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,
Trang 1Improved ecdysone receptor-based inducible gene regulation system
Subba R Palli1, Mariana Z Kapitskaya2, Mohan B Kumar2and Dean E Cress2
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-lusretinoid 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 melanogasterand 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
fumiferanaEcR) [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/ BombyxEcR (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)
Trang 2induction 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-vivodelivery
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 XbaI site on the 3¢ 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 XbaI site on the 3¢ 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 XbaI digested pM 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 pFRLUCreporter 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 pFRLUCwith 7X EcRE The 7X EcRE fragment was amplified from pMK43.2 [29] using primers containing PstI and XmaI sites in forward and reverse primers, respectively The TATAA sequence was included
in the reverse primer The PCR products were cloned into PstI and XmaI 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 pFRLUCwith SEAP
at KpnI and XbaI restriction sites
Ligands Ponasterone A and Muristerone A were purchased from Alexis Corporation (San Diego, CA, USA) RG-102240 also known as GSTM-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%
Trang 3Cells and transfections and reporter assays
CHO and A549 cells were grown in F12 medium containing
2 mM L-glutamine and 10% bovine calf serum 3T3, 293 and
CV1 cells were grown in Dulbecco’s modified Eagle’s
medium containing 4 mM L-glutamine, 1.5 gÆL)1 sodium
bicarbonate, 4.5 gÆL)1glucose 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 lg of
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-luciferaseTM
repor-ter assay system from Promega Corporation (Madison, WI,
USA) b-Galactosidase was measured using Gal-Screen
system from Applied Biosystems (Foster City, CA, USA)
The SEAP activity in the medium was quantified using
Phospha-LightTMSystem 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 pFRLUCreporter 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 lMor 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-taining 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 the Gal-Screen 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.
Trang 4The 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 lM or higher
concentration of RG-102240, 0.04 lMor higher
concentra-tion of RG-102317, 5 lMor higher concentration of PonA
and 25 lMor 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 lMconcentration of RG-102240 and by threefold in the presence of 5 lMconcentration 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), pFRLUCand 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.
Trang 5(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 pFRLUCwere transfected into 3T3 cells and the cells were grown in the presence of 1 lMRG-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), pFRLUCand 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.
Trang 6switches, 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 pFRLUCshowed 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),
pFRLUCand pTKRL The transfected cells were grown in the
medium containing 1 l M concentration of RG-102240 For the data
shown in A, cells were collected at 0, 1, 3, 6, 12, 24, 48 and 72 h after
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-LightTMSystem from Applied Biosystems FI, fold induction.
Ligand
RLU ± SD FI ± SD RLU ± SD FI ± SD RLU ± SD FI ± SD
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 25 lm 771 ± 86 14 ± 1 77 ± 12 1 2540 ± 187 313 ± 70
Trang 7construct 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 (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 nucleonucleo-tides 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] Bisacylhydrazine nonsteroidal ligands have undergone an extensive battery of toxicology tests for EPA registration as commercial insecticides [33] These chemicals are classified as green chemistry by EPA because
of their favorable environmental and toxicology profiles [36] Thus, this new CfEcR-based switch has most of the desirable properties of an optimal gene regulation system and is currently being evaluated for in vivo efficacy One limitation of the current version of this switch is the requirement of slightly higher concentration of ligands for maximum induction Experiments are in progress to improve the sensitivity of this switch by modifying both EcR and RXR
Acknowledgements
We thank M Padidam, D W Potter, P White and P Kumar for critical reading of the manuscript and M R Koelle of Stanford University for the gift of pMK43.2 reporter vector.
References
1 Riddiford, L.M., Cherbas, P & Truman, J.W (2000) Ecdysone receptors and their biological actions Vitam Horm 60, 1–73.
2 Bourguet, W., Ruff, M., C hambon, P., Gronemeyer, H & Moras, D (1995) Crystal-structure of the ligand-binding domain of the human nuclear receptor RXR-alpha Nature 375, 377–382.
3 Billas, I.M., Moulinier, L., Rochel, N & Moras, D (2001) Crystal structure of the ligand-binding domain of the ultraspiracle protein USP, the ortholog of retinoid X receptors in insects J Biol Chem.
276, 7465–7474.
