Tài liệu Báo cáo khoa học: Improved ecdysone receptor-based inducible gene regulation system doc

Improved ecdysone receptor-based inducible gene regulation system Subba R. Palli 1 , Mariana Z. Kapitskaya 2 , Mohan B. Kumar 2 and Dean E. Cress 2 1 Department of Entomology, College of Agriculture, University of Kentucky, KY, USA; 2 RHeoGene LLC, Spring House, PA, USA To develop an ecdysone receptor (EcR)-based inducible gene regulation system, several constructs were prepared by fusing DEF domains of Choristoneura fumiferana EcR (CfEcR), C. fumiferana ultraspiracle (CfUSP), Mus muscu- lus retinoid X receptor (MmRXR) to either GAL4 DNA binding domain (DBD) or VP16 activation domain. These constructs were tested in mammalian cells to evaluate their ability to transactivate luciferase gene placed under the control of GAL4 response elements and synthetic TATAA promoter. A two-hybrid format switch, where GAL4 DBD was fused to CfEcR (DEF) and VP16 AD was fused to MmRXR (EF) was found to be the best combination. It had the lowest background levels of reporter gene activity in the absence of a ligand and the highest level of reporter gene activity in the presence of a ligand. Both induction and turn- off responses were fast. A 16-fold induction was observed within 3 h of ligand addition and increased to 8942-fold by 48 h after the addition of ligand. Withdrawal of the ligand resulted in 50% and 80% reduction in reporter gene activity by 12 h and 24 h, respectively. Keywords: gene switch; ponasterone A; receptors; EcR; RXR. Twenty hydroxyecdysone (20E) is a steroid hormone that regulates molting, metamorphosis, reproduction and vari- ous other developmental processes in insects. Ecdysone functions through a heterodimeric receptor complex com- prised of ecdysone receptor (EcR) and ultraspiracle (USP). Both EcR and USP cDNAs have been cloned from Drosophila melanogaster and several other insects [1] and were shown to be members of the steroid hormone receptor superfamily. Members of this superfamily are characterized by the presence of five modular domains, A/B (transacti- vation), C (DNA binding/heterodimerization), D (hinge, heterodimerization), E (ligand binding, heterodimerization, transactivation) and F (transactivation). Crystallographic studies on the E domain structures of several nuclear receptors showed a conserved fold composed of 11 helices (H1 and H3–H12) and two short strands (s1 and s2) [2]. Recently, the crystal structure of USP was solved by two groups [3,4], both structures showed a long H1-H3 loop and an insert between H5 and H6. These structures appear to lock USP in an inactive conformation by displacing helix 12 from agonist conformation. In both crystal structures USP had a large hydrophobic cavity, which contained phos- pholipid ligands. The crystal structure of the EcR has yet to be determined; however, homology models for CtEcR (Chironomus tentans EcR) [5], and CfEcR (Choristoneura fumiferana EcR) [6] have been generated [7,8]. Ecdysone receptors are found in insects and other related invertebrates [1]. Ecdysteroids and related compounds have been identified in plants, insects and other related inverte- brates. EcR and its ligands are not detected in vertebrates such as humans, therefore they are very good candidates for developing gene regulation systems for use in vertebrates. Insect EcR can heterodimerize with retinoid X receptor (RXR) and transactivate genes that are placed under the control of ecdysone response elements (EcRE) in various cellular backgrounds including mammalian cells. The EcR- based gene switch is being developed for use in various applications including gene therapy, expression of toxic proteins in cell lines as well as for cell-based drug discovery assays [9–17]. After initial reports [18,19] on the function of EcR as an ecdysteroid dependent transcription factor in cultured mammalian cells, No et al. [20] used D. melanogaster EcR (DmEcR) and human RXRa to develop an ecdysone inducible gene expression system that can function in mammalian cells and mice. Later, Suhr et al. [21] showed that the nonsteroidal ecdysone agonist, tebufenozide, induced