Báo cáo khoa học: Functional characterization of ecdysone receptor gene switches in mammalian cells docx

Functional characterization of ecdysone receptor gene switches in mammalian cells Siva K Panguluri1, Prasanna Kumar2 and Subba R Palli1 Department of Entomology, College of Agriculture, University of Kentucky, Lexington, KY, USA RheoGene Inc., Norristown, PA, USA Keywords ChIP assay; EcR; gene switch; RXR; RNAi Correspondence S R Palli, Department of Entomology, College of Agriculture, University of Kentucky, Lexington, KY 40546, USA Fax: +1 859 323 1120 Tel: +1 859 257 4962 E-mail: RPALLI@UKY.EDU (Received 15 August 2006, revised October 2006, accepted 18 October 2006) doi:10.1111/j.1742-4658.2006.05545.x Regulated expression of transgene is essential in basic research as well as for many therapeutic applications The main purpose of the present study is to understand the functioning of the ecdysone receptor (EcR)-based gene switch in mammalian cells and to develop improved versions of EcR gene switches We utilized EcR mutants to develop new EcR gene switches that showed higher ligand sensitivity and higher magnitude of induction of reporter gene expression in the presence of ligand We also developed monopartite versions of EcR gene switches with reduced size of the components that are accommodated into viral vectors Ligand binding assays revealed that EcR alone could not bind to the nonsteroidal ligand, RH-2485 The EcR’s heterodimeric partner, ultraspiracle, is required for efficient binding of EcR to the ligand The essential role of retinoid X receptor (RXR) or its insect homolog, ultraspiracle, in EcR function is shown by RXR knockdown experiments using RNAi Chromatin immunoprecipitation assays demonstrated that VP16 (activation domain, AD):GAL4(DNA binding domain, DBD):EcR(ligand binding domain, LBD) or GAL4(DBD):EcR(LBD) fusion proteins can bind to GAL4 response elements in the absence of ligand The VP16(AD) fusion protein of a chimera between human and locust RXR could heterodimerize with GAL4(DBD):EcR(LBD) in the absence of ligand but the VP16(AD) fusion protein of Homo sapiens RXR requires ligand for its heterodimerization with GAL4(DBD):EcR(LBD) Inducible expression of transgene is desirable for various applications such as functional genomics, gene therapy, therapeutic protein production and tissue engineering [1–4] Many laboratories have designed a number of gene switches to achieve inducible expression However, many of these systems have their own advantages and disadvantages, such as differences in background, levels of induction, as well as unpredictability of their performance in vivo, mainly because of the inadequate understanding of molecular mechanisms of their function in vivo An insect hormone receptor based gene regulatory system, ecdysone receptor (EcR) gene switch, is gaining popularity due to its low background activity in the absence of ligand, and high inducible expression in the presence of synthetic nonsteroid ligands that have shown very good safety profiles (as reviewed in [1]) The EcR is a member of the nuclear receptor superfamily [2] which is composed of N-terminal A ⁄ B domain, the DNA binding C domain, the D domain (hinge region), ligand binding E domain, and C-terminal F domain EcR heterodimerizes with other Abbreviations AD, activation domain; Cf, Choristoneura fumiferana; ChIP, chromatin immunoprecipitation; DB, Drosophila ⁄ Bombyx; DBD, DNA binding domain; Dm, Drosophila melanogaster; EcR, ecdysone receptor; GE, GAL4:CfEcR; GST, glutathione S-transferase; Hs, Homo sapiens; LBD, ligand binding domain; Lm, Locusta migratoria; RE, response element; RXR, retinoid X receptor; USP, ultraspiracle 5550 FEBS Journal 273 (2006) 5550–5563 ª 2006 The Authors Journal compilation ª 2006 FEBS S K Panguluri et al nuclear receptors [1], noticeably with ultraspiracle (USP), an orthologue of the vertebrate retinoid X receptor (RXR) EcR functions as a ligand controlled transcription factor, which makes it a suitable constituent of gene switches The main advantage of EcR gene switches in mammalian system is that the ecdysteroids, which are used to induce the switch, are structurally different from mammalian steroids, and they are not expected to bind to vertebrate steroid receptors Moreover, humans eat large amounts of phytoecdysteroidcontaining vegetables without any detrimental effects [3] Many versions of EcR gene switches were developed in different laboratories to achieve high degree of induction Initially, Christopherson et al [4] transfected a HEK293 cell line with Drosophila melanogaster EcR (DmEcR) and a reporter gene to test various ecdysteroids and vertebrate steroids Ponasterone A and Muresterone A (MurA) were active and MurA could also induce reporter gene when used with estrogen response element (RE) No et al [5] used constructs for fusion protein containing N-terminal truncation of DmEcR attached to the VP16 activation domain and complete human RXRa and achieved 212-fold induction with low basal activity This EcR gene switch was shown to function in mammalian cells and mice Later, Suhr et al [6] reported high level transactivation of reporter gene in the absence of RXR by using Bombyx mori EcR with MurA or tebufenozide Hoppe et al [7] developed a hybrid Drosophila ⁄ Bombyx ecdysteroid receptor (DB-EcR) which has positive aspects of both receptors and independent of recombinant RXR and found to be efficient in both in vitro and in vivo applications The expression of EcR and RXR in a bicistronic vector [8] further improved EcR-based gene switches Although these versions of EcR-based switches have several desirable characteristics, they have high basal activity which causes low fold induction Recently Palli et al [9] developed a bipartite format of EcR-based inducible system by using the DEF domains of Choristoneura fumiferana EcR (CfEcR) fused to GAL4 DNA binding domain (DBD) and VP16 activation domain (AD) fused to Mus musculus RXR ligand binding domain [MmRXR(EF)] This version of EcR gene switch supported nearly 9000-fold induction of reporter gene by 48 h after treatment of a diacylhydrazine ligand (1 lm RG-102240) This switch was reported to have good performance in stable cell lines as well as in mouse tumors [10] Recently, Palli et al [11] and Karzenowski et al [12] reported further improvements to the bipartite EcR gene switch The current versions of bipartite EcR gene switch have very low background activity of reporter gene in the absence of ligand and Functional characterization of EcR gene switches are activated by subnanomolar concentrations of ligand One concern about this system for in vivo applications is the use of RXR, which is involved in large number of metabolic pathways In the light of the potential use of this EcR gene switch for in vivo applications such as gene therapy, understanding the mechanism of functioning of this switch in a mammalian system and improving the switch to reduce any potential pleiotropic effects are therefore necessary The data presented here showed that the use of two mutant versions of EcR (EcRtvy and EcRtvay) increased ligand sensitivity of EcR gene switch EcR gene switch in a monopartite format containing a fusion protein of VP16 activation domain, GAL4 DNA binding domain and the EcR DEF domains supported ligand inducible reporter activity levels that are higher than