Analysis of chimeric RXRs con-taining regions of LmRXR and HsRXR and point mutants of HsRXR showed that the amino acid residues present in helix 9 and in the two loops on either end of h
Trang 1Steroid hormones, ecdysteroids, regulate insect
develop-ment, reproduction and several other physiological
processes The most active form of ecdysteroids is
20-hy-droxyecdysone (20E) The 20E transduces its signal
through a heterodimeric complex of two nuclear
recep-tors, the ecdysone receptor (EcR) [1] and the
ultraspira-cle (USP), an ortholog of the vertebrate retinoid X
receptor (RXR) [2–4] Both EcR and USP are members
of the nuclear receptor superfamily [5] and exhibit a
typ-ical modular structure comprising the N-terminal
A⁄ B domain, the DNA-binding or C domain, the hinge
or D domain, the ligand-binding or E domain, and the
C-terminal F domain The ligand-binding domain
sup-ports ligand-dependent dimerization and transactivation functions A⁄ B and F domains support ligand-inde-pendent transactivation The DNA-binding domain and the N-terminal region of the hinge region are known to support dimerization of two receptors
The EcR:USP heterodimers bind to the ecdysteroid response elements (EcRE) present in the promoter regions of ecdysteroid response genes and regulate their transcription Most of the nuclear hormone receptors, including EcR, are modular and function as ligand-controlled transcription factors, a characteristic that renders these receptors or their key regions (e.g the ligand-binding domain) suitable for gene switches
Keywords
gene switch; ligand-binding domain; nuclear
receptor; steroid hormone
Correspondence
S R Palli, Department of Entomology, S225
Agricultural Science Center, College of
Agriculture, University of Kentucky,
Lexington, KY 40546, USA
E-mail: rpalli@uky.edu
(Received 14 July 2005, revised 26 August
2005, accepted 3 October 2005)
doi:10.1111/j.1742-4658.2005.05003.x
A pair of nuclear receptors, ecdysone receptor (EcR) and ultraspiracle (USP), heterodimerize and transduce ecdysteroid signals The EcR and its nonsteroidal ligands are being developed for regulation of transgene expres-sion in humans, animals and plants In mammalian cells, EcR:USP heterodimers can function in the absence of ligand, but EcR⁄ retinoid X receptor (EcR:RXR) heterodimers require the presence of ligand for activa-tion The heterodimer partner of EcR can influence ligand sensitivity of EcR so that the EcR⁄ Locusta migratoria RXR (EcR:LmRXR) heterodi-mers are activated at lower concentrations of ligand when compared with the concentrations of ligand required for the activation of EcR⁄ Homo sapiensRXR (EcR:HsRXR) heterodimers Analysis of chimeric RXRs con-taining regions of LmRXR and HsRXR and point mutants of HsRXR showed that the amino acid residues present in helix 9 and in the two loops
on either end of helix 9 are responsible for improved activity of LmRXR The EcR:Lm-HsRXR chimera heterodimer induced reporter genes with nanomolar concentration of ligand compared with the micromolar concen-tration of ligand required for activating the EcR:HsRXR heterodimer The EcR:Lm-HsRXR chimera heterodimer, but not the EcR:HsRXR hetero-dimer, supported ligand-dependent induction of reporter gene in a C57BL⁄ 6 mouse model
Abbreviations
CfEcR, Choristoneura fumiferana EcR; DMSO, dimethylsulfoxide; 20E, 20-hydroxyecdysone; ECD, ecdysteroid; EcR, ecdysone receptor; EcRE, ecdysone response element; G:CfEcR(DEF), GAL4:CfEcR(DEF); RLU, relative light units; RXR, retinoid X receptor; SEAP, secreted alkaline phosphatase; VP:Hs–LmRXR(EF), VP16:HsbRXR (helices 1–8) LmRXR (helices 9–12 + F); V:MmRXR(EF), VP16:MmRXR(EF); V:CfUSP(EF), VP16:CfUSP(EF); V:LmRXR(EF), VP16:LmRXR(EF); USP, ultraspiracle; WT, wild-type.
