The Experts below are selected from a list of 2712 Experts worldwide ranked by ideXlab platform
Subba Reddy Palli - One of the best experts on this subject based on the ideXlab platform.
-
Effect of Ecdysone Receptor gene switch ligands on endogenous gene expression in 293 cells.
FEBS Journal, 2007Co-Authors: Siva K. Panguluri, Robert E. Hormann, Subba Reddy PalliAbstract:Regulated gene expression may substantially enhance gene therapy. Correlated with structural differences between insect ecdysteroids and mammalian steroids, the ecdysteroids appear to have a benign pharmacology without adversely interfering with mammalian signaling systems. Consequently, the Ecdysone Receptor-based gene switches are attractive for application in medicine. In the present study, the effect of inducers of Ecdysone Receptor switches on the expression of endogenous genes in HEK 293 cells was determined. Four ligand chemotypes, represented by a tetrahydroquinoline (RG-120499), one amidoketone (RG-121150), two ecdysteroids [20-hydroxyEcdysone (20E) and ponasterone A (Pon A)], and four diacylhydrazines (RG-102240, RG-102277, RG-102398 and RG-100864), were tested in HEK 293 cells. The cells were exposed to ligands at concentrations of 1 µm (RG-120499) or 10 µm (all others) for 72 h and the total RNA was isolated and analyzed using microarrays. Microarray data showed that the tetrahydroquinoline ligand, RG-120499 caused cell death at concentrations ≥ 10 µm. At 1 µm, this ligand caused changes in the expression of genes such as TNF, MAF, Rab and Reprimo. At 10 µm, the amidoketone, RG-121150, induced changes in the expression of genes such as v-jun, FBJ and EGR, but was otherwise noninterfering. Of the two steroids tested, 20E did not affect gene expression, but Pon A caused some changes in the expression of endogenous genes. At lower concentrations pharmacologically relevant for gene therapy, intrinsic gene expression effects of ecdysteroids and amidoketones may actually be insignificant. A fortiori, even at 10 µm, the four diacylhydrazine ligands did not cause significant changes in expression of endogenous genes in 293 cells and therefore should have minimum pleiotropic effects when used as ligands for the Ecdysone Receptor gene switch.
-
The influence of heterodimer partner ultraspiracle/retinoid X Receptor on the function of Ecdysone Receptor
FEBS Journal, 2005Co-Authors: Subba Reddy Palli, Mariana Z. Kapitskaya, David W. PotterAbstract: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.
-
Heterodimerization of Ecdysone Receptor and ultraspiracle on symmetric and asymmetric response elements
Archives of Insect Biochemistry and Physiology, 2005Co-Authors: Srini C. Perera, Arthur Retnakaran, Peter J. Krell, Sichun Zheng, Qili Feng, Subba Reddy PalliAbstract:Heterodimerization of nuclear Receptors is facilitated by the interaction of two dimerization interfaces: one spanning the DNAbinding (C domain) region and the adjacent hinge (D domain) region, and the other in the ligand-binding (E domain) region. Ultraspiracle (USP) heterodimerizes with Ecdysone Receptor (EcR) and this complex participates in Ecdysone signal transduction. The natural Ecdysone response elements (EcREs) discovered so far are asymmetric elements composed of either imperfect palindromes or direct repeats. However, gel mobility shift assays have shown that both symmetric (perfect palindromes) and asymmetric (imperfect palindromes and direct repeats) elements can bind to the EcR/USP complex. Therefore, we analyzed EcR/USP domains involved in heterodimerization on different types of response elements (RE). Gel shift assays using fulllength and truncated EcR and USP proteins showed that heterodimerization of these two proteins in the presence of asymmetric RE (DR4 and the natural EcRE hsp27) requires both dimerization interfaces present in CD and E domains of both proteins. In contrast, the dimerization interface present in the E domain of either EcR or USP was not essential for heterodimerization on symmetric RE such as PAL1 or IR1. We conclude that the use of heterodimerization interfaces present in CD and E domains of EcR/USP depends on the nature of response elements they bind to. Arch. Insect Biochem. Physiol. 60:55–70, 2005. © 2005 Wiley-Liss, Inc.