those supported by the other versions of EcR gene switches reported so far We also discovered that EcR fusion proteins require heterodimeric partner for binding to ligand as well as for transactivation of reporter genes in cells An EcR gene switch that includes a fusion protein containing VP16 activation domain, GAL4 DNA binding domain and EcR DEF domains and a chimera containing helices 1–8 from human RXR (HsRXR) and helices 9–12 from locust (Locusta migratoria; LmRXR) supported lower background levels of reporter activity in the absence of ligand, higher inducible levels of reporter activity in the presence of ligand, and high ligand sensitivity We analyzed the DNA binding, ligand binding, heterodimerization and transactivation properties of the switch components by chromatin immunoprecipitation (ChIP) assay, RNAi and other methods to understand the mechanisms of the switch function in mammalian cells Results Monopartite EcR gene switches In order to develop a gene switch system with reduced size of the components while retaining the low background and robust induction levels; we constructed monopartite versions of EcR gene switches Such a system will be useful for incorporation into viral vectors, particularly adeno-associated viral vectors where the insert size is a major constraint For this purpose, we developed VGE, GVE and GEV monopartite formats (Fig 1A) by fusing VP16 activation domain with different truncations of GAL4 DNA binding domain (amino acids: 1–147, 1–93 and 1–65) and EcR (CDEF ⁄ DEF), and performed transactivation experiments in 3T3 cells The results clearly showed that in VG(147)E format (Fig 2A) containing GAL4 DBD FEBS Journal 273 (2006) 5550–5563 ª 2006 The Authors Journal compilation ª 2006 FEBS 5551 Functional characterization of EcR gene switches S K Panguluri et al A B Fig (A) Schematic diagram of constructs used in various experiments (B) Chemical structures of ligands used in various experiments (147) and EcR(CDEF) showed highest induction levels (a maximum fold induction of 481 at 25 lm ligand concentration) The VG(93)E format with GAL4 DBD (amino acids: 1–93) also showed good induction levels but were less than VG(147)E The VG(65)E format switch showed the lowest induction levels and very low basal activity The VG(65)E(CDEF) format switch showed highest fold induction (706-fold) at 25 lm concentration of ligand Similar results were observed with the GVE format switch (Fig 2B) except with higher background expression of reporter gene than that 5552 observed for VGE format switch The initial versions of GEV format switches were made by replacing helix 12 of EcR with VP16 activation domain This version of GEV supported constitutive expression of reporter gene expression in the absence of ligand (data not shown) We then modified GEV format switch to include helix 12 of EcR and this version supported ligand inducible reporter activity (see below) To understand the variation in the performance of monopartite switch in different cell lines, we tested the VGE version in four different cell lines, HEK293, FEBS Journal 273 (2006) 5550–5563 ª 2006 The Authors Journal compilation ª 2006 FEBS S K Panguluri et al Functional characterization of EcR gene switches A 70000 481 Normalised RLU 60000 50000 174 40000 310 30000 20000 10000 (105) ( 22 ) 47 (94) ( 91 ) (7 1) 706 (2 ) 49 0.04 0.2 25 0.04 0.2 25 0.04 0.2 25 0.04 0.2 25 0.04 0.2 25 0.04 0.2 25 VG(147)E(CDEF) VG(147)E(DEF) VG(93)E(CDEF) VG(93)E(DEF) VG(65)E(CDEF) VG(65)E(DEF) 30000 43 20000 21.6 27 7.8 15000 10000 (2363) (1613) 28 (881) (530) (547) G(147)VE(CDEF) G(147)VE(DEF) G(93)VE(CDEF) 25 0.04 0.2 5000 0.04 0.2 25 0.04 0.2 25 0.04 0.2 25 0.04 0.2 Normalised RLU 25000 G(93)VE(DEF) (149) 25 0.04 0.2 25 B G(65)VE(CDEF) G(65)VE(DEF) Fig (A) Induction of luciferase reporter gene by monopartite switches (A) Plasmid DNA samples of VP16(AD):GAL4(DBD)(1–147):EcR(CDEF) [VG(147)E(CDEF)] or VP16(AD):GAL4(DBD)(1–147):EcR(DEF) [VG(147)E(DEF)] or VP16(AD):GAL4(DBD)(1–93):EcR(CDEF) [VG(93)E(CDEF)] or VP16(AD):GAL4(DBD)(1–93):EcR(DEF) [VG(93)E(DEF)] or VP16(AD):GAL4(DBD)(1–65):EcR(CDEF) [VG(65)E(CDEF)] or VP16(AD):GAL4(DBD)(1–65):EcR(DEF) [VG(65)E(DEF)] and pFRLuc were transfected into 3T3 cells using superfect lipid reagent The transfected cells were grown in the medium containing 0, 0.04, 0.2, 1, and 25 lM concentrations of RG-102240 The cells were harvested at 48 h after adding ligand, and the reporter activity was measured using luciferase assay kit from Promega Total relative light units (RLU) presented are mean ± SD (n ¼ 3) Numbers above the bars represents the maximum fold induction observed for that particular combination The numbers in the parenthesis represent the RLU of the reporter activity in cells that exposed to dimethylsulfoxide (no ligand) (B) Plasmid DNA samples of GAL4(1–147):VP16:EcR(CDEF) [G(147)VE(CDEF)] or GAL4(1–147):VP16:EcR(DEF) [G(147)VE(DEF)] or GAL4(1– 93):VP16:EcR(CDEF) [G(93)VE(CDEF)] or GAL4(1–93):VP16:EcR(DEF) [G(93)VE(DEF)] or GAL4(1–65):VP16:EcR(CDEF) [G(65)VE(CDEF)] or GAL4(1–65):VP16:EcR(DEF) [G(65)VE(DEF)] and pFRLuc were transfected into 3T3 cells using superfect lipid reagent The transfected cells were grown in the medium containing 0, 0.04, 0.2, 1, and 25 lM concentrations of RG-102240 The cells were harvested at 48 h after adding ligand, and the reporter activity was measured The RLU presented are mean ± SD (n ¼ 3) Numbers above the bars represents the maximum fold induction observed for that particular combination The numbers in parentheses represents the RLU of the reporter activity in cells exposed to dimethylsulfoxide (no ligand) CHO, NIH 3T3 and CV1 Clear differences among four cell lines in induction levels as well as fold induction were observed (Fig 3) The 3T3 cells showed less basal expression in the absence of ligand and therefore high fold induction when compared to the other three cell lines To determine if the difference in the induction levels of reporter gene by EcR gene switch is due to the differences in levels of HsRXR isoforms, we transfected 3T3 cells with G:EcR(DEF) with V:HsRXRa or V:HsRXRb or V:HsRXRc and the transfected cells were exposed to various concentrations of RG-102240 As shown in Fig 4, G:EcR(DEF) in combination with RXRc or RXRb showed similar induction levels and these levels are higher than the induction levels supported by G:EcR(DEF) and V:HsRXRa combination Mutant EcRs improve the ligand sensitivity of both bipartite and monopartite EcR gene switches We tested the two mutant versions of CfEcR, EcRtvy (T335V, V390I and Y410E), EcRtvay (T335V, V390I, A393P and Y410E), along with RXR chimera (a chimera containing helices 1–8 from HsRXR and helices 9–12 from LmRXR) in NIH ⁄ 3T3 cells The gene FEBS Journal 273 (2006) 5550–5563 ª 2006 The Authors Journal compilation ª 2006 FEBS 5553 Functional characterization of EcR gene switches S K Panguluri et al 605 222 159 2000 145 1000 (2) ( 1.47) CV1 3T3 G: EcR +V :Hs- L mR Fig NIH ⁄ 3T3 cells showed high induction levels of reporter gene The plasmid DNA samples of VP16:GAL4:EcR(DEF) [VGE] and pFRLuc were transfected into 293, CHO, CV1 and 3T3 cells using superfect lipid reagent The transfected cells were grown in the medium containing 0, 0.1, 1, 5, 10 and 50 lM concentrations of RG-102240 The cells were harvested at 48 h after adding ligand, and the reporter activity was measured