4 Clayton, G.M., Peak-Chew, S.Y., Evans, R.M & Schwabe, J.W (2001) The structure of the ultraspiracle ligand-binding domain reveals a nuclear receptor locked in an inactive conformation Proc Natl Acad Sci USA 98, 1549–1554.
5 Imhof, M.O., Rusconi, S & Lezzi, M (1993) Cloning of a Chironomus tentans cDNA encoding a protein (cEcRH) homo-logous to the Drosophila melanogaster ecdysteroid receptor (dEcR) Insect Biochem Mol Biol 23, 115–124.
Trang 86 Kothapalli, R., Palli, S.R., Ladd, T.R., Sohi, S.S., Cress, D.,
Dhadialla, T.S., 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.
7 Wurtz, J.M., Guillot, B., Fagart, J., Moras, D., Tietjen, K &
Schindler, M (2000) A new model for 20-hydroxyecdysone and
dibenzoylhydrazine binding: a homology modeling and docking
approach Protein Sci 9, 1073–1084.
8 Kumar, M., Fujimoto, T., Potter, D.W., Deng, Q & Palli, S.R.
(2002) A single point mutation in ecdysone receptor leads to
increased ligand specificity: implications for gene switch
applica-tions Proc Natl Acad Sci USA 99, 14710–14715.
9 Albanese, C , Reutens, A.T., Bouzahzah, B., Fu, M., D’Amico,
M., Link, T., Nicholson, R., Depinho, R.A & Pestell, R.G (2000)
Sustained mammary gland-directed, ponasterone A-inducible
expression in transgenic mice Faseb J 14, 877–884.
10 Zhou, Y & Ratner, L (2001) A novel inducible expression system
to study transdominant mutants of HIV-1 Vpr Virology 287,
133–142.
11 Fussenegger, M (2001) The impact of mammalian gene regulation
concepts on functional genomic research, metabolic engineering,
and advanced gene therapies Biotechnol Prog 17, 1–51.
12 Yarovoi, S.V & Pederson, T (2001) Human cell lines expressing
hormone regulated T7 RNA polymerase localized at distinct
intranuclear sites Gene 275, 73–81.
13 Yam, J.W., Chan, K.W & Hsiao, W.L (2001) Suppression of the
tumorigenicity of mutant p53-transformed rat embryo fibroblasts
through expression of a newly cloned rat nonmuscle myosin heavy
chain-B Oncogene 20, 58–68.
14 Sparacio, S., Pfeiffer, T., Schaal, H & Bosch, V (2001)
Genera-tion of a flexible cell line with regulatable, high-level expression of
HIV Gag/Pol particles capable of packaging HIV-derived vectors.
Mol Ther 3, 602–612.
15 Patrick, C.W Jr, Zheng, B., Wu, X., Gurtner, G., Barlow, M.,
Koutz, C., Chang, D., Schmidt, M & Evans, G.R (2001)
Muri-sterone a-induced nerve growth factor release from genetically
engineered human dermal fibroblasts for peripheral nerve tissue
engineering Tissue Eng 7, 303–311.
16 Pacchia, A.L., Adelson, M.E., Kaul, M., Ron, Y & Dougherty,
J.P (2001) An inducible packaging cell system for safe, efficient
lentiviral vector production in the absence of HIV-1 accessory
proteins Virology 282, 77–86.
17 Lin, G & Stern, R (2001) Plasma hyaluronidase (Hyal-1)
pro-motes tumor cell cycling Cancer Lett 163, 95–101.
18 Christopherson, K.S., Mark, M.R., Bajaj, V & Godowski, P.J.
(1992) Ecdysteroid-dependent regulation of genes in mammalian
cells by a Drosophila ecdysone receptor and chimeric
transactiva-tors Proc Natl Acad Sci USA 89, 6314–6318.
19 Yang, G., Hannan, G., Lockett, T & Hill, R (1995) Functional
transfer of an elementary ecdysone gene regulatory system to
mammalian cells-transient transfections and stable cell-lines Eur.
J Entomol 92, 379–389.
20 No, D., Yao, T.P & Evans, R.M (1996) Ecdysone-inducible gene
expression in mammalian cells and transgenic mice Proc Natl
Acad Sci USA 93, 3346–3351.