high level of transactivation of reporter genes in mammalian cells through Bombyx mori EcR (BmEcR) [22] and endogenous RXR. Hoppe et al. [23] combined DmEcR and BmEcR systems and created a chimeric Drosophila/ Bombyx EcR (DBEcR) that had combined positive aspects of both systems, i.e. the chimeric receptor bound to modified ecdysone response elements and functioned without exogenous RXR. Recent improvements to the EcR-based gene switch include expression of both EcR and RXR in a bicistronic vector [24] and the discovery that the RXR ligands enhance the ligand dependent activity of the EcR-based gene switch [25]. An optimal gene regulation system should have the following characteristics: (a) low or no basal expression in the absence of an inducer (b) high induced expression in the presence of a wide range of inducer concentration (c) rapid Correspondence to S. R. Palli, Department of Entomology, College of Agriculture, University of Kentucky, Lexington KY 40546. Fax: + 1 859 3231120, Tel.: +1 859 2574962, E-mail: RPALLI@UKY.EDU Abbreviations: 20E, twenty hydroxyecdysone; EcR, ecdysone receptor; LBD, ligand binding domain; RXR, retinoid X receptor; USP, ultrapiracle. (Received 11 December 2002, revised 21 January 2003, accepted 5 February 2003) Eur. J. Biochem. 270, 1308–1315 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03501.x induction response after addition of an inducer (d) rapid switch-off response after removal of an inducer (e) repeated on and off responses (f) specific response to inducer with no pleiotropic effects and (g) the length of DNA constructs should be smaller for convenient packaging into viruses for in-vivo delivery. The current versions of EcR-based gene switches do not have some of the desirable characteristics of an optimal gene regulation system described above. For example, the background and induced levels of reporter gene activity are higher and lower, respectively, resulting in lower-fold induction. There was also no systematic analysis performed to find the optimum length of EcR and RXR required for the best performance of the switch. The studies presented here are designed to overcome some of the drawbacks associated with the EcR-based gene switch. We have constructed several gene switch plasmids by fusing C. fumi- ferana EcR (CfEcR) [6,26], C. fumiferana ultraspiracle (CfUSP) [27], Mus musculus RXR (MmRXR) [28], to either GAL4 DNA binding domain or VP16 activation domain. Combinations of these receptor constructs were analyzed in several mammalian cell lines using transient transactivation assays and selected a switch that had very low basal expression in the absence of a ligand and high- induced expression in the presence of a ligand. Both induction and turn-off of reporter gene in response to addition and withdrawal of ligand, respectively, were rapid. This CfEcR-based switch also showed differential sensiti- vity to steroids and nonsteroidal ligands. We have also identified that DEF domains of CfEcR and EF domains of MmRXR are required for the best performance of this gene switch. Materials and methods Constructs VgRXR (DmVgRXR) receptor plasmid and pIND b-galactosidase reporter plasmid were purchased from Invitrogen (Invitrogen Corporation, Carlsbad, CA, USA). This switch contains two receptors, DmEcR(CDEF) fused to VP16 activation domain [(V:DmE(CDEF)] and expressed under CMV promoter and full-length HsRXRa [HsR (A/BCDEF)] expressed under RSV promoter. In addition, the P-box in the DmEcR C (DNA binding) domain was altered to resemble that of glucocorticoid receptor (GR) recognizing a hybrid of EcR and GR response elements (E/GRE). These two receptors hetero- dimerize and bind to ligand and regulate b-galactosidase reporter placed under the control of 4X E/GRE-DMTV promoter (pIND b-galactosidase). CfVgRXR plasmid was constructed by replacing CDEF domains of DmEcR of DmVgRXR with CDEF domains of CfEcR. First the CfEcR fragment containing DNA binding domain C-terminal to the P-box and complete DEF domains was amplified using primers containing BamHI and BstXI sites on 5¢ and 3¢ ends, respectively. The PCR product