Proc. Natl. Acad. Sci. USAVol. 92, pp. 10477-10481, November 1995Biochemistry

Identification and characterization of a Drosophila nuclearreceptor with the ability to inhibit the ecdysone responseANDREW C. ZELHOF*tt, TSO-PANG YAO0§II, RONALD M. EvANSl, AND MICHAEL MCKEOWNt***Department of Biology, and §Graduate Program in Biomedical Science, University of California, San Diego, La Jolla, CA 92093; and IHoward Hughes MedicalInstitute, and tMolecular Biology and Virology Laboratory, The Salk Institute for Biological Studies, La Jolla, CA 92037

Communicated by Dan L. Lindsley, University of California, San Diego, La Jolla, CA, July 27, 1995

ABSTRACT In a search for retinoid X receptor-like mol-ecules in Drosophila, we have identified an additional memberof the nuclear receptor superfamily, XR78E/F. In the DNA-binding domain, XR78E/F is closely related to the mamma-lian receptor TR2, as well as to the nuclear receptors Coup-TFand Seven-up. We demonstrate that XR78E/F binds as ahomodimer to direct repeats of the sequence AGGTCA. Intransient transfection assays, XR78E/F represses ecdysonesignaling in a DNA-binding-dependent fashion. XR78E/F hasits highest expression in third-instar larvae and prepupae.These experiments suggest that XR78E/F may play a regu-latory role in the transcriptional cascade triggered by thehormone ecdysone in Drosophila.

Both the retinoid X receptor (RXR) and ultraspiracle (Usp)are responsible for coordinating networks of gene expression.Proper signaling by the thyroid hormone receptor, the retinoicacid receptor, the vitamin D receptor, and the peroxisomeproliferator-activated receptor is achieved through hetero-dimeric complexes with RXR (1-6). The ability of RXR-likemolecules to form heterodimers is evolutionarily conserved asdemonstrated by the fact that Usp, the Drosophila RXR, canreplace RXR in heterodimers with the above mammaliannuclear receptors (7). In Drosophila, the functional ecdysonereceptor complex is a heterodimer between the ecdysonereceptor (EcR) and Usp (8, 9).The EcR complex is responsible for mediating the transcrip-

tional response to the steroid hormone 20-hydroxyecdysoneleading to molting and metamorphosis. Analyses of the tran-scriptional cascade triggered by ecdysone, seen as puffingpatterns in the Drosophila salivary gland chromosomes, led toproposal of a regulated cascade of transcription factors nec-essary for proper metamorphosis (10). Molecular character-ization of the genes within the ecdysone-induced salivary glandpuffs revealed that several of the associated genes encodeorphan nuclear receptors. Orphan receptors have the charac-teristic DNA-binding domains (DBDs) and ligand-bindingdomains of the steroid/nuclear receptor superfamily but donot have identified ligands.The biological role of many orphan receptors is not clear. In

some cases, orphan receptors can modulate hormonal signal-ing of other nuclear receptors. Coup-TF and Seven-up canrepress RXR/Usp-based signaling pathways (11-13, 29),whereas ,BFTZ-F1 can enhance transcription from gene targetsof the EcR complex (14). Due to this potential of orphanreceptors to modulate signaling pathways or possibly respondto specific hormones, we want to identify additional membersof this family. This paper describes the orphan receptorXR78E/F and its possible role in modulating hormonal sig-naling pathways.tt

MATERIALS AND METHODSGenomic Southern Blotting and Isolation of cDNAs. Stan-

dard procedures were used for isolation of genomic DNA andSouthern blotting (15, 16). The stringent and nonstringenthybridization conditions have been described (17), except thatwashes were performed with 2x SSC/0.1% SDS two times atroom temperature and with 1 x SSC/0.1% SDS at 65°C. DNAblots and libraries were screened with DNA encoding themouse RXRI3 DBD. A size-selected genomic library was madeby digesting Canton S genomic DNA with EcoRI, size-frac-tionating the fragments on an agarose gel, and isolatingfragments of 2-5 kb. The DNA was ligated to A ZAP II,EcoRI-digested phage arms and packaged according to themanufacturer's instructions (Stratagene). Plasmids containingpositive inserts were derived from phage according to themanufacturer's instructions (Stratagene). An imaginal disccDNA library (provided by C. Zuker, University of California,San Diego) was screened with the cloned genomic fragmentCSR 2.5.