21 Suhr, S.T., Gil, E.B., Senut, M.C & Gage, F.H (1998) High level
transactivation by a modified Bombyx ecdysone receptor in
mammalian cells without exogenous retinoid X receptor Proc.
Natl Acad Sci USA 95, 7999–8004.
22 Swevers, L., Drevet, J.R., Lunke, M.D & Iatrou, K (1995) The
silkmoth homolog of the Drosophila ecdysone receptor (B1
isoform): cloning and analysis of expression during follicular cell differentiation Insect Biochem Mol Biol 25, 857–866.
23 Hoppe, U.C., Marban, E & Johns, D.C (2000) Adenovirus-mediated inducible gene expression in vivo by a hybrid ecdysone receptor Mol Ther 1, 159–164.
24 Wyborski, D.L., Bauer, J.C & Vaillancourt, P (2001) Bicistronic expression of ecdysone-inducible receptors in mammalian cells Biotechniques 31, 618–620,622,624.
25 Saez, E., Nelson, M.C., Eshelman, B., Banayo, E., Koder, A., Cho, G.J & Evans, R.M (2000) Identification of ligands and coligands for the ecdysone-regulated gene switch Proc Natl Acad Sci USA 97, 14512–14517.
26 Perera, S.C., Ladd, T.R., Dhadialla, T.S., Krell, P.J., Sohi, S.S., Retnakaran, A & Palli, S.R (1999) Studies on two ecdysone receptor isoforms of the spruce budworm, Choristoneura fumi-ferana Mol Cell Endocrinol 152, 73–84.
27 Perera, S.C., Palli, S.R., Ladd, T.R., Krell, P.J & Retnakaran, A (1998) The ultraspiracle gene of the spruce budworm, Chori-stoneura fumiferana: cloning of cDNA and developmental expression of mRNA Dev Genet 22, 169–179.
28 Leid, M., Kastner, P., Lyons, R., Nakshatri, H., Saunders, M., Zacharewski, T., Chen, J.Y., Staub, A., Garnier, J.M., Mader, S.
et al (1992) Purification, cloning, and RXR identity of the HeLa cell factor with which RAR or TR heterodimerizes to bind target sequences efficiently Cell 68, 377–395.
29 Koelle, M.R., Talbot, W.S., Segraves, W.A., Bender, M.T., Cherbas, P & Hogness, D.S (1991) The Drosophila EcR gene encodes an ecdysone receptor, a new member of the steroid receptor superfamily Cell 67, 59–77.
30 Perera, S.C., Sundaram, M., Krell, P.J., Retnakaran, A., Dhadialla, T.S & Palli, S.R (1999) An analysis of ecdysone receptor domains required for heterodimerization with ultra-spiracle Arch Insect Biochem Physiol 41, 61–70.
31 Tran, H.T., Askari, H.B., Shaaban, S., Price, L., Palli, S.R., Dhadialla, T.S., Carlson, G.R & Butt, T.R (2001) Reconstruc-tion of ligand-dependent transactivaReconstruc-tion of Choristoneura fumi-ferana ecdysone receptor in yeast Mol Endocrinol 15, 1140–1153.
32 Lezzi, M., Bergman, T., Henrich, V.C., 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.
33 Dhadialla, T.S., Carlson, G.R & Le, D.P (1998) New insecticides with ecdysteroidal and juvenile hormone activity Ann Rev Entomol 43, 545–569.
34 Martinez, A., Scanlon, D., Gross, B., Perara, S.C., Palli, S.R., Greenland, A.J., Windass, J., Pongs, O., Broad, P & Jepson, I (1999) Transcriptional activation of the cloned Heliothis virescens (Lepidoptera) ecdysone receptor (HvEcR) by muristeroneA Insect Biochem Mol Biol 29, 915–930.
35 Karns, L.R., Kisielewski, A., Gulding, K.M., Seraj, J.M & Theodorescu, D (2001) Manipulation of gene expression by an ecdysone-inducible gene switch in tumor xenografts BMC Bio-technol 1, 11.
36 Carlson, G.R., Dhadialla, T.S., Hunter, R., Jansson, R.K., Jany, C.S., Lidert, Z & Slawecki, R.A (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.