Trang 2(ligand-dependent regulation of transgenes) in various
biotechnology applications Several nuclear receptors,
including the glucocorticoid receptor (GR), the
pro-gesterone receptor (PR), the estrogen receptor (ER),
and the EcR are being used to develop gene switches
for applications in medicine and agriculture Because
the EcR and its ligands are not found in vertebrates,
they are attractive targets for the development of gene
switches for applications in humans The EcR gene
switch is being developed for use in various
applica-tions including gene therapy, expression of toxic
pro-teins in cell lines and cell-based drug discovery assays
[6–14]
EcRs function as an ecdysteroid-dependent
tran-scription factor in cultured mammalian cells [15,16]
No et al [17] used DmEcR and human RXRa to
develop an EcR gene switch and demonstrated its
function in mammalian cells and mice The EcR gene
switch was improved by using a nonsteroidal ecdysone
agonist, tebufenozide, to induce a high level of
repor-ter gene transactivation in mammalian cells through
Bombyx mori EcR (BmEcR) [18,19] and endogenous
RXR Later, Hoppe et al [20] combined DmEcR and
BmEcR systems and created a chimeric Drosophila⁄
Bombyx EcR (DBEcR) that had combined positive
aspects of both systems so that the chimeric receptor
was capable of binding to modified EcRE and also
functioned without exogenous RXR Saez and
coworkers discovered that the RXR ligands enhance
the ligand-dependent activity of EcR-based gene
swit-ches [21], and Wybroski and coworkers [22] developed
methods for expression of both EcR and RXR in a
bicistronic vector
Although the current versions of EcR gene switch
possess several fundamental features that confer great
potential for enhancement, they do not satisfy all of
the criteria desirable for a generally useful gene
regula-tion system To improve the EcR-based switch, we
tested several combinations of GAL4 DNA-binding
domain (GAL4 DBD), VP16 activation domain
(VP16 AD), EcR and RXR and found that a
two-hybrid format switch, in which GAL4 DBD was fused
to CfEcR (DEF) and VP16 AD was fused to Mus
musculus RXR (MmRXR) EF was the best
combina-tion in terms of low background levels of reporter gene
activity in the absence of a ligand and high levels of
reporter gene activity in the presence of a ligand [23]
However, the ligand sensitivity of this two-hybrid
for-mat EcR gene switch is not very high and requires a
micromolar concentration of ligand for induction of
genes To improve the ligand sensitivity of EcR gene
switch, we tested insect RXR and chimeras between
human and insect RXRs as partners for EcR and
discovered that the partner of EcR affects functioning
of EcR in gene switch applications The ligand sensi-tivity of EcR gene switch was improved by 100-fold
by replacing HsRXR with a chimera between HsRXR and an insect RXR from Locusta migrotoria (LmRXR)
Results
Use of invertebrate RXR improves the function
of EcR in mammalian cells Alignment of USP and RXR sequences showed that the RXR homologs, USPs from lepidopteran and dip-teran insects fall into one group and the RXR homo-logs identified from insects belonging to other orders (e.g Heteroptera, Locusta migratoria and Coleoptera, Tenebrio molitor, as well as from crab and tick group with vertebrate RXRs) (Fig 1) In other words, the RXR homologs identified in insects belonging to orders other than Lepidoptera and Diptera as well as from crab and tick are closer to vertebrate RXRs than
to their counterparts in lepidopteran and dipteran insects
As shown in Fig 2A, use of USP from the lepidop-teran insect Choristoneura fumiferana [V:CfUSP(EF)]
as a partner for EcR from Choristoneura fumiferana [G:CfEcR(DEF)] resulted in the expression of a repor-ter gene in the absence of ligand and showed low levels
of ligand-dependent induction In contrast, use of
Fig 1 Phylogenetic tree of USP ⁄ RXR ligand-binding domain sequences The phylogenetic tree was prepared using DNA STAR
(DNA star Inc., Madison, WI) The sequences used are Homo sap-iens retinoid X receptor (HsRXR) [26], Xenopus laevis retinoid X receptor (XlRXR) [27], fiddler crab Uca pugilator RXR homolog (UpRXR) [28], Locusta migratoria RXR homolog (LmRXR) [29], Amblyomma americanum RXR homolog (AmaRXR) [30], Bombyx mori USP (BmUSP) [31], Manduca sexta USP (MsUSP) [32], Choris-toneura fumiferana USP (CfUSP) [33], Drosophila melanogaster USP (DmUSP) [34–36], Aedes aegypti USP (AaUSP) [37], Chirono-mus tentans USP (CtUSP) [27].