-
813. Ecdysone Receptor-Based Inducible Gene Regulation System for Simultaneous Regulation of Multiple Genes
Molecular Therapy, 2004Co-Authors: Prasanna Kumar, Subba Reddy Palli, Anand K. Katakam, Mohan Basavaraju Kumar, Henry Hoff, Dean Ervin CressAbstract:An Ecdysone Receptor (EcR)-based inducible gene regulation system that functions in mammalian cells in a tightly regulated fashion is described. This gene switch functions efficiently in a two-hybrid format in which heterologous DNA binding and transcription activation domains are fused to the two heterodimeric Receptor partners EcR and RXR. Extensive mutagenesis of the Ecdysone Receptor along with the development and screening of a large number of non-steroid ligands has resulted in the discovery of several orthogonal ligand-Receptor pairs. Receptor mutants were obtained that are activated at low nanomolar concentrations of the ligand. The different components of the gene switch were assembled into a two-vector format or into a single cassette all-in-one vector for expression of genes of interest. Different Ecdysone Receptor mutants were fused to different DNA binding domains to create several independently acting gene switch combinations (multiplex). The orthogonal nature of the ligand-Receptor pairs was demonstrated in reporter assays of two-channel gene switch combinations. Experiments will be presented to demonstrate that the multiplex gene switch system works independently in single cells in response to different ligands. The utility of this system in gene therapy applications will be discussed.
-
improved Ecdysone Receptor based inducible gene regulation system
FEBS Journal, 2003Co-Authors: Subba Reddy Palli, Mariana Z. Kapitskaya, Mohan Basavaraju Kumar, Dean Ervin CressAbstract: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.
Haruhiko Fujiwara - One of the best experts on this subject based on the ideXlab platform.
-
Characterization of core promoter elements for Ecdysone Receptor isoforms of the silkworm, Bombyx mori
Insect Molecular Biology, 2007Co-Authors: H. Shirai, Manabu Kamimura, Haruhiko FujiwaraAbstract:Two Ecdysone Receptor (EcR) isoforms, EcR-A and EcR-B1, are expressed in a tissue- and stage-specific manner, although the details of their transcription mechanisms are unknown. We determined the transcription start sites of EcR-A and EcR-B1 isoforms of Bombyx mori and found that both core promoter regions consist of initiator (Inr) and downstream promoter elements (DPE) but not TATA boxes. Promoter truncation analysis performed using the luciferase reporter assays and BmN cells showed that, in both isoforms, the regions -296 to -74 for BmEcR-B1, -104 to -61 for BmEcR-A and downstream regions of +1 are essential for basal transcriptional activity. Mutation experiments revealed that both DPE and its 5'-flanking CGCGCG sequence are crucial but DPE of BmEcR-B1 is not important for BmEcR-A transcription. These results indicate that the basal promoter activities differ between the two BmEcR isoforms.
-
tissue specific and stage specific expression of two silkworm Ecdysone Receptor isoforms ecdysteroid dependent transcription in cultured anterior silk glands
FEBS Journal, 1997Co-Authors: Manabu Kamimura, Shuichiro Tomita, Makoto Kiuchi, Haruhiko FujiwaraAbstract:Previously, we isolated a cDNA clone for the Ecdysone Receptor B1 isoform of the silkworm, Bombyx mori (BmEcR-B1). Here we report the cloning of a cDNA that encodes the Bombyx Ecdysone Receptor A isoform (BmEcR-A) and mRNA expression of the two BmEcR isoforms during molting and metamorphosis. At larval-pupal transformation, mRNA expression of BmEcR-B1 was predominant in most tissues examined, including three larval tissues (midgut, epidermis, and fat body) and the wing imaginal disc. The anterior silk gland was the only tissue where BmEcR-A was predominant. These expression patterns were different from observations demonstrated in Drosophila. In the anterior silk gland, both EcR isoforms were expressed synchronously during the fifth larval instar, while expression of the A isoform preceded that of the B1 isoform by two days in the fourth instar. Precedence of BmEcR-A during the fourth instar and synchronization of both isoforms during the fifth instar were also observed in the middle and posterior silk glands, suggesting that transcription of BmEcR in the silk gland is regulated differently in these two instars. In the cultured anterior silk glands of day 0 of the fifth instar, transcription of BmEcR-A and BmEcR-B1 was induced dose dependently by more than 5 ng/ml 20-hydroxyEcdysone. BmEcR-A and BmEcR-B1 mRNAs were induced within 2 h and 1 h, respectively, of the addition of 20-hydroxyEcdysone. These results suggest that the increase of BmEcR mRNAs during the fifth instar is induced in vivo by a small increase in ecdysteroids.
-
Molecular cloning of an Ecdysone Receptor (B1 isoform) homologue from the silkworm, Bombyx mori, and its mRNA expression during wing disc development.
Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 1996Co-Authors: Manabu Kamimura, Shuichiro Tomita, Haruhiko FujiwaraAbstract:We reported the isolation and sequence of a clone encoding a putative Ecdysone Receptor B1 isoform of the silkworm, Bombyx mori. The predicted open reading frame encoded 543 amino acids, with 51%, 95% and 71% identities with the Drosophila melanogaster Ecdysone Receptor B1 isoform in the N terminal A/B region, DNA binding domain (C region) and ligand binding domain (E region), respectively. A single 6.2 kb message for the EcR gene was abundant in wing discs and fat bodies at the onset of metamorphosis. At the same stage, however, no or a tiny amount of mRNA was shown in posterior or middle silk glands, respectively. During the final instar, the mRNA expression in wing discs was maximal on the day of wandering. These data suggest the transcription of the Bombyx EcR gene is regulated in tissue-specific and stage-specific manner during metamorphosis.
Manabu Kamimura - One of the best experts on this subject based on the ideXlab platform.
-
Characterization of core promoter elements for Ecdysone Receptor isoforms of the silkworm, Bombyx mori
Insect Molecular Biology, 2007Co-Authors: H. Shirai, Manabu Kamimura, Haruhiko FujiwaraAbstract:Two Ecdysone Receptor (EcR) isoforms, EcR-A and EcR-B1, are expressed in a tissue- and stage-specific manner, although the details of their transcription mechanisms are unknown. We determined the transcription start sites of EcR-A and EcR-B1 isoforms of Bombyx mori and found that both core promoter regions consist of initiator (Inr) and downstream promoter elements (DPE) but not TATA boxes. Promoter truncation analysis performed using the luciferase reporter assays and BmN cells showed that, in both isoforms, the regions -296 to -74 for BmEcR-B1, -104 to -61 for BmEcR-A and downstream regions of +1 are essential for basal transcriptional activity. Mutation experiments revealed that both DPE and its 5'-flanking CGCGCG sequence are crucial but DPE of BmEcR-B1 is not important for BmEcR-A transcription. These results indicate that the basal promoter activities differ between the two BmEcR isoforms.
-
Binding affinity of nonsteroidal Ecdysone agonists against the Ecdysone Receptor complex determines the strength of their molting hormonal activity
European Journal of Biochemistry, 2003Co-Authors: Chieka Minakuchi, Yoshiaki Nakagawa, Manabu Kamimura, Hisashi MiyagawaAbstract:N-tert-Butyl-N,N'-dibenzoylhydrazine and its analogs are nonsteroidal Ecdysone agonists that exhibit insect molting hormonal and larvicidal activities. The interaction mode of those Ecdysone agonists with the heterodimer of the Ecdysone Receptor and ultraspiracle has not been fully elucidated. We expressed the Ecdysone Receptor B I and the ultraspiracle of the lepidopteran, Chilo suppressalis, using an in vitro transcription/translation system and confirmed, using gel-shift assays, that the proteins function as Ecdysone Receptors. We also analyzed their ligand-binding affinity. A potent ecdysteroid, ponasterone A, specifically bound to the Ecdysone Receptor with low affinity (K D = 55 nM), and the specific binding was dramatically increased (K D = 1.2 nM) in the presence of the ultraspiracle. For seven nonsteroidal Ecdysone agonists and five ecdysteroids, the binding activity to the in vitro-translated Ecdysone Receptor-ultraspiracle complex was linearly correlated with the binding activity to the inherent Receptor protein in the cell-free preparation of C. suppressalis integument. The binding to the Ecdysone Receptor-ultraspiracle complex for a series of compounds was highly correlated with their molting hormonal activity, indicating that the binding affinity of nonsteroidal Ecdysone agonists to the Ecdysone Receptor-ultraspiracle complex primarily determines the strength of their molting hormonal activity.
-
molecular cloning expression analysis and functional confirmation of two Ecdysone Receptor isoforms from the rice stem borer chilo suppressalis
Insect Biochemistry and Molecular Biology, 2002Co-Authors: Chieka Minakuchi, Yoshiaki Nakagawa, Shuichiro Tomita, Makoto Kiuchi, Manabu KamimuraAbstract:PCR techniques were used to clone and identify cDNAs for Ecdysone Receptor A and B1 (EcR-A and EcR-B1) isoforms from the rice stem borer, Chilo suppressalis. They differ only in the N-terminal A/B regions and show high sequence identities to other insects’ EcRs. At the wandering stage, EcR-B1 mRNA was expressed more abundantly in the midgut than in the epidermis and fat body, whereas expression levels of EcR-A mRNA were similar in the three tissues. In the epidermis of the last instar larvae, the maximal mRNA expression of both EcR-A and EcR-B1 was observed from the wandering to prepupal stages prior to the peak of ecdysteroid titer in the hemolymph. In gel mobility shift assays, in vitro translated C. suppressalis EcR-B1 (CsEcR-B1) and Bombyx mori ultraspiracle (BmUSP) proteins bound to the Pal 1 and Drosophila melanogaster hsp27 Ecdysone response element as a heterodimer. These results indicate that the cDNAs isolated here encode functional Ecdysone Receptors.
