共查询到20条相似文献,搜索用时 31 毫秒
1.
Chemical inducible gene regulation systems provide essential tools for the precise regulation of transgene expression in plants
and animals. Recent development of a two-hybrid ecdysone receptor (EcR) gene regulation system has solved some of the drawbacks
that were associated with the monopartate gene switch. To further improve the versatility of the two-hybrid EcR gene switch
for wide spread use in plants, chimeras between Homo sapiens retinoid X receptor (HsRXR) and insect, Locusta migratoria RXR (LmRXR) were tested in tobacco protoplasts as partners with Choristoneura fumiferana EcR (CfEcR) in inducing expression of the luciferase reporter gene. The RXR chimera 9 (CH9) along with CfEcR, in a two-hybrid
format gave the best results in terms of low-background expression levels in the absence of ligand and high-induced expression
levels of the reporter gene in the presence of nanomolar concentrations of the methoxyfenozide ligand. The performance of
CH9 was further tested in corn and soybean protoplasts and the data obtained was compared with the other EcR switches that
contained the wild-type LmRXR or HsRXR as EcR partners. In both transient expression studies and stable transformation experiments,
the fold induction values obtained with the CH9 switch were several times higher than the values obtained with the other EcR
switches containing LmRXR or HsRXR. The new CfEcR two-hybrid gene switch that uses the RXR CH9 as a partner in inducing reporter
gene expression provides an efficient, ligand-sensitive and tightly regulated gene switch for plants. 相似文献
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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 expression 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 activation. The heterodimer partner of EcR can influence ligand sensitivity of EcR so that the EcR/Locusta migratoria RXR (EcR:LmRXR) heterodimers are activated at lower concentrations of ligand when compared with the concentrations of ligand required for the activation of EcR/Homo sapiens RXR (EcR:HsRXR) heterodimers. Analysis of chimeric RXRs containing 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 concentration of ligand required for activating the EcR:HsRXR heterodimer. The EcR:Lm-HsRXR chimera heterodimer, but not the EcR:HsRXR heterodimer, supported ligand-dependent induction of reporter gene in a C57BL/6 mouse model. 相似文献
3.
Tavva VS Dinkins RD Palli SR Collins GB 《The Plant journal : for cell and molecular biology》2006,45(3):457-469
The ecdysone receptor (EcR) has been used to develop gene switches for conditional regulation of transgene expression in plants and humans. All EcR-based gene switches developed to date for use in plants are monopartate and require micromolar concentrations of ligand for activation of the transgene; this has limited the use of these gene switches. We have developed a Choristoneura fumiferana ecdysone receptor (CfEcR)-based two-hybrid gene switch that works through the formation of a functional heterodimer between EcR and the retinoid X receptor (RXR) upon application of the chemical ligand methoxyfenozide. Methoxyfenozide is already registered for field use with an excellent safety profile, and it has potential as a gene switch ligand for applications in the field. The receptor constructs were prepared by fusing DEF domains (hinge region plus ligand-binding domain) of CfEcR to the GAL4 DNA-binding domain and EF domains (ligand-binding domain) of ultraspiracle from Choristoneura fumiferana (CfUSP) or RXR from Locusta migratoria (LmRXR), Mus musculus (MmRXR) or Homo sapiens (HsRXR) to the VP16 activation domain. These receptor constructs were tested for their ability to induce expression of the luciferase gene placed under the control of 5x GAL4 response elements and -46 35S minimal promoter in tobacco, corn and soybean protoplasts and in transgenic Arabidopsis and tobacco plants. By adopting the two-hybrid format, the sensitivity of the CfEcR gene switch has been improved from micromolar to nanomolar concentrations of methoxyfenozide. The sensitivity of the CfEcR + LmRXR two-hybrid switch was 25 to 625 times greater than the monopartate gene switch, depending on the plant species tested. 相似文献
4.