and the RLU presented are mean ± SD (n ¼ 3) Numbers above the bars represents the maximum fold induction observed for that particular combination The numbers in parentheses represents the RLU of the reporter activity in cells that exposed to dimethylsulfoxide (no ligand) 80000 0.2 G : E tv y + V : Hs - L m R DMSO 0.1 10 50 0.1 10 50 0.1 10 50 0.1 10 50 CHO (1.5) ( 5) 293 2000 ( 12.8) 4000 0.2 (9.3) 6000 DMSO (8.3) 8000 500 4316 4621 1500 4917 10000 0.2 2500 12000 DMSO 3000 Normalised RLU Normalised RLU 3500 G :E t v a y + V : H s - L m R Fig Two mutant of CfEcR perform better than wild-type EcR Plasmid DNA samples of GAL4:CfEcR (GE) + VP16:Hs-LmRXR (VHs-LmR) or GAL4:CfEcRtvy (GEtvy) + VHs-LmR or GAL4:CfEcRtvay (GEtvay) + VHs-LmR and pFRLuc were transfected into 3T3 cells using superfect lipid reagent The transfected cells were grown in the medium containing 0, 0.2, and lM concentrations of RG-102240 The cells were harvested at 48 h after adding ligand and the reporter activity was measured The RLU presented are mean ± SD (n ¼ 3) Numbers above the bars represents the maximum fold induction observed for that particular combination The numbers in the parenthesis represents the RLU of the reporter activity in cells exposed to dimethylsulfoxide (no ligand) DMSO, dimethylsulfoxide 4555 Normalised RLU 70000 3054 60000 50000 40000 2765 30000 20000 10000 50 10 0.1 50 G:EcR V :R X R 10 (15 7) 0.1 50 G:EcR V: R X R 10 (19.2) 0.1 (11 5) 0 G:EcR V :R X R Fig The GAL4:EcR + VP16:RXRc switch showed higher induction than the switches containing a and b isoforms of RXR The plasmid DNA samples of GAL4:EcR (GE) + VP16:HsRXRa ⁄ RXRb ⁄ RXRc and pFRLuc 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 lM concentrations of RG-102240 The cells were harvested at 48 h after adding ligand The reporter activity was measured and the RLU presented are mean ± SD (n ¼ 3) Numbers above the bars represents the maximum fold induction observed for that particular combination The numbers in parentheses represents the RLU of the reporter activity in cells exposed to dimethylsulfoxide (no ligand) switches containing mutant EcR EcRtvy induced reporter activity to much higher levels at 0.2 lm concentration of ligand when compared to those supported by wild-type (wt) EcR (Fig 5) For example, at 0.2 lm RG-102240, EcRtvy switch showed 2226-fold induction of the reporter activity compared to 1133fold and 1313-fold induction supported by EcRtvay and wild-type EcR gene switches, respectively These 5554 differences in the induction of reporter activity were evident even at a higher concentration (5 lm) of ligand The gene switches containing mutant EcRtvy supported high fold induction of reporter activity (4917-fold) compared to the other mutant EcRtvay (4316-fold) or wtEcR (4621-fold) at lm RG-102240 (Fig 5) Similar differences in the activity of EcRtvy, EcRtvay and wtEcR gene switches were observed in HEK293 cells (data not shown) The monopartite gene switch format consists of the DEF domains of mutant CfEcR (EcRtvy and EcRtvay), GAL4 DNA binding domain and VP16 activation domain Various combinations of the EcR ligand binding domain, VP16 activation domain and GAL4 DNA binding domain were constructed (Fig 1A) and tested in NIH ⁄ 3T3 cells Out of six combinations tested, VGEtvy (VP16:GAL4:EcRtvy) showed the highest level of induction (1289-fold) in cells treated with lm RG-102240 (Fig 6A) The reporter gene induction was dose dependent and significant levels of reporter gene induction were observed even at lower concentration (0.2 lm) of RG-102240 The VGEtvay (VP16:GAL4: EcRtvay) also showed dose dependent induction with maximum induction of 53-fold in cells treated with lm RG-102240 (Fig 6B) The low levels of induction in VGEtvay, when compared to the VGEtvy, are not only due to low levels of induced reporter activity but also due to high background activity in the absence of ligand Although the FEBS Journal 273 (2006) 5550–5563 ª 2006 The Authors Journal compilation ª 2006 FEBS S K Panguluri et al 12000 EcR fusion proteins need heterodimeric partner for ligand binding 4917 10000 8000 6000 1289 4000 2.9 G:EcRtvy+ V:Hs-LmR GVEtvy 0.2 DMSO 0.2 VGEtvy 19.6 (3.7) GEtvyV 4316 8000 7000 6000 5000 4000 3000 2000 1000 (1.5) (1464) G:EcRtvay+ V:Hs-LmR VGEtvay GVEtvay 9.7 0.2 DMSO 0.2 DMSO (2.8) DMSO (36) 53 0.2 DMSO Normalised RLU 0.2 DMSO 0.2 DMSO B (522) (3) DMSO (2) 2000 0.2 Normalised RLU A Functional characterization of EcR gene switches GEtvayV Fig Induction of luciferase gene by monopartite switches with mutant EcRs (A) Plasmid DNA samples of GEtvy + VHs-LmR or VP16:GAL4:EcRtvy (VGEtvy) or GAL4:VP16:EcRtvy (GVEtvy) or GAL4:EcRtvy:VP16 (GEtvyV) and pFRLuc were transfected into 3T3 cells using superfect lipid reagent The transfected cells were grown in the medium containing 0, 0.2, and lM concentrations of RG-102240 The cells were harvested at 48 h after adding ligand and the reporter activity was measured and the RLU presented are mean ± SD (n ¼ 3) Numbers above the bars represents the maximum fold induction observed for that particular combination The numbers in parentheses represents the RLU of the reporter activity in cells exposed to dimethylsulfoxide (no ligand) (B) The experiments and procedures used are same as in (A), except EcRtvay mutant was used in place of EcRtvy mutant DMSO, dimethylsulfoxide GVE combinations GAL4:VP16:EcRtvy (GVEtvy) and GAL4:VP16:EcRtvay (GVEtvay) showed high sensitivity to the ligand (showed induced reporter activity even at low concentrations of 0.02 lm RG102240), they were leaky, showing high background expression of reporter gene in the absence of ligand The GEV combinations GAL4:EcRtvy:VP16 (GEtvyV) and GAL4:CfEcRtvay:VP16 (GEtvayV) showed low background in the absence of ligand, but they also showed very low levels of reporter induction in the presence of ligand (19.6-fold and 9.7-fold, respectively, at lm RG-102240 concentration) Thus, out of the three formats of monopartite switches tested, VGE format appeared to perform better than GVE or GEV Out of the two mutants tested, EcRtvy showed the highest ligand sensitivity, low background and high levels of ligand-induced reporter expression To determine whether VGE fusion protein requires heterodimeric partner for binding to ligand, we performed ligand binding assays The glutathione S-transferase (GST) fusion proteins of CfEcR DEF mutant (Etvy), VGEtvy, GVEtvy, GEtvyV and CfUSP(A–F) proteins were expressed in bacteria using GST expression vectors and purified Etvy, VGEtvy, GVEtvy and GEtvyE alone or in combination with CfUSP were tested for binding to [3H]RH-2485, a close analog of RG-102240 (Fig 1B) As shown in Fig 7A, [3H]RH-2485 showed very little binding to Etvy, VGEtvy, GVEtvy and GEtvyE However, in the presence of CfUSP, [3H]RH-2485 bound to all the four EcR proteins suggesting that EcR requires the USP partner for its high affinity binding to the ligand We then determined the dissociation constants (Kd) of RH-2485 binding to Etvy + CfUSP and VGEtvy + CfUSP As shown in Fig 7B,C, RH-2485 bound to Etvy + CfUSP and VGEtvy + CfUSP with high affinity with Kd values of 1.647 nm (confidence interval of )0.5963 to 3.891, R2 of 0.9292, degree of freedom of 10) and 1.447 nm (confidence interval of 0.9588–1.935, R2 of 0.9788, degree of freedom of 28), respectively These data showed that VGE fusion protein of EcR requires