was cloned into BamHI and BstXI digested DmVgRXR. CfVgRXRDel plasmid was constructed by removing HsRXRa from CfVgRXR. The CfVgRXR DNA was digested with EcoRV and NotI and the recessed 3¢ ends of NotI-digested fragments were filled using Klenow fragment of DNA polymerase I, ligated and transformed into E. coli. GAL4:CfEcR(DEF) [G:CfE(DEF)] and various trunca- tions of CfEcR were constructed by amplifying defined regions of CfEcR (NCBI accession number AAC36491) using primers containing a BamHI or EcoRI site on the 5¢ end and a XbaIsiteonthe3¢ end. The PCR products were cloned into BamHI/EcoRI and XbaI sites of pM vector (Clontech Inc. Palo Alto, CA, USA). VP16: CfEcR (CDEF) [V:CfE(CDEF)] and VP16:CfEcR(DEF) [V:CfE(DEF)] were constructed by transferring BamHI and XbaI frag- ments from G:CfE(CDEF) and G:CfE(DEF) to BamHI and XbaI digested pVP16 vector (Clontech Inc. Palo Alto, CA, USA). VP16:MmRXR (DEF) [V:MmR(DEF)] and various truncations of MmRXRa were constructed by amplifying defined regions of MmRXR (NCBI accession number NP035435) with primers containing EcoRI site on the 5¢ end and XbaIsiteonthe3¢ end. The PCR products were cloned into EcoRI and XbaI digested pVP16 vector. GAL4:MmRXR(DEF) [G:MmR(DEF)] was constructed by ligating EcoRI and XbaI digested fragment of MmRXR from V:MmR(DEF) to EcoRI and XbaIdigestedpM vector. V:MmR[DEF(H 4–12] was constructed by deleting BamHI fragment containing helices (H) 1–3 from V:MmR(EF). V:MmR(EF) was digested with BamHI and the larger fragment containing vector plus helices 4–12 of MmRXR was isolated, ligated and transformed. VP16:CfUSP(DEF) [V:CfU(DEF)] was constructed by amplifying DEF domains of CfUSP (NCBI accession number AAC31795) using primers containing EcoRI and BamHI sites in the forward and reverse primer, respectively, followed by cloning of the PCR product into EcoRI and BamHI digested pVP16 and pM vectors. pFRLUC reporter plasmid (5· GAL4 response element was fused to synthetic GGGTATATAAT sequence) was purchased from Strata- gene Cloning Systems (La Jolla, CA, USA). pFRLUCE- cRE was constructed by replacing 5· GAL4 response elements of pFRLUC with 7X EcRE. The 7X EcRE fragment was amplified from pMK43.2 [29] using primers containing PstIandXmaI sites in forward and reverse primers, respectively. The TATAA sequence was included in the reverse primer. The PCR products were cloned into PstIandXmaI digested pFRLUC. The pINDSEAP reporter vector was constructed by replacing b-galactosidase gene of pINDLaZ with SEAP from pSEAP2-basic vector (Clontech Inc. Palo alto, CA, USA) at HindIII and XbaI restriction enzyme sites. The pFRSEAP reporter vector was constructed by replacing luciferase of pFRLUC with SEAP at KpnIandXbaI restriction sites. Ligands Ponasterone A and Muristerone A were purchased from Alexis Corporation (San Diego, CA, USA). RG-102240 also known as GS TM -E [N-(1,1-dimethylethyl)-N¢-(2-ethyl- 3-methoxybenzoyl)-3,5-dimethylbenzohydrazide] and RG-102317 [N-(1,1-dimethylethyl)-N¢-(5-methyl-2,3-dihydro- benzo-1,4-dioxine-6-carbonyl)-3,5-dimethylbenzohydrazide] are synthetic stable bisacylhydrazine ecdysone agonists synthesized at Rohm and Haas Company. All ligands were supplied in dimethylsulfoxide and the final concentration of dimethylsulfoxide was maintained at 0.1%. Ó FEBS 2003 EcR-based gene switch (Eur. J. Biochem. 270) 1309 Cells and transfections and reporter assays CHO and A549 cells were grown in F12 medium containing 2m ML -glutamine and 10% bovine calf serum. 3T3, 293 and CV1 cells were grown in Dulbecco’s modified Eagle’s medium containing 4 m ML -glutamine, 1.5 gÆL )1 sodium bicarbonate, 4.5 gÆL )1 glucose and 10% bovine calf serum. All media and serum were purchased from Life Techno- logies, Rockville, MR, USA. One hundred thousand CHO or 293 cells or 50 000 of 3T3 or CV1 or A549 cells were plated per well of 12-well plates. The following day the cells were transfected with 0.25 lg of receptor(s) and 1.0 lgof reporter constructs using 4 lL of LipofectAMINE 2000 (Life Technologies, Rockville, MR, USA) for CHO and 293 cells, LipofectAMINE plus (Life Technologies, Rockville, MR, USA) for CV1 cells or