amide peptide family have been shown to be generated from a polyprotein precursor which is encoded by a single mRNA. We have cloned the genomic sequence encoding the precursor protein of diapause hormone-pheromone biosynthesis activating neuropeptide (DH-PBAN) by using a DH-PBAN cDNA as a probe, and analyzed its structure. The gene comprised six exons interspersed by five introns. The diapause hormone sequence along with a signal sequence was encoded in the first and second exons, and the pheromone biosynthesis activating neuropeptide was in the fourth and fifth exons. The major transcription initiation site of the gene was localized at 25 bp upstream from the translation start site. A single copy of this gene was present in a haploid genome. The 5′-upstream region of the gene contained a sequence similar to the ecdysone responsive element of Drosophila hsp 23 gene, and five decanucleotide motifs, which shared the homeodomain binding core sequence, TAAT. Genomic Southern analysis on DNA from some insect species other than the silkworm showed positive bands which hybridized with DH-PBAN cDNA of the silkworm. Thus, the DH-PBAN-like gene seems to be widely distributed in insects.

Cloning and developmental expression of the ecdysone receptor gene from the spruce budworm, choristoneura fumiferana

Degenerate oligonucleotides were designed on the basis of conserved amino acid sequences in the DNA and ligand-binding regions of the members of the steroid hormone receptor superfamily. Using these oligonucleotides in RNA-PCR, a cDNA fragment was isolated from the spruce budworm, Choristoneura fumiferana. Comparison of the deduced amino acid sequence of this cDNA fragment with the members of the steroid hormone receptor superfamily suggested that this PCR fragment is a region of the ecdysone receptor from C. fumiferana. Using this cDNA fragment as a probe, 10 clones were isolated from a cDNA library that was constructed using the RNA from 4- and 5-day old embryos of C. fumiferana. Two cDNA clones (1.3 and 3 kb) that overlap and show amino acid identity with Drosophila melanogaster ecdysone receptor B-1 isoform (DmEcR) were characterized and sequenced. The longest open reading frame had 539 codons and covered the complete EcR coding region. The deduced amino acid sequence of this open reading frame had all five of the regions typical for a steroid hormone nuclear receptor. The C domain or DNA binding region showed the highest identity wit EcR proteins from D. melanogaster, Chironomus tendons, Aedes aegypti, Manduca sexta, and Bombyx mori. The A/B region, D domain or hinge region, E domain, or ligand binding region also showed significant amino acid similarity with the EcR proteins from the five insects mentioned above. The C. fumiferana ecdysteroid receptor (CfEcR) cDNA probe detected a 6.0-kb mRNA that was present throughout the development of C. fumiferana. The CfEcR mRNA increases in abundance at the time of the ecdysteroid peak during the molting phase in the embryonic, larval and pupal stages but remains low during the intermolt period. In the 6th instar larvae, the 6-kb CfEcR mRNA was detected in the epidermis, fat body, and midgut and maximum expression was observed during the prepupal peak of ecdysteroids in the hemolymph. CfEcR mRNA was induced in ecdysone treated CF-203 cells as well in the epidermis and midgut of larvae that were fed the nonsteroidal ecdysteroid agonist, RH-5992. The induction occurred within an hour and reached maximum levels around 3 hr, after which it decreased to the basal level by 6 hr. In vitro transcription and translation of the CfEcR cDNA yielded a 67-Kda protein that bound to the ecdysone response element (EcRE) as a heterodimer, along with the ultraspiracle protein.

Isolation of proteins that interact specifically with the retinoid X receptor: two novel orphan receptors.

We have used a yeast genetic system to isolate cDNAs encoding proteins that specifically interact with the ligand-binding domain of human retinoid X receptor-alpha (RXR alpha). A number encoded portions of two known RXR heterodimer partners, the retinoic acid receptor (RAR) and the peroxisome proliferator activated receptor. Of four additional RXR-interacting proteins (RIPs) selected for further study two, RIP14 and RIP15, are previously unidentified orphan members of the nuclear receptor superfamily. Two others, RIP110 and RIP13, do not show significant similarities to previously reported proteins. RIP110 interacts with LexA-RXR only in yeast cells grown in the presence of the RXR ligand 9-cis-RA, while the interaction of the four receptor superfamily members and RIP13 is unaffected by the presence or absence of 9-cis-RA. RIP110 and RIP13 also interact in yeast with several other members of the receptor superfamily, but RIP14 and RIP15 interact only with RXR. Analysis of larger cDNA clones demonstrates that there are at least two isoforms of RIP14 that differ in the N-terminal (A and B) and hinge (D) domains. Northern blot analysis indicates that RIP14 is expressed specifically in liver and kidney, while RIP15 is expressed in every tissue tested. Both RIP14 and 15 bind as heterodimers with RXR to the RA response element (RARE) from the promoter of the RAR beta 2 isoform (the beta RARE), and RIP14 and RXR heterodimers also bind the ecdysone response element from the Drosophila heat shock protein 27 promoter. Both heterodimers also bind to several synthetic RAREs and other elements. In cotransfections, neither RIP14 nor RIP15 trans-activates a reporter containing multiple copies of the beta RARE under any of a variety of conditions, suggesting that their activities are dependent on the binding of as yet unidentified specific ligands or on activation by other processes.