heterodimeric partner for binding to the ligand Addition of RXR improves the performance of monopartite gene switch Because ligand binding assays showed that USP is required for binding of ligand to EcR, we decided to test whether RXR is required for efficient functioning of VGEtvy switch For this purpose we transfected pFRLuc reporter, VGEtvy and one of the RXRs (VHsRXR, VLmRXR or VP16 fusion of one of the Hs-LmRXR chimeras) into NIH ⁄ 3T3 cells and the transfected cells were exposed to various concentrations of RG-102240 Chimeras 6, 8, and VLmRXR increased the performance of VGEtvy switch Chimera 10, 11 and VHsRXR caused much lower enhancement of VGEtvy switch when compared to VLmRXR or VHs-LmRXR chimeras 6, 8, and (Fig 8A) Thus, inclusion of VP16 fusion of one of the three chimeras between HsRXR and LmRXR or LmRXR itself caused substantial increase in the performance of VGEtvy switch Next, we wanted to determine whether the presence of VP16 on both the receptors (VGEtvy and VRXR) is responsible for the improvement in the performance of VGEtvy switch in the presence of VP16:RXR VP16 domain was deleted from VHs- FEBS Journal 273 (2006) 5550–5563 ª 2006 The Authors Journal compilation ª 2006 FEBS 5555 Functional characterization of EcR gene switches S K Panguluri et al Hs-LmRXR with and without VP16 fusion protein performed equally well in improving the performance of both VGEtvy and GEtvyV switches Similar results were observed when Hs-LmRXR chimera with and without VP16 was compared as a partner for VGEtvay and GEtvayV (Fig 8C) Thus, RXR region but not the additional VP16 domain is responsible for improving the performance of VGEtvy and GEtvyV switches VGE fusion proteins utilize endogenous RXR for efficient transgene activation Fig Ligand binding assay of EcR fusion proteins (A) The GST fusion proteins of CfEcR DEF tvy mutant (Etvy), VGEtvy, GVEtvy and GEtvyV by themselves or in combination with GST fusion protein of CfUSP were incubated with [3H]-labeled RH-2485 (0.000822 pmol) in presence of excess RG-102240 (10 mM) The unbound ligand was removed by dextran coated activated charcoal treatment and radioactivity in the supernatant was counted using liquid scintillation counter The specific binding was expressed in disintegrations per minute and the error bars shown are the standard deviation of three replicates (B,C) Saturation kinetics of the binding of [3H]-labeled RH-2485 to mutant CfEcRtvy + CfUSP (B) or CfEcRtvy fusion protein in monopartite format (VGEtvy) plus CfUSP (C) Specific binding (d) which is the total binding (j) minus the nonspecific binding (m) and dissociation constants (Kd) are displayed LmRXR chimera and the construct was tested for its ability to improve the performance of VGEtvy and GEtvyV in NIH ⁄ 3T3 cells As shown in Fig 8B, 5556 The ligand binding experiments showed that the ligand binding to EcR improved in the presence of USP The transactivation experiments showed that the VGE fusion protein can support the reporter gene induction in the absence of exogenously supplied RXR but the reporter activity was higher in the presence of exogenously supplied RXR chimeras It is then possible that the endogenous RXRs in the cells are binding to VGE, enabling them to transactivate genes placed under their control We tested this hypothesis using double-stranded RNA prepared against HsRXRa and HsRXRb Microarray analysis of gene expression in HEK293 cells showed that only HsRXRa and HsRXRb are expressed in these cells and HsRXRc expression is not detected (data not shown) HEK293 cells were transfected with VGEtvy and pFRLuc or VGEtvy and pFRLuc and double-stranded RNA for RXRa and RXRb or bacterial malE gene In cells transfected with double-stranded RNA for HsRXRa and HsRXRb, there was 3.5-fold decrease in induction of reporter activity in the presence of RG-102240 when compared to the reporter induction levels in the cells transfected with malE double-stranded RNA or those that were transfected with only VGEtvy and pFRLuc (Fig 9A) We have also performed a parallel experiment to determine the levels of RXR mRNA in the cells transfected with double-stranded RNAs using quantitative RT-PCR The quantitative RT-PCR results showed that there is not much difference in RXR levels in maltose double-strand RNA treated and untreated (positive control) cells, but a significant decrease in RXR levels was observed in cells transfected with RXR double-stranded RNAs In these cells, the RXRa showed 95.7% and the RXRb showed 78.5% knockdown when compared to the levels in cells transfected with malE double-stranded RNA (Fig 9B) These data suggest that VGE fusion proteins bind to endogenous RXR and the EcR ⁄ RXR heterodimer binds to the response elements and transactivates genes placed under the control of the response elements FEBS Journal 273 (2006) 5550–5563 ª 2006 The Authors Journal compilation ª 2006 FEBS S K Panguluri et al Functional characterization of EcR gene switches Normalised RLU A 25000 1927 20000 1312 228 15000 1414 10000 5000 177 (5.3) (62) (11) (10) 98 (261) (5.3) 3.6 (10) VGEtvy+ V:HsR B VGEtvy+ VGEtvy+ VGEtvy+ V:Hs-LmR V:Hs-LmR 10 V:Hs-LmR 11 0.2 0.04 0.008 0.2 DMSO 0.04 0.008 0.2 DMSO 0.04 0.008 0.2 DMSO 0.04 0.008 0.2 VGEtvy+ V:Hs-LmR DMSO 0.04 0.008 0.2 VGEtvy+ V:Hs-LmR DMSO 0.04 0.008 0.2 DMSO 0.04 0.008 DMSO VGEtvy+ V:LmR 35000 2517 Normalised RLU 30000 25000 2897 2465 20000 15000 10000 1289 1425 5000 14000 12000 10000 8000 6000 4000 2000 (37) VGEtvy+ Hs-LmR GEtvyV+ V:Hs-LmR 0.2 5 0.2 DMSO VGEtvy+ V:Hs-LmR GEtvyV+ Hs-LmR 309 438 129 53 380 VGEtvay VGEtvay+ V:Hs-LmR VGEtvay+ Hs-LmR GEtvayV+ V:Hs-LmR 0.2 DMSO (5.8) 0.2 DMSO (40) 0.2 DMSO (40) 0.2 DMSO (21.4) 0.2 DMSO Normalised RLU C 0.2 DMSO 0.2 DMSO VGEtvy (3.6) (8.2) DMSO (11.4) 0.2 (6.5) DMSO (3) GEtvayV+ Hs-LmR Fig Induction of luciferase gene by monopartite switches with different RXRs (A) Plasmid DNA samples of VGEtvy + VP16:HsRXR (HsR) or VGEtvy + VP16:Hs(1–6)-LmRXR(7–12) (V:Hs-LmR-6) or VGEtvy + VHs(1–8)-LmR(9–12) (V:Hs-LmR-8) or VGEtvy + VHs(1–9)LmR(10–12) (V:Hs-LmR-9) or VGEtvy + VHs(1–10)-LmR(11–12) (V:Hs-LmR-10 or VGEtvy + VHs(1–11)-LmR (12) (V:Hs-LmR-11) or VGEtvy + VP16:LmRXR (VLmR) and pFRLuc were transfected into 3T3 cells using superfect lipid reagent The transfected cells were grown in the medium containing 0, 0.008, 0.04 and 0.2 lM concentrations of RG-102240 The cells were harvested at 48 h after adding ligand and the reporter activity was measured and the RLU presented are mean ± SD (n ¼ 3) Numbers above the bars represent the maximum fold induction observed for that particular combination The numbers in parentheses represent the RLU of the reporter activity in cells that exposed to dimethylsulfoxide (no ligand) (B) Plasmid DNA samples of VGEtvy or VGEtvy + VHs-LmR or VGEtvy + Hs-LmR or GEtvyV + VHs-LmR or GEtvyV + Hs-LmR and pFRLuc were transfected into 3T3 cells using superfect lipid reagent The transfected cells were grown in the medium containing 0, 0.2, and lM concentrations of RG-102240 The cells were harvested at 48 h after adding ligand and the reporter activity was measured and the RLU presented are mean ± SD (n ¼ 3) Numbers above the bars represent the maximum fold induction observed for that particular combination The numbers in parentheses represent the RLU of the reporter activity in cells that exposed to dimethylsulfoxide (no ligand) (C) The