Salivary Chromosome in Situ Hybridization. Chromosomein situ hybridizations were performed as described by Mc-Keown et al. (18) except a digoxigenin-labeled DNA probe wasused and detection was performed as recommended by themanufacturer (Boehringer Mannheim).

Developmental Northern Blot. Total RNA was isolated byhomogenizing the animals in RNA extraction buffer (200 mMNaCl/20 mM Tris-HCl, pH 7.5/20 mM EDTA/2% SDS) thatcontained proteinase K (2.0 mg/ml) (Merck). The homoge-nate was incubated at 50°C for 1 hr and then extracted twicewith phenol/chloroform (1:1) and once with chloroform/isoamyl alcohol (24:1). All nucleic acids were precipitated withethanol. After resuspending the nucleic acid pellet in diethylpyrocarbonate-treated water, an equal volume of 8 M LiCl wasadded and then the mixture was incubated for at least 1 hr at-20°C to precipitate the RNA. Standard procedures wereused for Northern blotting (15).

Plasmids. CMX XR78E/F was constructed by placing thecDNA into CMXPL1, a derivative of the CMX expressionvector (19). CMX GEcR and CMX Usp have been described(7). The luciferase reporter constructs AMTV-hsEcRE5-LUCand AMTV-Eip28/293-LUC have been described (29).

Transfection Assays. Monkey kidney CV-1 cells were grownin Dulbecco's modified Eagle's medium containing 10% resincharcoal-stripped bovine calf serum. Twenty-four hours beforetransfection, cells were plated into 48-well plates. Cells were

Abbreviations: RXR, retinoid X receptor; Usp, ultraspiracle; EcR,ecdysone receptor; DBD, DNA-binding domain; EMSA, electrophoreticmobility-shift assay; EcRE, ecdysone response element.tA.C.Z. and T.-P.Y. contributed equally to this work.ItPresent address: Dana-Farber Cancer Institute, Mayer Building,Room 652, 44 Binney Street, Boston, MA 02115-6084.* *To whom reprint requests should be addressed at: Molecular

Biology and Virology Laboratory, The Salk Institute for BiologicalStudies, P.O. Box 85800, San Diego, CA 92186-5800.

ttThe sequence reported in this paper has been deposited in theGenBank data base (accession no. U31517).

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The publication costs of this article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertisement" inaccordance with 18 U.S.C. §1734 solely to indicate this fact.

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transfected using DOTAP according to the manufacturer'sinstructions (Boehringer Mannheim). Transfection assay mix-tures contained (per well) 0.06 ,g of reporter, 0.02 ,tg of eachreceptor, and 0.1 ,g of CMX ,B-galactosidase to determinetransfection efficiency. After 2 hr, the liposomes were removedand the cells were incubated for 36 hr in medium either withoutor with the ligand muristerone A (Zambon, Bresso, Italy) at afinal concentration of 1 ,uM. Cell extracts were prepared andassayed for luciferase and f3-galactosidase activity as described(19). All experimental points represent the average of threetransfections.

Electrophoretic Mobility-Shift Assays (EMSAs). Protein forall EMSAs was generated by in vitro transcription/translationreactions using the TNT coupled reticulocyte lysate system(Promega) according to the manufacturer's instructions. Thesynthetic oligonucleotides were the same as described byZelhof et al. (29) as well as the conditions for the EMSAs. TheDrosophila embryo extract was a gift from J. Kadonaga (Uni-versity of California, San Diego). The monoclonal antibody forEcR was a gift from David Hogness (Stanford University) andthe Usp antisera have been described elsewhere (7).

Antisera Production. Amino acids 1-160 of XR78E/F werecloned by PCR into the glutathion S-transferase fusion vectorpGEX2T (Pharmacia). The subsequent glutathion S-transferasefusion protein was used to produce antisera as described by Yaoet al. (7).