Expression and Function of the ultraspiracle (usp) Gene during Development of Drosophila melanogaster

The usp locus encodes a member of the nuclear hormone receptor superfamily in Drosophila melanogaster that interacts with EcR (ecdysone receptor) to mediate ecdysteroid-induced gene expression. A 2.7-kb usp mRNA was detected at all developmental times tested, although its abundance varied. Among premetamorphic stages, both the 2.7-kb transcript and Usp protein attained their highest levels in the late third larval instar. The 2.7-kb usp transcript was also found in adult stages and a 1.2-kb transcript was detected in the polyadenylated RNA fraction of both mature adult females and early embryos. Aneuploids carrying two usp mutant alleles and a putative variegating usp+ allele often developed deformities of the adult wing disc that apparently resulted from mutational disruption of usp activity before metamorphosis and whose frequency was affected by maternal genotype. Both of the recessive lethal usp mutations associated with this "cleft thorax" phenotype involved substitutions of conserved arginine residues in the DNA-binding domain, although the frequency of the phenotype was not the same for the two alleles. Both mutant proteins retained the ability to form heterodimers with EcR in vitro but showed reduced affinity for an ecdysone response element.

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

The transcription of the Drosophila melanogaster Fbp1 gene is induced by the steroid hormone 20-hydroxyecdysone and restricted to the late-third-instar fat body tissue. In a previous study we showed that the -68 to -138 region relative to the transcription start site acts as an ecdysone-dependent third-instar fat body-specific enhancer in a transgenic assay. Here we report that seven nucleoprotein complexes are formed in vitro on this enhancer when a nuclear extract from late-third-instar fat body is used in a gel shift assay. Accurate mapping of the binding sites of the complexes revealed a remarkably symmetrical organization. Using specific antibodies, one of the complexes was identified as a heterodimer consisting of the ecdysone receptor (EcR) and Ultraspiracle (USP) proteins. The binding site of the heterodimer as defined by mutagenesis and methylation interference experiments bears strong sequence similarity to the canonical hsp27 ecdysone response element, including an imperfect palindromic structure. The two elements diverge at three positions in both half-sites, indicating that the structure of an active EcR/USP binding site allows considerable sequence variations. In vivo footprinting experiments using ligation-mediated PCR and wild-type or ecdysteroid-deficient larvae show that occupancy of the Fbp1 EcR/USP binding site and adjacent region is dependent on a high concentration of ecdysteroids. These results provide strong evidence for a direct role of the EcR/USP heterodimer in driving gene expression in response to changes of the ecdysteroid titer during Drosophila larval development.

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

The transcription of the Drosophila melanogaster Fbp1 gene is induced by the steroid hormone 20-hydroxyecdysone and restricted to the late-third-instar fat body tissue. In a previous study we showed that the -68 to -138 region relative to the transcription start site acts as an ecdysone-dependent third-instar fat body-specific enhancer in a transgenic assay. Here we report that seven nucleoprotein complexes are formed in vitro on this enhancer when a nuclear extract from late-third-instar fat body is used in a gel shift assay. Accurate mapping of the binding sites of the complexes revealed a remarkably symmetrical organization. Using specific antibodies, one of the complexes was identified as a heterodimer consisting of the ecdysone receptor (EcR) and Ultraspiracle (USP) proteins. The binding site of the heterodimer as defined by mutagenesis and methylation interference experiments bears strong sequence similarity to the canonical hsp27 ecdysone response element, including an imperfect palindromic structure. The two elements diverge at three positions in both half-sites, indicating that the structure of an active EcR/USP binding site allows considerable sequence variations. In vivo footprinting experiments using ligation-mediated PCR and wild-type or ecdysteroid-deficient larvae show that occupancy of the Fbp1 EcR/USP binding site and adjacent region is dependent on a high concentration of ecdysteroids. These results provide strong evidence for a direct role of the EcR/USP heterodimer in driving gene expression in response to changes of the ecdysteroid titer during Drosophila larval development.