experiments and procedures used are the same as in (B) except VGEtvay was used in place of VGEtvy DMSO, dimethylsulfoxide FEBS Journal 273 (2006) 5550–5563 ª 2006 The Authors Journal compilation ª 2006 FEBS 5557 Functional characterization of EcR gene switches 653 624 VGEtvy Relative Expression B 0.2 0.04 0.008 DMSO 0.2 0.04 VGEtvy + malE DsRNA 185 (3.1) 0.008 DMSO 0.2 (19) 0.04 0.008 16000 14000 12000 10000 8000 6000 4000 0 (19.8 ) DMSO RLU A S K Panguluri et al VGEtvy+ RXR DsRNA 0.900 0.800 0.700 0.600 0.500 0.400 0.300 0.200 0.100 95.7% 78 5% 0.000 Co n t ro l malE RXR α Primers RN A i C o n tr o l malE R NA i RXR β Primers Fig Endogenous RXR is involved in EcR gene switch functioning (A) Plasmid DNA samples of VGEtvy or VGEtvy plus doublestranded RNA for malE or VGEtvy plus double-stranded RNAs for RXRa and RXRb and pFRLuc were transfected into HEK293 cells using superfect lipid reagent The transfected cells were grown in the medium containing 0, 0.008, 0.04, 0.2, and lM concentrations of RG-102240 The cells were harvested at 48 h after adding ligand and the reporter activity was measured and the RLU mean ± SD (n ¼ 3) are presented Numbers above the bars represents the maximum fold induction observed for that particular combination The numbers in parentheses represents the RLU of the reporter activity in cells that exposed to dimethylsulfoxide (no ligand) DMSO, dimethylsulfoxide (B) Plasmid DNA samples of VGEtvy or VGEtvy plus double-strand RNA for malE or VGEtvy plus doublestrand RNAs for RXRa and RXRb were transfected into HEK293 cells using superfect lipid reagent The cells were harvested for RNA isolation after 48 h of transfection and normalized expression levels of RNA were estimated through quantitative RT-PCR by using RXRa and RXRb primers GAL4:EcR fusion proteins bind to GAL4 response element independent of ligand and RXR, but EcR binding to HsRXR is ligand-dependent It is not known whether GAL4:EcR (GE) fusion proteins bind to GAL4 response element prior to or after binding to ligand: ChIP assay was used to answer this question NIH ⁄ 3T3 cells were transfected with GEtvy + VHs-LmRXR, GEtvy + VHsRXR, VGEtvy + VHsLmRXR, or VGEtvy + VHsRXR The ligand, RG-102240 (5 lm) was added to the cells at h after transfection and the cells were incubated for 48 h The 5558 transfected cells were fixed with formaldehyde according to the manufacturer’s protocol (Active Motif, Carlsbad, CA, USA) and the sheared chromatin was pulled down with anti-VP16 IgG for bipartite switches, with anti-GAL4 IgG for monopartite gene switch complex An expected size PCR product was detected after amplification using GALRE primers and the template DNA eluted from cells transfected with VGE or GE fusion protein constructs plus VHs-LmRXR fusion protein constructs and exposed to either dimethylsulfoxide or RG-102240 and precipitated with GAL4 or VP16 antibodies suggesting that the ligand is not required for heterodimer of VGE or GE fusion proteins and VHs-LmRXR binding to the GAL4 response elements (Fig 10; lane 1–8) In contrast, an expected size PCR product was not detected when the template DNA eluted from cells transfected with GE + HsRXR and exposed to dimethylsulfoxide and precipitated with VP16 antibodies was used as a template (Fig 10; lane 9) On the other hand, an expected size PCR product was detected using the template DNA from cells transfected with GE + VHsRXR and exposed to ligand RG-102240 and precipitated with VP16 antibodies This suggested that VHsRXR can heterodimerize with GE and the heterodimer binds to GAL4 response elements only in the presence of ligand (Fig 10; lane 10) Fig 10 The fusion proteins of GE and VGE bind to GAL4 RE in the absence of ligand HsRXR but not chimera between Hs-LmRXR require ligand for heterodimerization with EcR Analysis of the binding of switch components to response element in ChIP assay Lane1: VGEtvy(dimethylsulfoxide), lane 2: VGEtvy(RG-102240), lane 3: VGEtvy + V:HsR(dimethylsulfoxide), lane 4: VGEtvy + V:HsR(RG-102240), lane 5: VGEtvy + V:Hs-LmR(dimethylsulfoxide), lane 6: VGEtvy + V:Hs-LmR(RG-102240), lane 7: GEtvy + V:HsLmR(dimethylsulfoxide), lane 8: GEtvy + V:Hs-LmR(RG-102240), lane 9: GEtvy + V:HsR(dimethylsulfoxide) and lane 10: GEtvy + V:HsR(RG-102240).The HEK293 cells were transfected with VGEtvy or VGEtvy + V:HsR or VGEtvy + V:Hs-LmR or GEtvy + V:HsR or GEtvy + V:Hs-LmR and pFRLUC dimethylsulfoxide or lM RG-102240 ligand was added to each combination after h of transfection and the cells were fixed with formaldehyde at 48 h after addition of ligand and precipitated with VP16 antibodies as described in Experimental procedures VP16 antibodies were able to precipitate DNA– protein complexes for all combinations tested except the GEtvy + V:HsR treated with dimethylsulfoxide (lane 9) suggesting that V:HsRXR binds to GEtvy only in the presence of ligand FEBS Journal 273 (2006) 5550–5563 ª 2006 The Authors Journal compilation ª 2006 FEBS S K Panguluri et al Discussion The most significant contributions of this study are better understanding of the molecular mechanism of EcR gene switches in mammalian cells and development of an efficient EcR gene switch that showed low background activity in the absence of ligand and high induced activity in the presence of ligand Many of the current EcR switches are in bipartite format, which requires RXR for efficient transactivation of the genes placed under their control Among these EcR switches, the RheoSwitchÒ system display the lowest background activity coupled with a strong inducibility (up to 10 000-fold) in cultured cells [11,12] These switches can be very good for applications where the size of the switch components is not a concern However, for certain applications in which the cells are sensitive to the slightest perturbations in the metabolic pathways involving RXR can be a drawback [13] In order to find the best possible monopartite switch format, we screened different formats of switches VG(147)E(CDEF), VG(147)E(DEF), VG(93)E(CDEF), VG(93)E(DEF), VG(65)E(CDEF) and VG(65)E(DEF) that include VP16 activation domain and three different truncations of GAL4 DNA binding domain (G147, G93 and G65) and two different EcR truncations (CDEF and DEF) Similar constructs were also made in GVE and GEV formats From the transactivation experiments it was concluded that VGE is the best format for monopartite switch that showed high induction levels which are dose responsive Out of all VGE formats tested, VG(65)E(CDEF) showed high fold induction (706-fold at 25 lm RG-102240) but with low reporter activity when compared to the reporter levels supported by other format switches Although the G(93) and E(CDEF) switch showed higher fold inductions among all the VGE and GVE switches tested, they are not preferred because of the concern that the EcR DNA binding domain present in them may bind to endogenous nuclear receptors and cause changes in endogenous gene expression The formats with G(147) and E(DEF) were found to have high fold induction as well as high induction levels, which can be useful for developing an efficient monopartite switches for in vivo studies To identify an efficient EcR monopartite switch, we screened three formats of these switches that had activation domain, DNA binding domain and two different mutant (tvy and tvay) EcR ligand binding domain arranged in different orders (VGE, GVE and