Trang 3RXR from Homo sapiens [V:HsRXR(EF)] or RXR
from orthopteran insect, Locusta migratoria
[V:Lm-RXR(EF)] as a partner for G:CfEcR(DEF) showed
low background levels of expression of the
GALRE-regulated luciferase reporter gene in the absence of
lig-and lig-and the luciferase activity increased after exposure
to the ligand, RG-102240 (Fig 2A) The increase in
lu-ciferase activity occurred in cells that were transfected
with the G:CfEcR(DEF) and V:HsRXR(EF)
con-structs and exposed to a 5 lm or higher concentration
of RG-102240 (Fig 2A) By contrast, the increase in
luciferase activity occurred at 200 nm or higher
con-centrations of RG-102240 in cells that were transfected
with the G:CfEcR(DEF) + V:LmRXR(EF) switch,
showing that the ligand sensitivity of the
G:CfEcR-(DEF) + V:LmRXR(EF) switch is higher than that
of the G:CfEcR(DEF) + V:HsRXR(EF) switch
Pro-teins isolated from 3T3 cells transfected with
V:LmRXR(EF), V:CfUSP(EF) or V:HsRXR(EF) were
analyzed using western blots and VP16 antibodies As
shown in Fig 2B, all three fusion proteins are
expressed in similar quantities suggesting that the
dif-ference observed in ligand sensitivity of LmRXR,
HsRXR and CfUSP switches is due to structure of
these proteins rather than due to differences in their
expression levels
When the green fluorescence protein (GFP; placed
under the control of GALRE) and G:CfEcR(DEF) +
V:CfUSP(EF), G:CfEcR(DEF) + V:HsRXR(EF) or
G:CfEcR(DEF) + V:LmRXR(EF) constructs were
transfected into 3T3 cells, the cells transfected with
G:CfEcR(DEF) + V:CfUSP(EF) switch constructs
showed GFP fluorescence in the cells treated with dimethylsulfoxide (DMSO), 1.0 or 10 lm RG-102240 (Fig 3) Low levels of GFP fluorescence were detected
in 3T3 cells transfected with G:CfEcR(DEF) + V:LmRXR(EF) constructs and exposed to DMSO However, upon exposure to 1.0 or 10 lm RG-102240, these cells showed higher GFP fluorescence (Fig 3) In contrast, the GFP activity was not observed in 3T3 cells transfected with G:CfEcR(DEF) + V:HsRX-R(EF) constructs and exposed to DMSO Upon expo-sure to 1.0 or 10 lm RG-102240, these cells showed GFP fluorescence (Fig 3) The data show that G:CfEcR(DEF) + V:CfUSP(EF) switch supports the expression of the GFP gene placed under the control
of GALRE even in the absence of ligand By contrast, G:CfEcR(DEF) + V:HsRXR(EF) and G:CfEcR-(DEF) + V:LmRXR(EF) switches induce the expres-sion of GFP placed under the control of GALRE in the presence of ligand, RG-102240 In addition, the G:CfEcR(DEF) + V:LmRXR(EF) switch is more sen-sitive to ligand than the G:CfEcR(DEF) + V:HsRXR-(EF) switch Thus, the data from these experiments confirm the results observed with the luciferase reporter
Amino acid residues present in helix 9 and in loops on either side of helix 9 of RXR are responsible for increased activity of LmRXR
As shown in Fig 2A, LmRXR performed better than HsRXR as a partner for EcR in ligand-dependent induction of reporter genes in 3T3 cells To determine
Fig 2 (A) Transactivation of reporter gene by EcR + HsRXR, EcR + LmRXR and CfEcR + CfUSP gene switches 3T3 cells were transfected with pRLUC, pFRLUC, G:CfEcR(DEF) and V:HsRXR(EF) or V:LmRXR(EF) or V:CfUSP(EF) for 4 h The transfected cells were grown in med-ium containing DMSO, 0.04, 0.2, 1 or 5 l M RG-102240 At 48 h after addition of ligand, cells were harvested and assayed for luciferase activity The fly luciferase activity was normalized using Renilla luciferase activity The values presented are mean ± SD (n ¼ 3) (B) Twenty micrograms of proteins from 3T3 cells transfected with V:LmRXR(EF), V:CfUSP(EF) or V:HsRXR(EF) constructs were separated on SDS ⁄ PAGE, transferred to nitrocellulose and analyzed using VP16 antibodies The position of 50 and 37 kDa bands form Bio-Rad Precision plus protein standards is shown on the left Arrows point to 32, 38 and 36 kDa fusion protein bands.
