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Ecdysteroid-Dependent Regulation of Genes in Mammalian Cells

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Ecdysteroid-Dependent Regulation of Genes in Mammalian Cells Proc. Natl. Acad. Sci. USA Vol. 89, pp. 6314-6318, July 1992 Genetics Ecdysteroid-dependent regulation of genes in mammalian cells by a Drosophila ecdysone receptor and chimeric transactivators (steroid hormone receptor/gene regulatlon/lnducble tansptin) KAREN S. CHRISTOPHERSON, MELANIE R. MARK, VIVEK BAJAJ*, AND PAUL J. GODOWSKIt Department of Cell Genetics, Genentech, Inc., 460 Point San Bruno Boulevard, South San Francisco, CA 94080 Communicated by Keith R. Yamamoto, April 6, 1992 (receivedfor review February 7, 1992) ABSTRACT Steroid receptors are members of a large was obtained by using chimeric receptors containing the EcR family of transcription factors whose activity is tightly regu- ligand-binding domains fused to heterologous DNA-binding lated by the binding of their cognate steroid ligand. Mam- domains. Finally, the activity of these chimeric proteins lian steroid hormone receptors have been exploited to obtain could be increased by inclusion of a potent viral transacti- the regulated expression of heterologous genes in mamUma vation domain. cells. However, the utility ofthese systems in cultured cells and transgenic animals is limited by the presence of endogeous AND METHODS steroids and their receptors. We show that a Drosophila MATERIALS ecdysone receptor can function in cultured mammalin cls as Hormones. All hormones were purchased from Sigma with an ecdysteroid~dependent tnscription factor. The avit of the exception of muristerone A, which was obtained from the ecdysone receptor was not induced by any of the mamma- Simes (Milan), and ponasterone A, a gift from David King lian steroid hormones tested. The DNA-binding and transac- (University of California, Berkeley). Hormones were dis- tivation activities of viral, aan, or bacterial proteins solved in ethanol at a final concentration of 1 mM. were rendered ecdysteroid-dependent when fused to the ligand- Co ion ofPlasmids. Reporter plasmids pEc4M--nCO, bng domain of the ecdysone receptor. The ecdysone recep- pG4M-77CO, and pE4M-77CO contain four copies of a 28- or tor may prove useful in selectively regulating the expressW of 29-base-pair DNA sequence containing an ecdysone re- endogenous or heterologous genes in mammalian cells. sponse element (EcRE) (5'-GATCCGACAAGGGTTCAAT- GCACTTGTCA-3'), a glucocorticoid response element (GRE) (5'-GATCCGTAGCTAGAAACAGACTGTTCTGA- The steroid hormone receptor superfamily represents an 3'), or an estrogen response element (ERE) (5'-GATCCG- evolutionarily conserved group of proteins that influence TAGCTAGGTCAGACTGACCTGA-3'), respectively, at po- developmental and metabolic processes primarily by func- sition -77 of a mouse mammary tumor virus (MMTV) tioning as ligand-dependent transcription factors (1-3). Struc- promoter-chloramphenicol acetyltransferase (CAT) gene fu- tural analysis of receptor proteins has identified domains of sion. The MMTV promoter in this reporter gene was con- these proteins that function to bind DNA or ligand and to structed by using a synthetic oligonucleotide and extends enhance transcription (4-7). Ligand binding is required for from position -77 to position -16 relative to the site of the interaction of some receptors with their cognate response transcription initiation within the MMTV long terminal re- elements in vivo (8) and may also control dimerization and peat (LTR). Reporter plasmid pG4M-_nGO was derived from transcriptional activation properties (9, 10). pG4M--nCO by replacing the CAT gene with a human growth The ability of transcription factors to function in heterol- hormone cDNA. To construct the reporter plasmid ogous species has provided a system to analyze the functional pOC-33CO, the GREs and MMTV promoter of pG4M-77CO domains of these factors and a novel mechanism to control were replaced with an oligonucleotide encoding the minimal the expression of heterologous genes (11, 12). Mammalian cytomegalovirus (CMV) immediate early (IE) promoter (-33 steroid hormone receptors have been shown to function when to -19). pX4C-33CO was derived from pOC-33CO by addi- expressed in yeast (13-15) and Drosophila cells (16). tion of four copies of a Lex operator (5'-TCGACGTACTG- We wished to develop a system to specifically regulate TATGTACATACAGTACC-3') immediately upstream ofthe heterologous genes in mammalian cells in tissue culture and minimal CMV IE promoter. in transgenic animals. Such a system could provide a pow- Receptor construct pRSV.GGG contains a Rous sarcoma erful approach to understanding the functions ofthe product virus (RSV) LTR driving the expression ofa cDNA encoding of that gene. Systems that exploit the ability of mammalian the rat glucocorticoid receptor (GR) (20). The receptor plas- steroid hormone receptors to function as ligand-dependent mid pRSV.GEG was derived from pRSV.GGG by replacing transcription factors have proved useful in regulating the a HindIII-Aha III fragment of the rat GR cDNA with a expression ofheterologous genes in mammalian cells (17, 18). double-stranded oligonucleotide (5'-AAGCTTCAGGATGT- However, the applications of these systems in cultured cells CATTACGGAGTACTGACATGTGAAGGCTGCAAAG- or transgenic animals are limited by the presence of endog- TATTCTTT-3') incorporating two amino acid substitutions enous steroid receptors and their ligands. In this report we (G458E, S459G) that alter the DNA-binding specificity to show that the Drosophila ecdysone receptor (EcR) protein, allow ERE recognition (21, 22). In constructs denoted "V," a member of the steroid hormone receptor superfamily (19), can function as a ligand-dependent transcription factor in Abbreviations: EcR, ecdysone receptor; GR, glucocorticoid recep- mammalian cells. The activity of the EcR is induced upon tor; EcRE, ecdysone response element; GRE, glucocorticoid re- administration of certain ecdysteroids but not by any of the sponse element; ERE, estrogen response element; MMTV, mouse mammalian hormones tested. Novel target-gene specificity mammary tumor virus; RSV, Rous sarcoma virus; LTR, long ter- minal repeat; CMV, cytomegalovirus; IE, immediate early; CAT, chloramphenicol acetyltransferase. The publication costs ofthis article were defrayed in part by page charge *Present address: Graduate School of Arts and Sciences, Harvard payment. This article must therefore be hereby marked "advertisement" University, Cambridge, MA 02138. in accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 6314 Downloaded by guest on September 24, 2021 Genetics: Christopherson et al. Proc. Natl. Acad. Sci. USA 89 (1992) 6315 the sequence encoding amino acids 153-406 of the rat GR is probes were prepared and the RNase digestion was carried replaced by the sequence encoding the transcriptional acti- out as described (24). Protected fragments were fractionated vation domain of the herpes simplex virus VP16 protein in a 6% polyacrylamide sequencing gel and visualized by (amino acids 411-490). Receptor expression plasmid pRSV.- autoradiography. GGEc was constructed by replacing the sequence encoding the GR ligand-binding domain in pRSV.GGG (amino acids 528-795) with a sequence coding for the ligand-binding RESULTS domain of the EcR (amino acids 329-878). Similarly, to Only Specific Ecdysteroids Are Agonists for the EcR in construct GXEc, the cDNA encoding the ligand-binding Mammalian Cells. A cDNA clone for the Drosophila EcR was domain of GXG (referred to as NLxC in ref. 5) was replaced obtained by screening a cDNA library prepared from Dro- with DNA encoding the EcR ligand-binding domain. The sophila early pupal larvae with oligonucleotide probes based expression of the RSV LTR used in the receptor expression on the partial EcR sequence (referred to as DHR23 in ref. 25). plasmids was not affected by muristerone A. Sequence files The deduced amino acid sequence of the clone used here of all reporter and receptor genes in this study are available matches the published sequence ofthe EcR (19). The highest upon request. region of homology between the EcR and other members of Cell Culture, Transfection, and Analysis of Reporter Gene the steroid receptor superfamily is found in a cysteine-rich Activity. Human 293 cells (embryonic kidney cell line) were region (residues 264-329) that corresponds to the DNA- cultured in a 1:1 mixture of Dulbecco's modified Eagle's binding domain. The putative ligand-binding domain shares medium and Ham's nutrient mixture F12, supplemented with limited homology with retinoic acid and thyroid hormone 10% fetal bovine serum. Subconfluent cultures grown in receptors and the vitamin D receptor. The N-terminal domain 60-mm culture dishes were transfected (5) with 2.0 pug of the (residues 1-263) does not share significant homology with any receptor expression plasmid, or the parental expression steroid receptor superfamily member plasmid pRSV as a control, and 0.5 ,ug of the reporter (19). plasmid. As an internal control for transfection efficiency, 0.5 The EcR has been reported to regulate transcription of ,ug of the control plasmid pRSV.hGH was included in the genes containing EcREs in Drosophila tissue culture cells transfection mixture. After transfection, cells were treated treated with 20-hydroxyecdysone (19). We determined with hormone as indicated. Cells were harvested 36-40 hr whether the EcR could function in mammalian cells treated after transfection, and extracts were prepared by freezing with ecdysteroids to enhance the transcription of a reporter three times in dry ice and thawing at 37TC. CAT assays were gene containing EcREs
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