GEV) All the three monopartite switch formats supported ligand dependent transactivation of reporter gene activation However, the performance of the VGE format Functional characterization of EcR gene switches was the best as in the case of VGE format with wildtype EcR GVE format showed higher background levels of induction in the absence of ligand and GEV showed low induction levels as well as low background expression of reporter gene The differences in the performance of these monopartite switches could be due to the difference in ligand induced conformational changes as well as displacement of corepressors and recruitment of coactivators by EcR fusion proteins upon addition of ligand The placement of EcR relative to GAL4 and VP16 probably influence the behavior of fusion protein upon binding to ligand Apparently, having activation domain (VP16) and DNA binding domain (GAL4), in that order, on the NH2-terminal end of EcR is better than the other two formats Interestingly, this is the arrangement in natural EcRs [activation domain A ⁄ B, DNA binding domain (C) and ligand binding (E) domain] The difference in induction levels between cell lines were found when we transfected VGE monopartite switch The difference might be due to the difference in the levels of endogenous RXR isoforms among cell lines This in turn can be explained by the results obtained in the transfection experiments with G:CfEcR with V:RXRa or V:RXRb or V:RXRc The data showed that the induction levels and fold induction are higher in transfections containing V:RXRc rather than with RXRa or RXRb Our microarray data showed that very low levels of cRXR in HEK293 cells, the isoform that supported higher induction levels This could be the possible reason for low induction levels observed for reporter activity supported by EcR gene switches in HEK293 cells when compared to 3T3, CHO and CV1 cell lines In addition to the difference in levels of RXRs, it could also be due to difference in levels of human carbon catabolite repressor protein complex, which is reported to interact with RXR and its heterodimers in ligand dependent manner and represses its transactivation function [14] There are very few reports on EcR-based monopartite switches Suhr et al [6] developed a monopartite switch by fusing B mori ecdysone receptor CDEF domains with VP16 activation domain The luciferase reporter gene was placed under the control of four tandem repeats of EcREs inserted upstream of a thymidine kinase gene minimal promoter This switch was reported to have high level of ligand induced transgene expression without exogenous RXR The present study indicates that the endogenous RXR participates in the functioning of this VP16-EcR gene switch However, the use of EcR DNA binding domain and EcRE raises the possibilities of endogenous steroid receptors of the host cells binding to the EcRE Recently, Karzenowski FEBS Journal 273 (2006) 5550–5563 ª 2006 The Authors Journal compilation ª 2006 FEBS 5559 Functional characterization of EcR gene switches S K Panguluri et al et al [12] reported that monopartite gene switch VGE that contained a mutant CfEcR (CfEcRvy) showed higher background activity in the absence of ligand, resulting in only four to nine-fold inductions To reduce the background activity in the absence of ligand Fourteen different activation domains were tested in place of VP16 and achieved a maximum induction of 152-fold in NIH ⁄ 3T3 and 207-fold in HEK293 cells exposed to lm RG-102240 In this study, we also compared the performance of two mutant EcRs with high ligand sensitivity due to three (T335V, V390I and Y490E) or four (T335V, V390I, A393P and Y490E) mutations in the ligand binding domain EcRtvy binds to both steroids and nonsteroids while EcRtvay binds to nonsteroids but not to ecdysteroids ([15] and data not shown) Both these mutants performed much better than the wildtype EcR in both bipartite and monopartite switches EcRtvy performed better than EcRtvay, but because of steroid insensitivity, EcRtvay may be useful for applications where exposure to ecdysteroids may be a problem EcRtvy is one of the most efficient EcR monopartite switches developed by the present work The ligand induced reporter activity supported by this switch is as good as that reported for bipartite switch [11] However, background activity in the absence of ligand is slightly higher than that of bipartite switch, which needs improvement prior to widespread use of this format in various applications Ligand binding assays showed that very little [3H]RH-2485 bound to GAL4:EcR, VGE, GVE and GEV fusion proteins However, addition of USP resulted in dramatic increase (between 7238 and 15 164-fold) in [3H]RH-2485 binding to these EcR fusion proteins Lezzi et al [16] reported that extracts of yeast cells expressing fusion protein of GAL4 actvation domain and DmEcR showed low level binding to [3H]ponasterone A and the specific binding increased dramatically with the addition of USP Similar observations were reported by Minakuchi et al [17] Our RNAi experiments also clearly demonstrated the requirement of RXR for efficient functioning of VGE switch Taken together, these observations clearly demonstrate the requirement of heterodimeric partner for efficient functioning of EcR As explained above, VGE fusion protein requires USP or RXR for efficient ligand binding and transactivation If this is the case, addition of RXR should improve the function of VGE switch When VGE or GEV construct was cotransfected with VP16:HsRXREF or VP16:LmRXR or a chimera between these two RXRs, both VP16:LmRXR and chimeras 6, and increased both ligand sensitivity and ligand induced 5560 reporter activity of EcR switch VP16:HsRXR did not cause much improvement in the performance of VGE or GEV switches This may be due to the fact that these switches already use endogenous RXR and additional RXR does not improve their performance VP16:LmRXR and the chimeras between HsRXR and LmRXR heterodimerize more efficiently with EcR than does HsRXR [11], therefore these receptors are able to improve switch performance Our data also showed that it is the RXR but not the additional VP16 domains present in VP16:RXR that is responsible for improvement in the performance of these switches The ligand sensitivity of VGE + Hs-LmR is similar to the sensitivity of bipartite switch, GE + V:Hs-LmRXR But, the induction levels of VGE + Hs-LmRXR switch are higher when compared to those of bipartite switch GE + V:Hs-LmRXR The major advantages of these new versions of VGEtvy + Hs-LmRXR and VGEtvay + Hs-LmRXR switches are the following: (a) because of use of mutant EcRs, ligand sensitivity of these switches is much higher than the earlier versions of EcR gene switches, (b) VGEtvay + Hs-LmRXR switch can be used in applications in which potential exposure to plant derived ecdysteroids could be a concern, and (c) the removal of VP16 activation domain from Hs-LmRXR chimera reduces the chances of interference with endogenous gene expression due to potential interaction of V:Hs-LmRXR with endogenous nuclear receptors The major disadvantage of this VGEtvy + Hs-LmR switch is the slightly higher background activity in the absence of ligand when compared to earlier versions of EcR bipartite switches Further modifications are needed to reduce the background activity of this switch The results from ChIP