In plants, regulation of transgene expression is typically accomplished through the use of inducible promoter systems. The ecdysone receptor (EcR) gene switch is one of the best inducible systems available to regulate transgene expression in plants. However, the monopartite EcR gene switches developed to date require micromolar concentrations of ligand for activation. We tested several EcR mutants that were generated by changing one or two amino acid residues in the highly flexible ligand-binding domain of Choristoneura fumiferana EcR (CfEcR). Based on the transient expression assays, we selected a double mutant, V395I + Y415E (VY), of CfEcR (CfEcR(VY)) for further testing in stable transformation experiments. The CfEcR(VY) mutant only slightly improved the induction characteristics of the two-hybrid gene switch, whereas the CfEcR(VY) mutant significantly improved the induction characteristics of the monopartite gene switch (VGCfE(VY)). The ligand sensitivity of the VGCfE(VY) switch was improved by 125-15 625-fold in different transgenic lines analyzed, compared to the VGCfE(Wt) switch. The utility of the VGCfE(VY) switch was tested by regulating the expression of an Arabidopsis zinc finger protein gene (AtZFP11) in both tobacco and Arabidopsis plants. These data showed that the VGCfE(VY) switch efficiently regulated the expression of AtZFP11 and that the phenotype of AtZFP11 could be induced by the application of ligand. In addition, the affected plants recovered after withdrawal of the ligand, demonstrating the utility of this gene switch in regulating the expression of critical transgenes in plants. 相似文献
5.
Tavva VS Palli SR Dinkins RD Collins GB 《Archives of insect biochemistry and physiology》2007,65(3):164-179
Genetic engineering of plants using transgenic technology is targeted to enhance agronomic performance or improved quality traits in a wide variety of plant species, and has become a fundamental tool for basic research in plant biotechnology. Constitutive promoters are presently the primary means used to express transgenes in plants. However, inducible gene regulation systems based on specific chemicals have many potential applications in agriculture and for enhancing the basic understanding of gene function. As a result, several gene switches have been developed. The ecdysone receptor gene switch is one of the best inducible gene regulation systems available, because the chemical, methoxyfenozide, required for its regulation is registered for field use. An EcR gene switch with a potential for use in large-scale field applications has been developed by adopting a two-hybrid format. In a two-hybrid switch format, the GAL4 DNA binding domain (GAL4 DBD) was fused to the ligand binding domain (LBD) of the Choristoneura fumiferana ecdysone receptor (CfEcR); and, the VP16 activation domain (VP16 AD) was fused to the LBD of Locust migratoria retinoid X receptor (LmRXR). The sensitivity of the CfEcR gene switch was improved from micromolar to nanomolar concentrations of ligand by using the CfEcR:LmRXR two-hybrid switch. In this report, we demonstrate the utility of CfEcR:LmRXR two-hybrid gene switch in functional genomics applications for regulating the expression of a Superman-like single zinc finger protein 11 (ZFP11) gene in both Arabidopsis and tobacco transgenic plants. 相似文献
6.
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). 相似文献
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Kumar MB Potter DW Hormann RE Edwards A Tice CM Smith HC Dipietro MA Polley M Lawless M Wolohan PR Kethidi DR Palli SR 《The Journal of biological chemistry》2004,279(26):27211-27218
The insect steroid hormone 20-hydroxyecdysone works through a ligand-activated nuclear receptor, the ecdysone receptor (EcR), which plays critical roles in insect development and reproduction. The EcR has been exploited to develop insecticides to control pests and gene switches for gene regulation. Recently reported crystal structures of the EcR protein show different but partially overlapping binding cavities for ecdysteroid (ECD) and diacylhydrazine (DAH) ligands, providing an explanation for the differential activity of DAH ligands in insects. 1-Aroyl-4-(arylamino)-1,2,3,4-tetrahydroquinoline (THQ) ligands were recently discovered as ecdysone agonists. Mutagenesis of the EcR (from Choristoneura fumiferana, CfEcR) ligand binding domain followed by screening in a reporter assay led to the identification of CfEcR mutants, which responded well to THQ ligands but poorly to both ECD and DAH ligands. These mutants were further improved by introducing a second mutation, A110P, which was previously reported to cause ECD insensitivity. Testing of these V128F/A110P and V128Y/A110P mutants in a C57BL/6 mouse model coactivator interaction assay and in insect cells showed that this mutant EcR is activated by THQ ligands but not by ECD or DAH ligands. The CfEcR and its V128F/A110P mutant were used to demonstrate simultaneous regulation of two reporter genes using THQ and DAH ligands. 相似文献
9.