SuperFect (Qiagen Inc. Valencia, CA, USA) for 3T3 cells or A549 cells. After transfection, the cells were grown in the medium containing ligands for 24–48 h. A second reporter, Renilla luciferase (0.1 lg), expressed under a thymidine kinase constitutive promoter was cotransfected into cells and was used for normalization. The cells were harvested, lyzed and the reporter activity was measured in an aliquot of lyzate. All transfection experiments were performed in triplicate and the experiments were repeated at least three times. Luciferase was measured using Dual-luciferase TM repor- ter assay system from Promega Corporation (Madison, WI, USA). b-Galactosidase was measured using Gal-ScreenÒ system from Applied Biosystems (Foster City, CA, USA). The SEAP activity in the medium was quantified using Phospha-Light TM System from Applied Biosystems. Results The fold inductions were lower for VgRXR-based switch formats We first tested DmVgRXR, CfVgRXR and CfVgRXRDel switches (Fig. 1A) for their ability to transactivate pIND- b-galactosidase reporter gene in 3T3 cells. CfVgRXR, DmVgRXR and CfVgRXRDel switches showed dose dependent induction of reporter gene activity upon addition of RG-102240 and supported maximum induced levels of 26-fold, 21-fold and sixfold, respectively (Fig. 1B). The lower fold inductions observed were mainly due to high background levels of reporter gene activity in the absence of ligand. The CfVgRXRDel switch, where no exogenous RXR was added, showed both higher background levels and lower induced levels of reporter gene activity and as a result the fold induction was lower for this switch when compared to DmVgRXR and CfVgRXR switches. Similar results were also observed in CHO, 293 and CV1 cells (data not shown). In all these cell lines, a maximum of 100-fold induction and an average of 25-fold induction were observed for CfVgRXR and DmVgRXR switches. CfVgRXR and DmVgRXR switches performed better than the CfVgRXRDel switch in all four cell lines tested. Two-hybrid switch formats showed high fold induction In order to develop a switch that has lower background and higher induced levels, we prepared receptor constructs where DEF domains of CfEcR, CfUSP and MmRXR were fused to either GAL4 DNA binding domain or VP16 activation domain. Different combinations of a GAL4 fusion receptor, a VP16 fusion receptor (Fig. 2A) and pFRLUC reporter were tested in 3T3 cells. Out of the four combinations tested, the G:CfE(DEF) + V:MmR(DEF) switch showed the highest level of induction (1014-fold; Fig. 2B). The reporter gene induction was RG-102240 dose- dependent and significant levels of reporter gene induction were observed at 1 l M or higher concentration of ligand. The G:MmR(DEF) + V:CfE(DEF) switch format also showed ligand-dependent induction of reporter gene acti- vity, but the induction was lower at 80-fold when compared to 1014-fold observed for the G:CfE(DEF) + V:MmR (DEF) switch. Use of CfUSP in place of MmRXR resulted in high background levels of reporter gene activity in the absence of ligand, as a result the induction was only twofold (Fig. 2B). These four switches performed in a similar way in CHO, CV1, 293 and A549 cells (data not shown). Fig. 1. Induction of b-galactosidase reporter gene by CfVgRXR, DmVgRXR and CfVgRXRdel switches. (A) Schematic diagram of constructs used in the experiment. (B) Plasmid DNA samples of CfVgRXR or DmVgRXR or CfVgRXRDel and pINDLacZ were transfected into 3T3 cells using Superfect (Qiagen Inc., Valencia, CA) lipid reagent. The transfected cells were grown in the medium con- taining0,0.1,1,5,10and50m M concentration of RG-102240. The cells were harvested at 48 h after adding ligand and the reporter activity was measured using the Gal-ScreenÒ system (Applied Bio- systems. Total relative light units (RLU) presented are mean ± SD (n ¼ 3). Numbers above the bars show the maximum fold induction observed for that particular combination. Fold induction was calcu- lated by dividing total RLUs in the presence of ligand by total RLUs in the absence of ligand. 1310 S. R. Palli et al.(Eur. J. Biochem. 