Trang 4which regions of LmRXR are responsible for this
improved activity, we prepared five chimeras of
LmRXR and HsRXR by sequentially replacing helix 6
with helix 12 of HsRXR with the corresponding
regions of LmRXR The chimeric RXRs, HsRXR and
LmRXR were assayed as partners for CfEcR in
ligand-dependent induction of reporter activity in 3T3
cells The luciferase reporter gene regulated by
GAL-RE (pFRLUC), G:CfEcR(DEF) and V:HsRXR(EF)
or V:LmRXR(EF) or VP6 fusion of each Hs–
LmRXR(EF) chimera shown in Fig 4A were
trans-fected into 3T3 cells and the transtrans-fected cells were
exposed to RG-102240 Luciferase activity was
measured at 48 h after addition of ligand The
G:CfEcR(DEF) + V:HsRXR(EF) switch induced
lu-ciferase activity at 1 lm or higher concentration of
RG-102240 and the G:CfEcR(DEF) +
V:LmRXR-(EF) switch induced luciferase activity at 0.2 lm or
higher concentration of RG-102240 (Fig 4A)
Repla-cing RXR with Hs–LmRXR(EF) chimera containing
helices 1–7 of HsRXR and 8–12 of LmRXR or helices
1–8 of HsRXR and 9–12 of LmRXR resulted in an
increase in ligand sensitivity of the EcR switch
Luci-ferase activity was induced with a 0.04 lm or higher
concentration of RG-102240 (Fig 4A) in the presence
of these chimeras The other three chimeras performed
similar to HsRXR Proteins isolated from 3T3 cells that were transfected with chimera constructs were analyzed using Western blots and VP16 antibodies As shown in Fig 4B, fusion proteins for all five chimeras expressed well, suggesting that the differences observed
in ligand sensitivity of gene switches containing chime-ras are due to structure of these proteins rather than
to differences in their expression levels The data sug-gest that the amino acid residues present in the region
of LmRXR that spans helices 8–9 and loops between helices 7–8, 8–9 and 9–10 are responsible for the increased activity of LmRXR
Comparison of amino acid sequences present in the ligand-binding domains of HsRXR and LmRXR showed that most of the differences in the amino acids between HsRXR and LmRXR are found in helix 9 and in the loops on either side of helix 9 To confirm the results observed in analysis of chimeras as well as
to identify the precise region of LmRXR that is responsible for the increase in its activity when com-pared with HsRXR, we performed site-directed muta-genesis on HsRXR and changed the amino acid residues of HsRXR that are different from LmRXR to the corresponding amino acid residues present in LmRXR The amino acids changed are shown in Fig 5 The performance of the mutants was compared
Fig 3 Differences in the transactivation of GFP reporter gene by EcR + HsRXR (HsRXR), EcR + LmRXR (LmRXR) and CfEcR + CfUSP (CfUSP) gene switches 3T3 cells were transfected with pFRGFP, G:CfEcR(DEF) and V:HsRXR(EF) or V:LmRXR(EF) or V:CfUSP(EF) for 4 h The transfected cells were treated with DMSO, 1 l M or 10 l M RG-102240, the cells were photographed 48 h after addition of ligand.
Trang 5with the parent RXRs in supporting EcR gene switch
activity in 3T3 cells As shown in Fig 5, the mutants
in which HsRXR amino acid residues were replaced
with LmRXR residues in helix 9 as well as in loops on
either side of helix 9 performed better than wild-type
HsRXR as partners of EcR in supporting
ligand-dependent induction of reporter activity One partic-ular mutant, in which three amino acids present in the loop between helix 8 and 9 of HsRXR were replaced with three amino acids present in the same region of LmRXR (D450E⁄ A451V ⁄ K452R), performed even better than LmRXR as a partner for EcR in
ligand-CH6 CH8 CH9 CH10 CH11
B
Fig 4 (A) 3T3 cells were transfected with
pRLUC, pFRLUC, G:CfEcR(DEF) and
V:HsRXR(EF) or V:LmRXR(EF) or VP6 fusion
of one the Hs–LmRXR(EF) chimeras
Trans-fected cells were exposed to DMSO, 0.04,
0.2, 1 or 5 l M RG-102240 for 48 h The cells
were harvested and assayed for luciferase
activity The fly luciferase activity was
nor-malized using Renilla luciferase activity The
values presented are mean ± SD (n ¼ 3).
(B) Twenty micrograms of proteins isolated
from 3T3 cells transfected with V:Hs–
LmRXR(EF) chimera constructs were
separ-ated on SDS ⁄ PAGE, transferred to
nitrocel-lose and analyzed using VP16 antibodies.
Arrow points to fusion protein bands.
Fig 5 Sequence of chimeras between HsRXR and LmRXR The amino acids that are from HsRXR are shown with a pink background The amino acids from LmRXR are shown with a green background The amino acids that were mutated are shown with a yellow background.
Trang 6dependent induction of reporter activity (Fig 6A).