RESULTSIdentification and Isolation of XR78E/F. To examine

whether genes for additional RXR-like molecules exist inDrosophila, we probed a blot of genomic DNA with DNAencoding the mouse RXRI3 DBD. Under moderate stringency,two prominent signals were seen (Fig. 1A, lane 1). The 8-kbband corresponds to ultraspiracle (usp) (20), and another bandis detected at -2.7 kb. To isolate this DNA, a size-selectedgenomic library was screened with the murine RXR,B DBDprobe. Several positive clones were isolated and containedidentical inserts. They were given the isolation name CSR 2.5.In situ hybridization maps CSR 2.5 to salivary chromosomeregion 78E/F (Fig. 1B). CSR 2.5 encodes a protein with adeduced amino acid sequence containing the zinc fingerscharacteristic of nuclear receptor DBDs (Fig. 2). Based on theposition of CSR 2.5, we refer to this locus as XR78E/F (Xreceptor at 78E/F, where X indicates an unknown ligand). Thisis a separate locus from the E78 locus, encoding E78A andE78B, at salivary chromosome position 78C (21).To further characterize this gene, we used CSR 2.5 to screen

an imaginal disc cDNA library. Several clones were isolatedand sequenced to obtain a complete, full-length cDNA se-

A

usp i 8. 0 kb

XR78 E/F'_ 2.7 kb

1.6 kb

16 .. ..

1 MDGVKVETFIKSEENRAMPLIGGGSASGGTPLPGGGVGMGAGASATL 4758 SVELCLVCGDRASGRHYGAISCEEGI IRKQLG,YQCRGMNC 9495 EVTMHHRRCQF RLQKCLASGIRSDSVQHERKPIVDRKEGIIAAAG 141142 SSSTSGGGNGSSTYLSGKSGYQQGRGKGHSVKAESAPRLQCTARQQR 188189 AFNLNAEYIPMGLNFAELTQTLMFATQQQQQQQQQHQQSGSYSPDIP 235236 KADPEDDEDDSMDNSSTLCLQLLANSASNNNSQHLNFNAGEVPTALP 282283 TTSTMGLIQSSLDMRVIHKGLQILQPIQNQLERNGNLSVKPECDSEA 329330 EDSGTEDAVDAELEHMELDFECGGNRSGGSDFAINEAVFEQDLLTDV 376377 QCAFHVQPPTLVHSYLNIHYVCETGSRIIFLTIHTLRKVPVFEQLEA 423424 HTQVKLLRGVWPALMAIALAQCQGQLSVPTIIGQFIQSTRQLADIDK 470471 IEPLKISKMANLTRTLHDFVQELQSLDVTDMEFGLLRLILLFNPTLF 517518 QHRKERSLRGYVRRVQLYALSSLRRQGGIGGGEERFNVLVARLLPLS 564565 SLDAEAMEELFFANLVGQMQMDALIPFILMTSNTSGL 601

FIG. 2. Amino acid sequence of XR78E/F. cDNA sequence en-codes a protein of 601 amino acids with a characteristic DBD andligand-binding domain of nuclear receptors. DBD is in boldface.

quence. Except for introns, the cDNA sequence matches thegenomic sequence and has an open reading frame of 601 aminoacids (Fig. 2). Several in-frame stop codons are found up-stream of the putative start codon. The open reading framepredicts a polypeptide with all the characteristics of a nuclearreceptor. There is a two-zinc finger DBD and the C-terminalregion contains sequences characteristic of nuclear receptorligand-binding domains (22). The DBD shows significanthomology to the DBD of the human orphan receptors TR2 andTR4/TAK1 (23-26). In the DBDs, XR78E/F and TR2 are74% identical. XR78E/F also shares homology with the mu-rine RXRf3 DBD (68%) as well as with the DBDs of Coup-TFand Seven-up (63%). The ligand-binding domain is moredivergent and cannot be clearly placed within one receptorsubfamily.