270) Ó FEBS 2003 The G:CfE(DEF) + V:MmR(DEF) switch performs better through nonsteroidal ligands when compared to steroids We tested dose–response of two nonsteroids (RG-102240 and RG-102317) and two steroids (PonA and MurA) for the G:CfE(DEF) + V:MmR(DEF) switch. This switch induced the luciferase gene expression at 1 l M or higher concentration of RG-102240, 0.04 l M or higher concentra- tion of RG-102317, 5 l M or higher concentration of PonA and 25 l M or higher concentration of MurA (Fig. 3A). The G:CfE(DEF) + V:MmR(DEF) switch seems to be more sensitive to nonsteroidal ligands when compared to steroids. Similar differential sensitivity between nonsteroidal ligands and steroids was also observed in CHO, 293 and CV1 cells (data not shown). To determine whether this difference in ligand sensitivity is due to the two-hybrid switch format or due to CfEcR itself, we have evaluated the dose–response of RG-102240 and PonA to the V:CfE(CDEF) switch. In this switch format, only V:CfE(CDEF) and pFRLUCEcRE reporter were transfected and V:CfE(CDEF) heterodimer- izes with endogenous RXR. As shown in Fig. 3B, the V:CfE(CDEF) switch induced the reporter gene activity by 45-fold in the presence of 5 l M concentration of RG-102240 and by threefold in the presence of 5 l M concentration of PonA. These results suggest that CfEcR is the most likely contributor to the differences in dose–response of non- steroidal ligands and steroids. Truncation analysis of MmRXR The optimum fragment of RXR required for a two-hybrid switch was identified by preparing VP16 activation domain fusions of MmRXR A/BCEDF, CDEF, DEF, EF, DEF (H4–12), DEF (H1–11) (Fig. 4A) and analyzing them in 3T3 cells in combination with G:CfE(CDEF) or G:CfE(DEF) and pFRLUC. As shown in Fig. 4(B), the V:MmR(EF) + G:CfE(CDEF) combination showed the highest fold induc- tion (13 881). Deleting the first three helices or the 12th helix of V:MmR(EF) reduced its activity significantly. A similar pattern was observed when G:CfE(DEF) was used as a partner for MmRXR truncations. Out of all truncations tested, V:MmR(EF) was the best partner for G:CfE(DEF) Fig. 3. The G:CfE(DEF) + V:MmR(EF) switch works better through nonsteroidal ligands than steroids. (A) Dose–response of the two-hybrid switch to four ligands. 3T3 cells were transfected with G:CfE(DEF), V:MmR(EF), pFRLUC and pTKRL. The transfected cells were grown in the medium containing 0, 0.04, 0.2, 1, 5 and 25 l M concen- tration of RG-102240 or RG-102317 or PonA or MurA. (B) Dose– response of V:CfE(CDEF) switch to two ligands. 3T3 cells were transfected with V:CfE(CDEF), pFRLUCEcRE and pTKRL. The transfected cells were grown in the medium containing 0, 0.04, 0.2, 1. 5 and 25 l M concentration of RG-102240 or PonA. Fig. 2. Induction of luciferase reporter gene by two-hybrid switches. (A) Schematic diagram of constructs used in the experiment. (B) Plasmid DNA samples of pFRLUC, pTKRL and G:CfE(DEF) + V:MmR (DEF) or G:CfE(DEF) + V:CfU(DEF) or G:MmR(DEF) + V:CfE (DEF) or G:CfU(DEF) + V:CfE(DEF) were transfected into 3T3 cells using Superfect lipid reagent. The transfected cells were grown in the medium containing 0, 0.1, 1, 5, 10 and 50 m M concentration of RG-102240. The cells were harvested at 48 h after adding ligand and the reporter activity was measured using a dual luciferase assay kit from Promega Corporation (Madison, WI, USA). Total relative light units (RLU) presented are mean ± SD (n ¼ 3). Numbers above the bars show the maximum fold induction observed for that particular combination. Ó FEBS 2003 EcR-based gene switch (Eur. J. Biochem. 