However, the performance of this mutant is not as
good as Hs–LmRXR (EF) chimera 9 (Fig 4A)
sug-gesting that not only these three amino acids but also
other amino acids that are different between HsRXR
and LmRXR in helix 9 as well as in the loops on
either side of helix 9 contribute to the improved
activ-ity of LmRXR Western blot analysis of proteins
iso-lated from 3T3 cells that were transfected with RXR
mutant constructs showed that the fusion proteins for
all nine mutants expressed well suggesting that the
dif-ferences observed in ligand sensitivity of gene switches
containing chimeras are due to structure of these
pro-teins rather than due to differences in their expression
levels (Fig 6B)
To confirm that the amino acid residues present in
helix 9, as well as in the loops on either side of helix 9,
are responsible for improved performance of LmRXR,
we produced a chimera in which the region of
LmRXR containing helix 9 and the two loops on either side of helix 9 were replaced with the corres-ponding region present in HsRXR The performance
of this chimera and two parent RXRs, LmRXR and HsRXR was evaluated as partners of EcR in ligand-dependent induction of reporter activity in 3T3 cells
As shown in Fig 7, The G:CfEcR(DEF) + V:Lm-HsRXR(EF) chimera switch induced the luciferase activity with 1 lm or higher concentration of
RG-102240 This is similar to the ligand sensitivity of the G:CfEcR(DEF) + V:HsRXR(EF) switch, but lower than that of the G:CfEcR(DEF) + V:LmRXR(EF) switch in which the luciferase activity was induced with 0.2 lm or higher concentration of RG-102240 (Fig 7) These data confirmed the results that the region of LmRXR containing helix 9 and the two loops on either side of helix 9 is responsible for improved per-formance of LmRXR as a partner for EcR in ligand-dependent induction of reporter activity
A
B
Fig 6 3T3 cells were transfected with pRLUC, pFRLUC, G:CfEcR(DEF) and V:HsRXR(EF) or V:LmRXR(EF) or mutants of HsRXR (A) Trans-fected cells were exposed to DMSO, 0.04, 0.2, 1 or 5 l M RG-102240 for 48 h The cells were harvested and assayed for luciferase activity The fly luciferase activity was normalized using Renilla luciferase activity The values presented are mean ± SD (n ¼ 3) (B) Twenty micro-grams of proteins isolated from 3T3 cells transfected with V:HsRXR(EF) mutant constructs were separated on SDS ⁄ PAGE, transferred to nitrocellulose and analyzed using VP16 antibodies The arrow points to fusion protein bands Mutant 1, D450E ⁄ A451V ⁄ K452R; mutant 2, S455K ⁄ N456S ⁄ P457A ⁄ S458Q; mutant 3, V462L; mutant 4, S470A; mutant 5, T473E; mutant 6, C475T ⁄ K476R ⁄ Q477T ⁄ K478T ⁄ Y475H; mutant 7, E481D ⁄ Q482E ⁄ 483P; mutant 8, A495S; mutant 9, A528S.
Trang 7To determine whether the region of LmRXR
(helix 9 and two loops either side of it) that improved
EcR performance would also affect RXR
perform-ance mediated through 9-cis-retinoic acid, we
com-pared the performance of HsRXR, LmRXR and the
two chimeras, Hs–LmRXR (helix 1–8 of HsRXR and
9–12 of LmRXR) and Lm-HsRXR (helix 9 and two
loops on either side of helix 9 of LmRXR were
replaced with the corresponding regions form
HsRXR) in transactivation assays As shown in
Fig 8, 9-cis-retinoic acid induced reporter genes
through G:CfEcR(DEF) + V:HsRXR(EF) switch at
0.2 lm or higher concentration of ligand However,
25 lm concentration of 9-cis-retinoic acid was needed
to induced reporter gene via the G:CfEcR(DEF)
+ V:LmRXR(EF) switch The two chimeras
per-formed similar to parents in this assay The
G:CfEcR(DEF) + V:Hs–LmRXR(EF) chimera switch
supported reporter gene induction at 0.2 lm or higher
concentration of 9-cis-retinoic acid and the
G:CfEcR-(DEF) + V:Lm-HsRXR(EF) chimera switch
suppor-ted reporter induction at 25 lm concentration of
9-cis-retinoic acid The data suggest that the chimeras
prepared by swapping helix 9 and the two loops on
either side of helix 9 do not affect 9-cis-retioic acid
activity through RXR
To determine whether the influence of USP⁄ RXR
on the EcR function is mediated at the level of
heterodimerization between EcR and USP⁄ RXR, we
performed pull-down assays Bacterially expressed fusion protein of GST and CfEcR(DEF) was used
to pull down in vitro translated HsRXR(EF), LmRXR(EF), CfUSP(EF) and Hs–LmRXR(EF) chi-mera in the absence and presence of 1 lm
RG-102240 There was no difference in the amount of CfUSP(EF) pulled down by EcR in the presence of DMSO or 1 lm RG-102240, suggesting that EcR and USP can heterodimerize in the absence of ligand (Fig 9) In contrast, the amount of HsRXR,
Fig 7 Comparison of two parent RXRs and Lm-HsRXR(EF)
chi-mera in transactivation assays 3T3 cells were transfected with
pRLUC, pFRLUC, G:CfEcR(DEF) and V:HsRXR(EF) or V:LmRXR(EF)
or VP6 fusion of Lm–HsRXR(EF) chimera (LmRXR helix 9 and loops
on either side of it were replaced with the corresponding region of
HsRXR) The transfected cells were exposed to DMSO, 0.04, 0.2,
1 or 5 l M RG-10240 for 48 h The cells were harvested and
assayed for the luciferase activity The fly luciferase activity was
normalized using Renilla luciferase activity The values presented
are mean ± SD (n ¼ 3).