Expression Pattern ofXR78E/F. To identify possible func-tions for XR78E/F, we examined its expression profile. Fig. 3shows a blot of total RNA from various stages of Drosophiladevelopment. Although XR78E/F is not expressed strongly inembryos, its expression increases after the second- to third-instar molt. Quantification of the ratio of XR78E/F RNA torp49 RNA shows a fairly constant expression level in theprepupal and pupal stages (data not shown). Based on studiesof salivary glands cultured in ecdysone, XR78E/F expressiondoes not appear to be strongly activated by ecdysone (data notshown). RNA in situ hybridization to embryos and imaginaldiscs show ubiquitous expression at low levels (data notshown).

Binding Profile of XR78E/F. Based on the homology withRXR and Coup-TF/Seven-up, XR78E/F may share similarDNA-binding specificity. Using EMSAs and in vitro translatedprotein, we find that XR78E/F binds to hormone responseelements consisting of direct repeats of the sequence AG-GTCA, in particular a direct repeat spaced by 1 nucleotide(DR1). To identify the relative affinity of XR78E/F for a DR1compared to other direct repeats with different spacing, a

B

1 2

FIG. 1. Drosophila genomic Southern blot and chromosome in situ hybridization. (A) DNA encoding the mouse RXRI3 DBD was used to probeEcoRI-digested Canton S genomic DNA under moderate stringency. Two prominent bands are present in lane 1. Lane 2 shows hybridization ofthe isolated CSR 2.5 fragment to genomic DNA. (B) In situ hybridization to polytene chromosome position 78 E/F.

10478 Biochemistry: Zelhof et al.

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FIG. 3. Developmental Northern blot ofXR78E/F. Total RNA was isolated at the stages indicated and probed with a full-length cDNA clone.Only one band is detected and relative levels of expression were compared using rp49 levels as analyzed on a Molecular Dynamics PhosphorImager(data not shown).

competition analysis was performed (Fig. 4A). In this assay, aradiolabeled DR1 was combined with a 10-fold molar excess ofeach unlabeled competitor oligonucleotide during the periodof protein binding. XR78E/F has the highest affinity for a DR1(Fig. 4A, lane 1), but other direct repeats compete for binding(lanes 4-7). An oligonucleotide of equal size but containingonly one of the two half sites does not compete (lane 3),suggesting that XR78E/F binds as a homodimer and not as amonomer. An inverted repeat, represented by the hs27 ecdy-sone response element (EcRE) (27), also does not compete(lane 8). Interestingly, this binding profile is similar to that ofSeven-up I and II. Heterodimerization with Usp and EcR hasnot been detected.To check whether endogenous XR78E/F protein can bind

to a DR1, we examined binding of proteins from Drosophilaembryo nuclear extracts to a CRBPII element (28) (Fig. 4B).This element contains four AGGTCA half sites separated by1 nucleotide. Several binding complexes are observed. Appli-cation of antisera against XR78E/F caused one complex to

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decrease or disappear (Fig. 4B, compare lanes 1 and 4, complexis indicated by arrow), while preimmune serum and antibodiesto EcR and Usp have no effect on this complex (Fig. 4B, lanes2 and 3). Control experiments with in vitro translated XR78E/Fprotein show that XR78E/F complexes comigrate with theputative XR78E/F CRBPII complex from embryos (data notshown) and that the XR78E/F complex is also disrupted uponaddition of XR78E/F antisera (Fig. 4C).XR78E/F Inhibits Ecdysone Signaling in a DNA-Dependent

Fashion. Both Seven-up and XR78E/F appear to have thesame DNA binding specificity and may possibly display over-lapping functions in vivo. However, because the expressionpattern of XR78E/F is different from seven-up, they areunlikely to perform redundant functions. XR78E/F RNAincreases in third-instar larvae and is slightly influenced byecdysone; thus, XR78E/F may contribute to regulation of thetranscriptional cascade induced by ecdysone. We used tran-sient transfection assays to test whether XR78E/F can alterecdysone signaling. CV-1 cells respond to ecdysone only after