270) 1311 (Fig. 4C). In this case also deleting the first three helices or the 12th helix of V:MmR(EF) reduced the performance of the switch significantly. Truncation analysis of CfEcR To identify the optimum fragment of CfEcR required for the best performance of the two-hybrid switch, we con- structed GAL4 DNA binding domain fusions of CfEcR A/ BCDEF, CDEF, 1/2CDEF (half of the DNA binding domain containing second zinc finger was included), DEF, EF and DE(H1-11) domains and assayed them in 3T3 cells in the presence of V:MmR(EF) and pFRLUC. Among the truncations tested (Fig. 5A), G:CfE(CDEF) + V:MmR (EF) showed the highest fold induction (Fig. 5B). The G:CfE(DEF) + V:MmR(EF) was the most sensitive com- bination (Fig. 5B). Deleting the D domain or the 12th helix and F domain reduced the activity of receptor gene significantly. Thus, the CfE(DEF) truncation showed the maximum ligand sensitivity and the CfE(CDEF) truncation showed the maximum induction. Rapid induction and turn off of reporter gene activity through the G:CfE(DEF) + V:MmR(EF) switch The best two-hybrid switch combination, G:CfE(DEF) + V:MmR(EF), was used to study the time-course of induc- tion and subsequent decline of reporter gene activity in 3T3 cells. Increase in reporter gene activity was observed one hour after adding ligand and the reporter activity increased steadily until 72 h after the addition of ligand (Fig. 6A). To study the time-course of decrease in reporter gene activity after withdrawal of ligand, G:CfE(DEF) + V:MmR(EF) and pFRLUC were transfected into 3T3 cells and the cells were grown in the presence of 1 l M RG-102240 for 24 h. Then the cells were washed with medium containing no ligand and were grown in the same medium for 72 h. As shown in Fig. 6B a 50% and 80% decrease in reporter gene activity was observed by 12 h and 24 h, respectively, after withdrawal of ligand. Thus, both the induction and decline of reporter gene activity are rapid for this switch. Comparison of the performance of the G:CfE(DEF) + V:MmR(EF) switch with other EcR-based switches To compare the performance of the two-hybrid switch developed with previous versions of EcR-based gene Fig. 4. Truncation analysis on MmRXR. (A) Truncations of MmRXR tested. The numbers above the horizontal bars indicate amino acid boundaries between domains of RXR. Ligand-binding domain and locations of helices were identified based on Egea et al.[37].(B)VP16 fusions of six MmRXR truncations were transfected into 3T3 cells along with G:CfE(CDEF), pFRLUC and pTKRL. The transfected cells were grown in the medium containing 0, 1, 5 and 25 m M RG- 120240. The cells were harvested at 48 h after adding ligands and the reporter activity was quantified. The numbers shown above the zero for each panel represent the mean RLUs observed in DMSO-treated cells. (C) Same as in B except G:CfE(DEF) was used in place of G:CfE(CDEF). Fig. 5. Truncation analysis on CfEcR. (A) Truncations of CfEcR tes- ted. The numbers on the top of horizontal bars indicate amino acid boundaries between domains of EcR. Helices within the LBD were identified based on CtEcR [7] and CfEcR [8] homology models. (B) GAL4 fusions of six CfEcR truncations were transfected into 3T3 cells along with V:MmR(EF), pFRLUC and pTKRL. The transfected cells were grown in the medium containing 0, 1, 5 and 25 m M RG-102240. The cells were harvested at 48 h after adding ligands and the reporter activity was quantified. 1312 S. R. Palli et al.(Eur. J. Biochem. 270) Ó FEBS 2003 switches, we modified previous versions of EcR-based switches so that our comparisons are carried out with the same receptor (CfEcR) and reporter (SEAP). As shown in Table 1, the G:CfE(DEF) + V:HsR(EF) version of the switch being reported here performed better than the two previous versions of switches. The fold induction with this new switch is higher when compared to the fold inductions observed for CfVgRXR and CfVgRXRdel versions of switches. The lower fold induction in the case of earlier versions of switches is mainly due to the higher background levels of reporter activity in the absence of ligand. Discussion The most significant contribution of this study is the development of an EcR-based gene switch that has over- come most of the drawbacks associated with the earlier versions [18,20,21,23]. This two-hybrid format EcR-based gene switch showed the lowest levels of background reporter gene activity in the absence of ligand and the highest levels of induced reporter gene activity in the presence of ligand, resulting in a strikingly high fold induction. There are three major differences between the two-hybrid switch developed in this study and the previous versions of EcR-based switches [18,20,21,23]. First, in the two-hybrid switch, we used the heterologous GAL4 DNA binding domain in place of the EcR DNA binding domain or its modified form used in the previous EcR-based switches. Second, DNA binding and activation domains were placed on two different proteins instead of on a single protein as carried out for previous versions of the EcR-based switch. Third, in the reporter construct, we have used a synthetic TATAA element in the place of minimal promoters used in the previous versions of EcR-based switches. We have constructed some switches where GAL4 DNA binding and VP16 activation and EcR ligand binding domains were placed in the same molecule. These switches in combination with pFRLUC showed higher background reporter activity in the absence of a ligand and as a result the fold induction was lower (data not shown). These results indicate that changing GAL4 DNA binding domain alone would not have significantly improved the performance of the EcR-based switch. The V:CfE(CDEF) switch that used EcRE and synthetic TATAA showed lower fold induction (maximum 45-fold; Fig. 3B) when compared to the G:CfE(DEF) + V:MmR(EF) switch that used GALRE synthetic TATAA (maximum 1014-fold; Fig. 2B), indica- ting that the use of synthetic TATAA in the reporter Fig. 6. Time-course of induction (A) and turn-off (B) of two-hybrid switch. 3T3 cells were transfected with G:Cf(DEF), V:MmR(EF), pFRLUC and pTKRL. The transfected cells were grown in the medium containing 1 l M concentration of RG-102240. For the data showninA,cellswerecollectedat0,1,3,6,12,24,48and72hafter adding ligand. The reporter activity was quantified and plotted. For the data shown in B and 24 hours after adding ligand, the cells were washed with ligand-free medium, grown in the medium containing dimethylsulfoxide for 0, 1, 3, 6, 12, 24, 48 and 72 h, then harvested and reporter activity was quantified and plotted. Table 1. Comparison of performance of CfVgRXR, CfVgRXRdel and G:CfE(DEF) + V:HsR(EF) switches. Plasmid DNAs of pINDSEAP and CfVgRXR or CfVgRXRdel, pFRSEAP and G:CfE(DEF) + V:HsR(EF) constructs were transfected into 3T3 cells plated into 96-well plates. After transfection, the cells were exposed to 0, 0.2, 1 and 5 l M RG-102240 and 1, 5 and 25 l M PonA for 48 h. The SEAP activity in the medium was quantified using the Phospha-Light TM System from Applied Biosystems. FI, fold induction. Ligand CfVgRXR CfVgRXRdel G:Cf(DEF) + V:HsR(EF) RLU ± SD FI ± SD RLU ± SD FI ± SD RLU ± SD FI ± SD Dimethylsulfoxide 55 ± 10 1 59 ± 2 1 9 ± 1 1 RG-102240 0.2 l M 108 ± 10 2 53 ± 22 1 9 ± 1 1 RG-102240 1 l M 1230 ± 89 29 ± 9 136 ± 24 2 525 ± 164 62 ± 10 RG-102240 5 l M 1478 ± 249 41 ± 21 355 ± 106 6 ± 2 2356 ± 73 288 ± 48 RG-102240 25 l M 2572 ± 470 47 ± 2 713 ± 138 12 ± 3 2582 ± 149 316 ± 49 PonA 1 l M 253±73 5±1 60±16 1 76±69 8±6 PonA 5 l M 185 ± 16 3 ± 1 70 ± 12 1 194 ± 90 23 ± 6 PonA 25 lm 771 ± 86 14 ± 1 77 ± 12 1 2540 ± 187 313 ± 70 Ó FEBS 2003 EcR-based gene switch (Eur. J. Biochem. 270) 1313 construct alone would not have improved the performance of the EcR-based switch to the extent observed in these studies. We have also tested switch formats using either CfE(A/BCDEF) or CfE(CDEF) in combination with V:MmR(A/BCDEF) or V:MmR(CDEF) or V:MmR(DEF) or V:MmR(EF) and pFRLUCEcRE. None of these switch formats supported the high fold inductions observed for two-hybrid format switches (data not shown) indicating that merely separating the DNA binding domain and the activation domain onto two molecules is not sufficient to improve the performance of this switch. It appears that a combination of several different factors contributed to the dramatic improvement in the performance of this two- hybrid switch. The mechanism of action of this two-hybrid format switch is not entirely understood. Truncation analyses showed that the helix 12 of both CfEcR and MmRXR are required for efficient transactivation. Deletion of either of these domains resulted in drastic reduction in transactiva- tion of reporter genes through these receptors indicating that C-terminal activation domains of these receptors are involved in transactivation