Fig 8 Comparison of two parent RXRs, Lm-HsRXR(EF) and Hs–LmRXR(EF) chimeras in 9-cis-retinoic acid induced transactiva-tion assays 3T3 cells were transfected with pRLUC, pFRLUC, G:CfEcR(DEF) and V:HsRXR(EF) or V:LmRXR(EF) or VP6 fusion of Lm-HsRXR(EF) chimera (LmRXR helix 9 and loops on either side of
it were replaced with the corresponding region of HsRXR) or Hs–LmRXR(EF) chimera 9 The transfected cells were exposed to DMSO, 0.04, 0.2, 1, 5 or 25 l M 9-cis-retinoic acid for 48 h The cells were harvested and assayed for the luciferase activity The fly luciferase activity was normalized using Renilla luciferase activity The values presented are mean ± SD (n ¼ 3) Asterisks on top of the bars indicate significant difference from DMSO-treated cells at
P < 0.5 determined by t-test.
Fig 9 GST:CfEcRDEF and [ 35 S]-methionine labeled Hs–LmRXR chi-mera (C) or HsRXREF (HsR) or LmRXREF (LmR) or CfUSP (CfU) were incubated in binding buffer containing DMSO or one lM RG-102240 and the complexes were precipitated with glutathione agarose beads The pellet was washed and resolved on SDS ⁄ PAGE and the gel was dried and exposed to X-ray film.
Trang 8LmRXR and Hs–LmRXR chimera pulled down by
EcR increased after addition of 1 lm RG-102240
The increase in amount of RXR pulled down by
EcR in the presence of ligand was maximum in the
case of HsRXR and minimum in the case of the
Hs–LmRXR chimera, and LmRXR was between
these two (Fig 9) These data suggest that the EcR
and USP can heterodimerize in the absence of
lig-and In contrast, EcR:RXR heterodimer stability is
increased by the presence of ligand
The EcR:Hs–LmRXR chimera switch is ligand sensitive and functions in mice
Based on the data, we selected a RXR chimera that contains helices 1–8 from HsRXR and 9–12 from LmRXR and evaluated its performance as a partner
of EcR in gene switch applications The EcR:Hs– LmRXR chimera switch initiated the induction of the luciferase reporter activity beginning at 0.04 lm RG-102240 and the luciferase activity reached peak levels in the presence of 1 lm RG-102240 (Fig 10A) This is a significant improvement in ligand sensitivity when compared with the EcR:HsRXR switch that requires 1 lm RG-102240 to initiate induction of the luciferase reporter gene and the reporter activity
rea-Fig 10 Dose-dependent induction of reporter gene by gene
switch receptors (A) 3T3 cells were transfected with G:Cf(DEF),
V:Hs–LmRXR(EF), pFRLUC and pRLUC The transfected cells were
grown in the medium containing 0, 0.04, 0.2, 1 or 5 l M
concentra-tion of RG-102240 The cells were collected at 48 h after adding
ligand and reporter activity was quantified The fly luciferase activity
was normalized using Renilla luciferase activity The values
presen-ted are mean ± SD (n ¼ 3) (B) 3T3 cells were transfected with
G:CfEcR(DEF) + V:HsRXR(EF) gene switch 3T3 cells were
trans-fected with G:Cf(DEF), V:HsRXR(EF), pFRLUC and pRLUC The
transfected cells were grown in the medium containing 0, 0.2, 1, 5
and 25 l M concentration of RG-102240 The cells were collected at
48 h after adding ligand and reporter activity was quantified The fly
luciferase activity was normalized using Renilla luciferase activity.
The values presented are mean ± SD (n ¼ 3) Fig 11 (A) Time course of induction of reporter gene by gene
switch plasmids (A) 3T3 cells were transfected with G:Cf(DEF), V:Hs–LmRXR(EF), pFRLUC and pRLUC The transfected cells were grown in the medium containing 1 l M concentration of RG-102240 The cells were collected at 0, 1, 3, 6, 12, 24, 48 and 72 h after add-ing ligand and reporter activity was quantified The fly luciferase activity was normalized using Renilla luciferase activity The values presented are mean ± SD (n ¼ 3) (B) 3T3 cells were transfected with G:Cf(DEF), V:Hs–LmRXR(EF), pFRLUC and pRLUC The trans-fected cells were grown in the medium containing 1 l M concentra-tion of RG-102240 At 48 h after addiconcentra-tion of ligand, the cells were washed with fresh medium and maintained in the fresh medium The cells were collected at 0, 1, 3, 6, 12, 24, 48 and 72 h after transfer to the fresh medium and the luciferase activity was quanti-fied The fly luciferase activity was normalized using Renilla luci-ferase activity The values presented are mean ± SD (n ¼ 3).