assay showed that VGE and GE can bind to GAL4 response elements in the absence of ligand Hs-LmRXR can heterodimerize with GE in the absence of ligand In contrast, HsRXR can bind to GE only in the presence of ligand The ChIP assay using anti-VP16 IgG suggests that EcR ⁄ Hs-LmRXR can heterodimerize in the absence of ligand On the other hand, EcR ⁄ HsRXR requires ligand for their heterodimerization These data support our earlier findings using pulldown assays that EcR can interact with Hs-LmRXR in the absence of ligand but needed ligand to pull down HsRXR [11] These findings indicate that it may be possible to modify EcR such that it no longer binds to endogenous HsRXR even in the presence of ligand Creation of an EcR mutant that no longer requires binding of heterodimeric partner for its high affinity binding to ligand may become more attractive choice for applications in vivo FEBS Journal 273 (2006) 5550–5563 ª 2006 The Authors Journal compilation ª 2006 FEBS S K Panguluri et al In summary, the research described here contributed to the development of an improved version of EcR gene switch with more desirable properties than the earlier versions These studies also contribute to understanding the function of EcR gene switch in mammalian cells, particularly the role of RXR or USP in the EcR function This in turn should help in designing more improved EcR gene switches in the future Experimental procedures Constructs To make VGE, pM vector (Clonetech Inc., Palo Alto, CA, USA) carrying DEF domains of CfEcR [9] was used as a template to amplify GAL4:CfEcR (GE) with restriction sites SalI on the 5¢ end and NotI on the 3¢ end, and the PCR product was cloned into pACT vector (Promega, Madison, WI, USA) Similarly, to make GVE, the CfEcR was excised with restriction sites BamHI and XbaI from pM vector and cloned in pACT vector This pACT vector containing EcR DEF domain was used as a template to amplify VP16:CfEcR (VE) with restriction sites SalI on 5¢ end and Not I on the 3¢ end and cloned into pBIND vector (Promega) We have also prepared VG(93)E, VG(65)E, G(93)VE and G(65)VE with truncated GAL4 DBD (amino acids: 1–93 and 1–65) VGEtvy, GVEtvy, VGEtvay and GVEtvay were prepared by replacing KpnI fragment in the E domain of CfEcR with the KpnI fragments from CfEcR containing T335V, V390I and Y410E mutations (tvy) and T335V, V390I, A393P and Y410E mutations (tvay) To prepare GEtvyV and GEtvayV, CfEcR DEF domains were amplified using VGEtvy and VGEtvay as a templates using primers containing restriction sites BamHI on the 5¢ end and HindIII on the 3¢ end and VP16AD encoding 44 amino scids from pVP16 with restriction sites HindIII on the 5¢ end and NotI on the 3¢ end, and these fragments were cloned in pBIND vector (Promega) Construction of VP16:HsRXR (a fusion between VP16 activation domain and Homo sapiens RXR EF domains, VHsRXR), VP16LmRXR (a fusion between VP16 activation domain and Locusta migratoria RXR EF domains, VLmRXR) and Chimeras between human and locust RXR (VHs-LmRXR) used here have been described previously [11] Hs-LmRXR chimera without VP16 AD (Hs-LmRXR) was prepared by PCR amplifying RXR chimera using primers containing EcoRI and XbaI restriction sites and cloning into the same sites in pACT (Promega) All constructs were assembled by standard cloning methods and confirmed by DNA sequencing Inducible luciferase reporter plasmid pFRLuc containing five copies of the GAL4 response element (5· GALRE) and synthetic minimal promoter was purchased from Stratagene Cloning Systems (La Jolla, CA, USA) Functional characterization of EcR gene switches Transient transfections NIH ⁄ 3T3 and HEK293 cells were maintained at 37 °C with 5% CO2 in DMEM supplemented with 10% fetal bovine serum and 1% penicillin ⁄ streptomycin (Invitrogen, CA, USA) One day before transfection, cells were plated in 48-well plates at a density of 12 500 cellsỈwell)1 On the second day, the medium from the plates were replaced with 100 lL of fresh serum free DMEM and the cells were transfected with 63 ng of receptor constructs and 250 ng of reporter construct pFRluc using SuperFect lipid (Qiagen Inc., Valencia, CA, USA) according to manufacturer’s instructions A second reporter, Renilla luciferase, expressed under a thymidine kinase constitutive promoter was cotransfected into cells and used for normalization After h of transfection, 125 lL of DMEM containing 20% fetal bovine serum and double the concentration of inducer, RG-102240 also known as GSTM-E or RSL was added Two days after transfection, the medium was discarded and the cells were lysed in 50 lL passive lysis buffer (Promega) Twenty microliters of extract were transferred to 96-well opaque plates and the luciferase and Renilla luciferase reporter activities were measured using the Dual luciferaseTM reporter assay system from Promega and Fluoroskan Ascent FL (ThermoLab Systems, Helsinki, Finland) All the transfection experiments were performed in triplicate and the experiments were repeated at least three times Ligand binding To determine whether CfEcR in VGE, GVE and GEV (with EcRtvy and EcRtvay mutants) format, needs heterodimeric partner to bind to ligand, VGE, GVE and GEV constructs were cloned in pGEX vector (Promega) in the sites SalI and NotI and expressed in BL-21 Escherichia coli cells Fifteen milliliters of LB medium containing 50 lgỈL)1 ampicillin was inoculated and grown overnight at 30 °C The overnight cultures were diluted into 500 mL of LB medium with 50 lgỈL)1 ampicillin and grown to D600 of 0.5 The cells were induced with 100 lm isopropyl thio-b-d-galactoside for h and harvested by centrifugation at 7700 g for 10 at °C (Eppendorf Microcentrifuge 5415D; Eppendorf AG, Hamburg, Germany) The cell pellet was resuspended in NaCl ⁄ Pi containing protease inhibitors, sonicated and subjected to centrifugation at 12 000 g for 10 at °C (Eppendorf Microcentrifuge 5415D) The clarified lysates were analyzed on a polyacrylamide gel and the proteins were used for ligand binding assays These conditions have been previously used to determine the binding of EcRs to ligands [18,19] The proteins were prepared in T-buffer (10 mm Tris pH 7.2 and mm dithiothreitol) to a final volume of FEBS Journal 273 (2006) 5550–5563 ª 2006 The Authors Journal compilation ª 2006 FEBS 5561 Functional characterization of EcR gene switches S K Panguluri et al 100 lL with 0.1 lL (84 323 disintegrations per minute) of 3H RH-112485 [Methoxy-3H] with specific activity of 83 CiỈmmol)1 and 99% pure (HPLC purified) is competed with 10 mm RG-102240 A similar reaction mixture was made for Scatchard analysis with lL of different dilutions of [3H]RH-112485 The reaction mixture was kept at °C for h; the unbound [3H]RH-2485 was removed by activated charcoal:dextran (10 : 1) and the bound radioactivity was measured in liquid scintillation counter The background radioactivity was measured by adding all the ingredients except proteins and was deducted from the hot and cold values The counts for liquid scintillation cocktail were also measured and were taken as reference or blank The counts of the radioactive ligand alone in the scintillation liquid cocktail were also measured and these values were taken in the y-axis as total added The reactions were done in triplicate The dissociation constant, Kd, was evaluated from the saturation curve for radioligand binding using nonlinear