through this switch. Previous studies showed that neither ligand nor F domain of CfEcR is required for heterodimerization of CfEcR with CfUSP [26,30]. Taken together, these studies indicate that this new two-hybrid format switch functions through heterodimeri- zation and ligand binding followed by conformational change in both receptors resulting in transactivation of genes placed under the control of this switch. It is interesting that RXR-based two-hybrid switches are highly inducible because of their low background reporter activity in the absence of ligand. On the other hand, USP- based switches are not inducible mainly because of high levels of reporter activity in the absence of ligand. We have observed similar results in yeast, where expression of full- length C. fumiferana EcR and USP led to transactivation of reporter gene in the absence of a ligand [31]. Deletion of A/B domains from both EcR and USP abolished the constitutive activation in this assay. Recently, Lezzi et al. [32] reported ligand-induced heterodimerization between the ligand bind- ing domains of D. melanogaster EcR and USP in yeast. The differences in performance of RXR- and USP-based switches are most likely due to the differences in the requirement of ligand for formation of heterodimers with CfEcR. Previous studies showed that CfEcR and CfUSP [26,30], BmEcR and D. melanogaster USP (DmUSP) can form heterodimers in the absence of ligand [21], whereas both DmEcR and BmEcR require the presence of ligand for formation of heterodimers with RXR. We observed differential sensitivity of the CfEcR-based two-hybrid switch to steroids and nonsteroidal ligands. Dose–response studies using two steroids (PonA and MurA) and two nonsteroids (RG-102240 and RG- 102317) in four cells lines (3T3, CHO, 293 and CV1) showed that the CfEcR-based two-hybrid switch is more sensitive to nonsteroidal ligands when compared to steroids. Previous studies also showed similar differences in binding of steroid and nonsteroidal ligands to CfEcR and CfUSP. RH-5992 and RH-2485 (bisacylhydrazines) bound to CfEcR and CfUSP at 10-fold higher affinity than the steroids, PonA and MurA [33]. Earlier versions of EcR- based gene switches also showed higher activity with nonsteroidal ligands when compared to the activity with steroids [23,25,34]. Previous published versions of EcR-based gene switches used CDEF domains of EcR and full-length RXR. In this study, we performed a systematic analysis and identified regions of both EcR and RXR required for optimum performance. The two-hybrid version of the CfEcR-based gene switch uses only 1072 nucleotides of CfEcR and 725 nucleotides of MmRXR when compared to 1973 nucleo- tides of DmEcR and 1388 nucleotides of RXR used in the commercially available version of EcR-based gene switch (Invitrogen Corporation, Carlsbad, CA, USA). The size of receptors used in gene switches are very important due to size limitations in packaging gene regulation and target gene constructs into various viruses for in vivo delivery. In transactivation assays, the CfEcR-based two-hybrid format switch showed very low reporter gene activity in the absence of ligand and high reporter gene activity in the presence of ligand. Both induction and switch-off responses were rapid. Recently, our collaborators confirmed the performance of this switch in stable cell lines as well as in mouse tumors [35]. 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(2001) The chemical and bio- logical properties of methoxyfenozide, a new insecticidal ecdy- steroid agonist. Pest Manag. Sci. 57, 115–119. 37. Egea, P.F., Mitschler, A., Rochel, N., Ruff, M., Chambon, P. & Moras, D. (2000) Crystal structure of the human RXRalpha ligand-binding domain bound to its natural ligand: 9-cis retinoic acid. EMBO J. 19, 2592–2601. Ó FEBS 2003 EcR-based gene switch (Eur. J. Biochem. 270) 1315 . Improved ecdysone receptor-based inducible gene regulation system Subba R. Palli 1 , Mariana Z. Kapitskaya 2 ,. Kentucky, KY, USA; 2 RHeoGene LLC, Spring House, PA, USA To develop an ecdysone receptor (EcR)-based inducible gene regulation system, several constructs