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FIG. 4. XR78E/F DNA-binding competition assay. (A) EMSAs were used to determine the relative affinities of XR78E/F for different hormoneresponse elements indicated. In vitro translated XR78E/F was incubated with 32P-labeled oligonucleotide representing a direct repeat spaced byone nucleotide (DR1) with or without (lane 1) a 10-fold excess of unlabeled competitor. EcRE represents an oligonucleotide based on the hs27EcRE. Competitors are listed above each lane. (B) EMSA of a 32P-labeled CRBPII element (27) using Drosophila embryonic nuclear extract, withthe addition of antibodies directed against specific nuclear receptors (Usp, lane 2; EcR, lane 3; XR78E/F, lane 4; preimmune serum, lane 5). Theputative XR78E/F complex (arrows) is disrupted only in lane 4. (C) EMSA using in vitro translated XR78E/F to demonstrate specificity of theXR78E/F antiserum. In A-C, arrows indicate position of the XR78E/F complex.

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FIG. 5. XR78E/F repression of EcR/Usp hormonal signaling. (A) Averaged data obtained from triplicate transfections using the reporterconstruct AMTV-hsEcRE5-LUC in CV-1 cells. (B) Data obtained for reporter construct AMTV-Eip28/293-LUC. Plasmids encoding XR78E/F,Usp, and EcR were transfected with the reporter plasmids as indicated. Luciferase activity for each transfection was standardized against3-galactosidase activity as an internal transfection control. A final concentration of 1 ,LM muristerone was used as ligand. SD is shown for each

point.

transfection of DNAs encoding both Usp and EcR (7). Usingreporter constructs containing either five copies of the hs27EcRE (A&MTV-hsEcRE5-LUC) or three copies of the Eip28/29 EcRE (AMTV-Eip28/293-LUC), we tested whetherXR78E/F could alter ecdysone signaling.As previously shown (7, 29), the transfection of DNA

encoding Usp and GEcR (a modified EcR with the N-terminaltransactivation domain of the glucocorticoid receptor) rendersCV-1 cells competent for ecdysone-induced transcriptionalactivation (Fig. 5, compare columns 1 and 2 with columns 3).The difference in activation between the two reporters isprobably due to the difference in copy number of the tworesponse elements. Cotransfection of DNA encodingXR78E/F in addition to the DNAs encoding Usp and GEcRhas different effects on hormone-induced transcriptional ac-

tivity depending on the response elements. For the AMTV-Eip28/293-LUC reporter, the presence of XR78E/F substan-tially inhibited transcriptional activity. We see 29.8-fold in-duction of transcriptional activity in the absence of XR78E/Fand only 4.1-fold induction in the presence of XR78E/F (Fig.SB, columns 3 and 4). In the case of the AMTV-hsEcRE5-LUCreporter, the presence of XR78E/F did not affect transcrip-tional activity (Fig. 5A, columns 3 and 4). Thus, XR78E/Falters ecdysone signaling in a response element-dependentmanner. With the AMTV-Eip28/293-LUC reporter, there is a

slight increase in luciferase activity in response to XR78E/Fand muristerone. This increase is slight and may overlap levelsseen in controls (Fig. SB, compare columns 5 and 6).Why does XR78E/F inhibit ecdysone signaling on the Eip

28/29 EcRE but not the hs27 EcRE? One possibility is thatXR78E/F competes with EcR/Usp for binding sites on theresponse elements and that XR78E/F binds to the Eip 28/29EcRE and not the hs27 EcRE. Examination of the hs27 EcREand the Eip 28/29 EcRE revealed that a degenerate directrepeat with 3-nucleotide spacing (DR3) is present in the Eip28/29 EcRE but not in the hs27 EcRE. One half site of thisDR3 is essential for EcR/Usp heterodimer binding to thiselement (29). From Fig. 4A, we know that a DR3 is a potentialXR78E/F binding site, while the hs27 EcRE appears to be a

poor binding site. To test whether XR78E/F has differentialbinding to the two EcREs, we used a competitive EMSA (Fig.6) to determine the relative affinity of XR78E/F for thesequences present in both reporter constructs, as well as theimportance of the direct repeat present in the Eip 28/29EcRE. XR78E/F was bound to a radiolabeled DR1 elementin the presence of a 25-fold molar excess of specific competitorDNAs. As expected, a DR1 competes for binding. Interest-

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10480 Biochemistry: Zelhof et al.