Nieva C Spindler-Barth M Spindler KD 《Archives of insect biochemistry and physiology》2008,68(1):40-48
Initially, nuclear import of the ecdysteroid receptor (EcR) in vertebrate cells (CHO-K1 and COS-7) does not afford a heterodimerization partner. Later on, EcR is retained in the nucleus only in the presence of a heterodimerization partner. Ultraspiracle (Usp) is more efficient compared to its vertebrate orthologue RXR and leads to an exclusively nuclear localization of EcR even in the absence of ligand. The DNA binding domain of the heterodimerization partner is important for retainment of EcR in the nucleus as shown by Usp4 (Usp(R130C)), which has lost its DNA binding capability. The C-terminal end of Usp (Usp(Delta205-508)) encompassing the C-terminal part of the D-domain and the E- and F-domains are essential for retainment of EcR in the nucleus. Nuclear localization is further influenced by cell-specific factors, since hormone and heterodimerization stabilizes the EcR protein in a cell-specific way. 相似文献
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11.
Drosophila ultraspiracle modulates ecdysone receptor function via heterodimer formation. 总被引:29,自引:0,他引:29
The vertebrate retinoid X receptor (RXR) has been implicated in the regulation of multiple hormonal signaling pathways through the formation of heteromeric receptor complexes that bind DNA with high affinity. We now demonstrate that ultraspiracle (usp), a Drosophila RXR homolog, can substitute for RXR in stimulating the DNA binding of receptors for retinoic acid, T3, vitamin D, and peroxisome proliferator activators. These observations led to the search and ultimate identification of the ecdysone receptor (EcR) as a Drosophila partner of usp. Together, usp and EcR bind DNA in a highly cooperative fashion. Cotransfection of both EcR and usp expression vectors is required to render cultured mammalian cells ecdysone responsive. These results implicate usp as an integral component of the functional EcR. By demonstrating that receptor heterodimer formation precedes the divergence of vertebrate and invertebrate lineages, these data underscore a central role for RXR and its homolog usp in the evolution and control of the nuclear receptor-based endocrine system. 相似文献
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Subba R Palli Mariana Z Kapitskaya Mohan B Kumar Dean E Cress 《European journal of biochemistry》2003,270(6):1308-1315
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 musculus retinoid X receptor (MmRXR) to either GAL4 DNA binding domain (DBD) or VP16 activation domain. These constructs were tested in mammalian cells to evaluate their ability to transactivate luciferase gene placed under the control of GAL4 response elements and synthetic TATAA promoter. A two-hybrid format switch, where GAL4 DBD was fused to CfEcR (DEF) and VP16 AD was fused to MmRXR (EF) was found to be the best combination. It had the lowest background levels of reporter gene activity in the absence of a ligand and the highest level of reporter gene activity in the presence of a ligand. Both induction and turn-off responses were fast. A 16-fold induction was observed within 3 h of ligand addition and increased to 8942-fold by 48 h after the addition of ligand. Withdrawal of the ligand resulted in 50% and 80% reduction in reporter gene activity by 12 h and 24 h, respectively. 相似文献
15.