regression analysis with one site binding (hyperbola) equation in the graphpad prism program (GraphPad Software, San Diego, CA, USA) RNAi RNAi experiments were performed in HEK293 cells transfected with double-strand RNA for RXRa and RXRb to determine whether endogenous RXR is helping the monopartite gene switches in the transactivation of reporter gene HEK293 cells were transfected at 40% confluence with lg each of RXRa and RXRb double-stranded RNAs per well in 12-well plate, and the cells were harvested three days after transfection Bacterial malE gene double-stranded RNA was used as a control Total RNA was isolated using Trizol reagent (Molecular Research Center Inc., Cincinnati, OH, USA) First strand cDNA was prepared by using M-MuLV Reverse Transcriptase (New England Biolabs, Ipswich, MA, USA) Quantitative PCR analysis was performed to determine whether the endogenous RXR was knockdown or not Monopartite gene switch VGEtvy, pFRLuc and double-stranded RNA for RXRa and RXRb were transfected The transfected cells were exposed to ligand for 48 h and the luciferase activity was measured Acknowledgements ChIP assay To determine whether the receptor proteins need ligand to bind to response element, ChIP assay was performed by using ChIP-ITTM kit (Active Motif) HEK293 cells were cultured in mL DMEM with 10% fetal bovine serum and the cells were transfected with GEtvy:VHs-LmRXR, GEtvy:VHsRXR, VGEtvy alone, VGEtvy:VHsRxR and VGEtvy:VHs-LmRXR The transfected cells were exposed to dimethylsulfoxide or lm RG-102240 for 48 h Then the cells were fixed with formaldehyde and collected by scraping with rubber policeman and centrifuged at 510 g for 10 at °C (Eppendorf Microcentrifuge 5415D) The cells were homogenized and the chromatin was sheared by using enzyme cocktail for 30 The sheared chromatin was run on an agarose gel (2%) to ensure that all the sheared chromatin was concentrated below 200 bp The sheared chromatin was pretreated with protein-G-agarose beads for h at °C The DNA–protein complexes were treated with GAL4 or VP16 antibodies (Sigma-Aldrich, St Louis, MO, USA) following the manufacturer’s instructions The mixture was then incubated with proteinG beads to collect the DNA–protein complexes Finally, the proteinase K treated DNA was eluted using DNApurification columns Five microliters of final DNA elute was used in a PCR using primers that amplify 5· GAL4 response element under the following conditions: 95 °C initial denaturation for min, 95 °C cycle denaturation for min, 60 °C annealing for min, 72 °C extension for and a final extension of at 72 °C and a total of 42 cycles were carried out The PCR amplified products were analyzed using 2% agarose gel 5562 This work was supported in part by RheoGene Inc research grant to SRP This is contribution number 06-08-115 from the Kentucky Agricultural Experimental Station We thank Dr Dean Cress of RheoGene Inc for support throughout the study and critical reading of the manuscript References Palli SR, Hormann RE, Schlattner U & Lezzi M (2005) Ecdysteroid receptors and their applications in agriculture and medicine Vitam Horm 73, 59–100 Mangelsdorf DJ, Thummel C, Beato M, Herrlich P, Schutz G, Umesono K, Blumberg B, Kastner P, Mark M & Chambon P (1995) The nuclear receptor superfamily: the second decade Cell 83, 835–839 Lafont R & Dinan L (2003) Practical uses for ecdysteroids in mammals including humans: an update J Insect Sci 3, Christopherson KS, Mark MR, Bajaj V & Godowski PJ (1992) Ecdysteroid-dependent regulation of genes in mammalian cells by a Drosophila ecdysone receptor and chimeric transactivators Proc Natl Acad Sci USA 89, 6314–6318 No D, Yao TP & Evans RM (1996) Ecdysone-inducible gene expression in mammalian cells and transgenic mice Proc Natl Acad Sci USA 93, 3346–3351 Suhr ST, Gil EB, Senut MC & Gage FH (1998) High level transactivation by a modified Bombyx ecdysone receptor in mammalian cells without exogenous reti- FEBS Journal 273 (2006) 5550–5563 ª 2006 The Authors Journal compilation ª 2006 FEBS S K Panguluri et al 10 11 12 13 14 noid X receptor Proc Natl Acad Sci USA 95, 7999– 8004 Hoppe UC, Marban E & Johns DC (2000) Adenovirusmediated inducible gene expression in vivo by a hybrid ecdysone receptor Mol Ther 1, 159–164 Wyborski DL, Bauer JC & Vaillancourt P (2001) Bicistronic expression of ecdysone-inducible receptors in mammalian cells Biotechniques 31, 618–620, 622, 624 Palli SR, Kapitskaya MZ, Kumar MB & Cress DE (2003) Improved ecdysone receptor-based inducible gene regulation system Eur J Biochem 270, 1308–1315 Karns LR, Kisielewski A, Gulding KM, Seraj JM & Theodorescu D (2001) Manipulation of gene expression by an ecdysone-inducible gene switch in tumor xenografts BMC Biotechnol 1, 11 Palli SR, Kapitskaya MZ & Potter DW (2005) The influence of heterodimer partner ultraspiracle ⁄ retinoid X receptor on the function of ecdysone receptor FEBS J 272, 5979–90 Karzenowski D, Potter DW & Padidam M (2005) Inducible control of transgene expression with ecdysone receptor: gene switches with high sensitivity, robust expression, and reduced size Biotechniques 39, 191–192, 194, 196 passim Toniatti C, Bujard H, Cortese R & Ciliberto G (2004) Gene therapy progress and prospects: transcription regulatory systems Gene Ther 11, 649–657 Winkler GS, Mulder KW, Bardwell VJ, Kalkhoven E & Timmers HT (2006) Human Ccr4-Not complex is a Functional characterization of EcR gene switches 15 16 17 18 19 ligand-dependent repressor of nuclear receptor-mediated transcription Embo J 25, 3089–3099 Kumar MB, Fujimoto T, Potter DW, Deng Q & Palli SR (2002) A Single Point Mutation in Ecdysone Receptor Leads to Increased Ligand Specificity: Implications for Gene Switch Applications Proc Natl Acad Sci USA 99, 14710–14715 Lezzi M, Bergman T, Henrich VC, Vogtli M, Fromel C, Grebe M, Przibilla S & Spindler-Barth M (2002) Ligand-induced heterodimerization between the ligand binding domains of the Drosophila ecdysteroid receptor and ultraspiracle Eur J Biochem 269, 3237–3245 Minakuchi C, Nakagawa Y, Kiuchi M, Tomita S & Kamimura M (2002) Molecular cloning, expression analysis and functional confirmation of two ecdysone receptor isoforms from the rice stem borer Chilo suppressalis Insect Biochem Mol Biol 32, 999–1008 Perera SC, Ladd TR, Dhadialla TS, Krell PJ, Sohi SS, Retnakaran A & Palli SR (1999) Studies on two ecdysone receptor isoforms of the spruce budworm, Choristoneura fumiferana Mol Cell Endocrinol 152, 73–84 Kothapalli R, Palli SR, Ladd TR, Sohi SS, Cress D, Dhadialla TS, Tzertzinis G & Retnakaran A (1995) Cloning and developmental expression of the ecdysone receptor gene from the spruce budworm, Choristoneura fumiferana Dev Genet 17, 319–330 FEBS Journal 273 (2006) 5550–5563 ª 2006 The Authors Journal compilation ª 2006 FEBS 5563 ... DNA binding domain and VP16 activation domain Various combinations of the EcR ligand binding domain, VP16 activation domain and GAL4 DNA binding domain were constructed (Fig 1A) and tested in NIH... probably in? ??uence the behavior of fusion protein upon binding to ligand Apparently, having activation domain (VP16) and DNA binding domain (GAL4), in that order, on the NH2-terminal end of EcR... formats Interestingly, this is the arrangement in natural EcRs [activation domain A ⁄ B, DNA binding domain (C) and ligand binding (E) domain] The difference in induction levels between cell lines