Trang 9ches peak levels in the presence of 25 lm RG-102240
(Fig 10B) The reporter gene regulated by the
EcR:Hs–LmRXR chimera switch was induced
begin-ning at 1 h after addition of ligand and reached peak
levels of 19 000-fold induction by 48 h after addition
of ligand (Fig 11A) The turn off of reporter activity
after withdrawal of ligand is also fast More than 50%
of reporter activity was reduced by 12 h after
with-drawal of ligand and by 24 h after withwith-drawal of
lig-and, most of the reporter activity disappeared
(Fig 11B)
To evaluate the performance of the G:CfEcR(DEF):
V:Hs–LmRXR(EF) switch in vivo in mice, reporter
(secreted alkaline phosphatase regulated by GALRE),
G:CfEcR(DEF) and V:Hs–LmRXR(EF) chimera or
V:HsRXR(EF) plasmids were electroporated into the
quadriceps of C57BL⁄ 6 mice The animals were
treated with 5 mg RG-102240⁄ 50 lL DMSO⁄
mouse by intraperitoneal injection at three days after
electroporation of plasmids Secreted alkaline
phos-phate (SEAP) in mouse sera was evaluated at various
time points after ligand administration The
G:CfEcR-(DEF) + V:Hs–LmRXR(EF) chimera switch induced
SEAP activity that reached peak levels at five days
after the administration of ligand (Fig 12) By
con-trast, the G:CfEcR(DEF) + V:HsRXR(EF) switch did
not cause induction of SEAP activity up to 25 days
after the administration of ligand (Fig 12) Thus, the
G:CfEcR(DEF) + V:Hs–LmRXR(EF) chimera switch
but not G:CfEcR(DEF) + V:HsRXR(EF) switch is
ments and regulates the expression of ecdysteroid responsive genes Because ecdysteroids and their lig-ands are absent in vertebrates, including humans, they are being developed to regulate transgenes in various applications including gene therapy, functional genom-ics, drug discovery, and biopharmaceutical production [24] In mammalian cells, the CfEcR and CfUSP het-erodimer induces the expression of reporter genes regu-lated by EcRE even in the absence of ligand; therefore, they are not useful for gene switch applications Ver-tebrate RXR such as human or mouse RXR have been used in the place of USP in all EcR gene switches developed to date One of the major limitations of cur-rent versions of EcR gene switches is the requirement
of micromolar concentrations of ligand for induction
of gene expression In phylogenetic analysis, Locusta migratoria RXR (LmRXR) falls into vertebrate RXR but not insect USP group (Fig 1) We hypothesized that unlike the CfEcR:CfUSP heterodimer, the CfEcR:LmRXR heterodimer does not induce reporter genes regulated by EcRE in the absence of ligand Furthermore, compared with the CfEcR:HsRXR het-erodimer, CfEcR:LmRXR heterodimers may induce reporter genes regulated by response elements at lower concentrations of ligand We tested these hypotheses
by comparing the performance of CfUSP, LmRXR and HsRXR as partners for CfEcR in induction of reporter genes regulated by GALRE in 3T3 cells and found that both hypotheses are true Pull-down experi-ments showed that the CfUSP heterodimerizes with the CfEcR in the absence of ligand (Fig 9), there-fore, CfEcR:CfUSP heterodimers can induce gene expression in the absence of ligand However, CfEcR:HsRXR heterodimers are at very low levels in the absence of ligand and upon addition of ligand, increased quantities of HsRXRs were pulled down by CfEcR suggesting that CfEcR:HsRXR heterodimers increase in the presence of ligand and induce gene expression
We created chimeric receptors comprised of regions from HsRXR and LmRXR and mutants of HsRXR, and evaluated their performance as partners of CfEcR
in ligand-dependent induction of gene expression These analyses showed that the amino acids present in helix 9 and in the two loops present on either side of helix 9 are responsible for improved performance of
Fig 12 In vivo comparison of human RXRb and
Hs-RXRb–Lm-RXRb fusion protein in a C57BL ⁄ 6 mouse model The gene
swit-ches, composed of plasmids containing pCMV⁄ GAL4-pCfEcR(DEF),
pCMV ⁄ VP16-Hs-RXR(EF) (s) or pCMV ⁄
VP16-HsRXRb(H1-8)-LmRXRb(H9-12) fusion (m), and 6xGAL4RE-TTR-SEAP, were
elec-troporated into the quadriceps of C57BL ⁄ 6 mice Animals were
treated with 5 mg RG-102240 ⁄ 50 lL DMSO ⁄ mouse by IP injection
three days after electroporation of plasmid SEAP in mouse sera
was evaluated for up to 17 days after ligand administration Values
are the average from seven animals ± SD.