Structural and functional characterization of a novel type of ligand-independent RXR-USP receptor 总被引:3,自引:0,他引:3
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Iwema T Billas IM Beck Y Bonneton F Nierengarten H Chaumot A Richards G Laudet V Moras D 《The EMBO journal》2007,26(16):3770-3782
Retinoid X receptor (RXR) and Ultraspiracle (USP) play a central role as ubiquitous heterodimerization partners of many nuclear receptors. While it has long been accepted that a wide range of ligands can activate vertebrate/mollusc RXRs, the existence and necessity of specific endogenous ligands activating RXR-USP in vivo is still matter of intense debate. Here we report the existence of a novel type of RXR-USP with a ligand-independent functional conformation. Our studies involved Tribolium USP (TcUSP) as representative of most arthropod RXR-USPs, with high sequence homology to vertebrate/mollusc RXRs. The crystal structure of the ligand-binding domain of TcUSP was solved in the context of the functional heterodimer with the ecdysone receptor (EcR). While EcR exhibits a canonical ligand-bound conformation, USP adopts an original apo structure. Our functional data demonstrate that TcUSP is a constitutively silent partner of EcR, and that none of the RXR ligands can bind and activate TcUSP. These findings together with a phylogenetic analysis suggest that RXR-USPs have undergone remarkable functional shifts during evolution and give insight into receptor-ligand binding evolution and dynamics. 相似文献
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Many members of the type II nuclear receptor subfamily function as heterodimers with the retinoid X receptor (RXR). A permissive heterodimer (e.g. peroxisome proliferator-activated receptor/RXR) allows for ligand binding by both partners of the receptor complex. In contrast, RXR has been thought to be incapable of ligand binding in a nonpermissive heterodimer, such as that of thyroid hormone receptor (TR)/RXR, where it has been referred to as a silent partner. However, we recently presented functional evidence suggesting that RXR in the TR/RXR heterodimer can bind its natural ligand 9-cis-RA in cells. Here we extended our study of the interrelationship of TR and RXR. We examined the potential modulatory effect of RXR and its ligand on the activity of TR, primarily using a Gal4-TR chimera. This study led to several novel and unexpected findings: 1) heterodimerization of apo-RXRalpha (in the absence of 9-cis-RA) with Gal4-TR inhibits T3-mediated transactivation; 2) the inhibition of Gal4-TR activity by RXRalpha is further enhanced by 9-cis-RA; 3) two different RXR subtypes (alpha and beta) differentially modulate the activity of Gal4-TR; 4) the N-terminal A/B domains of RXR alpha and beta are largely responsible for their differential modulation of TR activity; and 5) the RXR ligand 9-cis-RA appears to differentially affect T3-mediated transactivation from the Gal4-TR/RXRalpha (which is inhibited by 9-cis-RA) and TRE-bound TR/RXRalpha (which is further activated by 9-cis-RA) heterodimers. Taken together, these results further support our recent proposal that the RXR component in a TR/RXR heterodimer is not silent and, more importantly, reveal novel aspects of regulation of the activity of the TR/RXR heterodimer by RXR and RXR ligand. 相似文献
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Bielska K Seliga J Wieczorek E Kędracka-Krok S Niedenthal R Ożyhar A 《The Journal of steroid biochemistry and molecular biology》2012,132(3-5):227-238
The ultraspiracle protein (Usp), together with an ecdysone receptor (EcR) forms a heterodimeric ecdysteroid receptor complex, which controls metamorphosis in Drosophila melanogaster. Although the ecdysteroid receptor is considered to be a source of elements for ecdysteroid inducible gene switches in mammals, nothing is known about posttranslational modifications of the receptor constituents in mammalian cells. Up until now there has been no study about Usp sumoylation. Using Ubc9 fusion-directed sumoylation system, we identified Usp as a new target of SUMO1 and SUMO3 modification. Mutagenesis studies on the fragments of Usp indicated that sumoylation can occur alternatively on several defined Lys residues, i.e. three (Lys16, Lys20, Lys37) in A/B region, one (Lys424) in E region and one (Lys506) in F region. However, sumoylation of one Lys residue within A/B region prevents modification of other residues in this region. This was also observed for Lys residues in carboxyl-terminal fragment of Usp, i.e. comprising E and F regions. Mass spectrometry analysis of the full-length Usp indicated that the main SUMO attachment site is at Lys20. EcR, the heterodimerization partner of Usp, and muristerone A, the EcR ligand, do not influence sumoylation patterns of Usp. Another heterodimerization partner of Usp - HR38 fused with Ubc9 interacts with Usp in HEK293 cells and allows sumoylation of Usp independent of the direct fusion to Ubc9. Taken together, we propose that sumoylation of DmUsp can be an important factor in modulating its activity by changing molecular interactions. 相似文献