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ingly, the wild-type Eip 28/29 element competes nearly asefficiently as the DR1 (Fig. 6, compare lanes 2 and 3). If eitherhalf site of the DR3 present in Eip 28/29 is mutated (repre-sented by oligonucleotides Eip 28/29 1 and Eip 28/29 2),competition for binding is lost (lanes 4 and 5). The het-erodimer of EcR and Usp will bind to the Eip 28/29 1 element,with lower affinity, but binding to the Eip 28/29 2 element iseliminated (29). The hs27 EcRE 2X-1 and 2x-2 oligonucle-otides represent the two orientations of the individual hs27EcREs existing in the reporter construct AMTV-hsEcRE5-LUC. Neither of these oligonucleotides competes for DNAbinding (lanes 6 and 7). These results suggest that XR78E/Finhibits ecdysone responsiveness from the Eip 28/29 elementby direct competition for binding to the regulatory DNA sitesand that XR78E/F fails to inhibit ecdysone responsiveness onthe hs27 EcRE because it cannot bind to this element.

DISCUSSIONHere we have described the isolation and characterization ofa Drosophila orphan nuclear receptor, XR78E/F. Even thoughXR78E/F was isolated with a RXR DBD-based probe, thereceptor has higher homology to the DBD of the mammalianTR2 receptors. The mammalian TR2 receptors were isolatedfrom human testis and prostate libraries and belong to theCoup subfamily of nuclear receptors (23-26). They haveidentical DBDs but are diverse in their ligand-binding do-mains. Their function is not known. Like TR2, XR78E/Fshows homology with mammalian Coup-TF and DrosophilaSeven-up. The ligand-binding domain of XR78E/F does notshow any significant homology to other ligand-binding do-mains and does not have an identified ligand.XR78E/F expression appears to be ubiquitous and low, with

higher expression during the third-larval, prepupal, and pupalstages. The increase of expression during the third-larval instaris reminiscent of ecdysone-inducible genes, but little inductionis seen in ecdysone organ cultures. Although mRNA levels inembryos are very low, functional protein appears to be presentas judged by EMSAs and antibody shifting with embryonuclear extracts. The expression pattern ofXR78E/F does notsuggest a specific in vivo function, but biochemical data suggestthat XR78E/F may modulate transcriptional activity inducedby the ecdysone receptor complex or other hormone receptorsvia interactions with shared DNA-binding sites.The close homology between the DBDs of XR78E/F and

the Drosophila receptor Seven-up is reflected in their similarDNA-binding specificities. Like Svp, XR78E/F binds as ahomodimer to variously spaced direct repeats, with the highestaffinity for a DR1. Although XR78E/F shares similar DNA-binding specificities with Seven-up, XR78E/F is unlike Svp inthat it represses ecdysone-induced transcription only on anEcRE to which it binds. Thus, XR78E/F regulates transcrip-tion via DNA binding, whereas Seven-up can regulate ecdy-sone-induced transcription independent ofDNA binding (29).Based on the biochemical data and expression pattern ofXR78E/F, it is possible that XR78E/F may modulate certaingenes by raising the threshold level of the EcR complexnecessary for transcriptional activation. Under this model,XR78E/F may act to dampen responses to any prematurefluctuations in ecdysone titers to ensure proper spatial andtemporal expression of ecdysone-inducible genes.

We thank Dr. B. Forman, J. Chen, and H. Juguilon for technicaladvice and guidance on the transient transfection assays and P. Edeen

for technical support. We thank Dr. B. Forman and A. L. Scully forcritically reading this manuscript. This work was supported by grantsfrom the National Institutes of Health and American Cancer Societyto M.M. R.M.E. is an investigator of the Howard Hughes MedicalInstitute at The Salk Institute for Biological Studies. A.C.Z. is aChapman Fellow of The Salk Institute and was supported in part bya grant from the American Cancer Society to M.M., T.-P.Y. wassupported by the Howard Hughes Medical Institute.

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