Trang 10LmRXR when compared with the performance of
HsRXR as a partner for CfEcR Structural studies on
EcR:USP and RAR:RXR heterodimers showed that
helices 7 and 10, present in two nuclear receptors,
form heterodimerization interfaces and play critical
roles in heterodimerization [25] The data presented
here showed that besides helices 7 and 10, amino acid
residues present in helix 9 and in the two loops present
on either side of helix 9 play critical roles in
hetero-dimerization of EcR:USP⁄ RXR In fact, there is only
one amino acid each in helix 7 and helix 10 that is
dif-ferent between HsRXR and LmRXR Replacing these
two amino acids in HsRXR with the corresponding
amino acids present in LmRXR did not increase the
performance of HsRXR as a partner for CfEcR In
contrast, replacing HsRXR amino acids present in
helix 9 and in the loops on either side of helix 9 with
the amino acids present in corresponding positions in
LmRXR resulted in an increase in the performance of
HsRXR as a partner for CfEcR Particularly,
repla-cing three amino acids (DAK) located in the loop
between helices 8 and 9 of HsRXR with the amino
acids (EVR) present in the corresponding positions of
LmRXR resulted in a HsRXR mutant that performed
even better than LmRXR as CfEcR partner
Examina-tion of structures of EcR and RXR indicated that,
when compared with the aspartic acid (D) residue
pre-sent in the loop between helices 8 and 9 of HsRXR,
the glutamic acid (E) residue present in the
corres-ponding region of LmRXR is located at a more
favo-rable distance to interact with the arginine (R) residue
present in helix 7 of EcR [25] Taken together, these
studies conclusively show that the amino acid residues
present in helix 9 and in the two loops on either side
of helix 9 contribute to the heterodimerization of EcR
and RXR
The diacylhydrazine nonsteroidal ligands of EcR
are not highly polar, therefore; higher concentrations
of these ligands or highly sensitive gene switches are
required for in vivo applications Because the
CfEcR:HsRXR switch requires micromolar
concen-trations of ligand for the transactivation of genes, it
does not function very well for in vivo applications
However, the CfEcR:Hs–LmRXR chimera switch
requires only nanomolar concentrations of ligands
for the transactivation of genes and functions well
in vivo The RXR chimeras containing most of
HsRXR and helix 9 and the loops on either side of
helix 9 from HsRXR or mutants of HsRXR with a
change in just three amino acids (D450E⁄
A451V⁄ K452R) will definitely help in the
develop-ment of gene switches, especially those that require
in vivo applications
Experimental procedures
Constructs
The construction of GAL4:CfEcR(DEF) [G:CfEcR(DEF)], VP16:MmRXR(EF) [V:MmRXR(EF)] and VP16:CfUS-P(EF) [V:CfUSVP16:CfUS-P(EF) has been described previously [23] VP16:LmRXR(EF) [V:LmRXR(EF)] was constructed by amplifying EF domains of LmRXR using primers contain-ing EcoRI and BamHI sites in the forward and reverse pri-mer, respectively, followed by cloning of the PCR product into EcoRI and BamHI digested pVP16 vector (Clontech Inc Palo Alto, CA) pFRLUC reporter plasmid was pur-chased from Stratagene (La Jolla, CA) pRLUC is reporter plasmid expressing Renilla luciferase under the control of thymidine kinase promoter (Promega, Madison, WI) The GST fusion construct of MmR(EF) was made by cloning MmR(EF) domain into pGEX-5X-1 vector (Amersham Pharmacia Biotech, Piscataway, NJ) forward and reverse primers, respectively
Ligands
RG-102240 [N-(1,1-dimethylethyl)-N¢-(2-ethyl-3-methoxy-benzoyl)-3,5-dimethylbenzohydrazide] also known as
GSTM-E and RheoSwitch ligand 1 (RSL1) is a synthetic stable diacylhydrazine ecdysone agonist synthesized by RheoGene Inc.; 9-cis-retinoic acid was purchased from
Sig-ma Chemical Co (St Louis, MO, USA) The ligands were applied in DMSO and the final concentration of DMSO was maintained at 0.1% in both controls and treatments
Cells and transfections and reporter assays
3T3 cells were grown to 60% confluency Fifty thousand cells were plated per well of 12-well plates The next day, cells were transfected with 0.25 lg of receptor(s) and 1.0 lg of reporter constructs using 4 lL of SuperFect (Qiagen Inc., Valencia, CA) A second reporter, Renilla luciferase, expressed under a thymidine kinase constitutive promoter was cotransfected into cells and used for normal-ization After transfection, cells were grown in a medium containing ligands for 24–48 h The cells were harvested, lyzed and the reporter activity was measured in an aliquot
of lysate All transfection experiments were performed in triplicate and the experiments were repeated at least three times Luciferase and Renilla luciferase activities were measured using the Dual-luciferaseTM reporter assay sys-tem (Promega)
Construction of chimeras and mutants
Site-directed mutagenesis was carried out using the Quikchange site directed mutagenesis kit (Stratagene) Mutations were verified by sequencing RXR chimeras