-
tissue specific and stage specific expression of two silkworm Ecdysone Receptor isoforms ecdysteroid dependent transcription in cultured anterior silk glands
FEBS Journal, 1997Co-Authors: Manabu Kamimura, Shuichiro Tomita, Makoto Kiuchi, Haruhiko FujiwaraAbstract:Previously, we isolated a cDNA clone for the Ecdysone Receptor B1 isoform of the silkworm, Bombyx mori (BmEcR-B1). Here we report the cloning of a cDNA that encodes the Bombyx Ecdysone Receptor A isoform (BmEcR-A) and mRNA expression of the two BmEcR isoforms during molting and metamorphosis. At larval-pupal transformation, mRNA expression of BmEcR-B1 was predominant in most tissues examined, including three larval tissues (midgut, epidermis, and fat body) and the wing imaginal disc. The anterior silk gland was the only tissue where BmEcR-A was predominant. These expression patterns were different from observations demonstrated in Drosophila. In the anterior silk gland, both EcR isoforms were expressed synchronously during the fifth larval instar, while expression of the A isoform preceded that of the B1 isoform by two days in the fourth instar. Precedence of BmEcR-A during the fourth instar and synchronization of both isoforms during the fifth instar were also observed in the middle and posterior silk glands, suggesting that transcription of BmEcR in the silk gland is regulated differently in these two instars. In the cultured anterior silk glands of day 0 of the fifth instar, transcription of BmEcR-A and BmEcR-B1 was induced dose dependently by more than 5 ng/ml 20-hydroxyEcdysone. BmEcR-A and BmEcR-B1 mRNAs were induced within 2 h and 1 h, respectively, of the addition of 20-hydroxyEcdysone. These results suggest that the increase of BmEcR mRNAs during the fifth instar is induced in vivo by a small increase in ecdysteroids.
-
Molecular cloning of an Ecdysone Receptor (B1 isoform) homologue from the silkworm, Bombyx mori, and its mRNA expression during wing disc development.
Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 1996Co-Authors: Manabu Kamimura, Shuichiro Tomita, Haruhiko FujiwaraAbstract:We reported the isolation and sequence of a clone encoding a putative Ecdysone Receptor B1 isoform of the silkworm, Bombyx mori. The predicted open reading frame encoded 543 amino acids, with 51%, 95% and 71% identities with the Drosophila melanogaster Ecdysone Receptor B1 isoform in the N terminal A/B region, DNA binding domain (C region) and ligand binding domain (E region), respectively. A single 6.2 kb message for the EcR gene was abundant in wing discs and fat bodies at the onset of metamorphosis. At the same stage, however, no or a tiny amount of mRNA was shown in posterior or middle silk glands, respectively. During the final instar, the mRNA expression in wing discs was maximal on the day of wandering. These data suggest the transcription of the Bombyx EcR gene is regulated in tissue-specific and stage-specific manner during metamorphosis.
Peter Cherbas - One of the best experts on this subject based on the ideXlab platform.
-
Cryptocephal, the Drosophila melanogaster ATF4, is a specific coactivator for Ecdysone Receptor isoform B2.
PLoS Genetics, 2012Co-Authors: Sebastien A. Gauthier, Eric Vanhaaften, Lucy Cherbas, Peter Cherbas, Randall S. HewesAbstract:The Ecdysone Receptor is a heterodimer of two nuclear Receptors, the Ecdysone Receptor (EcR) and Ultraspiracle (USP). In Drosophila melanogaster, three EcR isoforms share common DNA and ligand-binding domains, but these proteins differ in their most N-terminal regions and, consequently, in the activation domains (AF1s) contained therein. The transcriptional coactivators for these domains, which impart unique transcriptional regulatory properties to the EcR isoforms, are unknown. Activating transcription factor 4 (ATF4) is a basic-leucine zipper transcription factor that plays a central role in the stress response of mammals. Here we show that Cryptocephal (CRC), the Drosophila homolog of ATF4, is an Ecdysone Receptor coactivator that is specific for isoform B2. CRC interacts with EcR-B2 to promote Ecdysone-dependent expression of ecdysis-triggering hormone (ETH), an essential regulator of insect molting behavior. We propose that this interaction explains some of the differences in transcriptional properties that are displayed by the EcR isoforms, and similar interactions may underlie the differential activities of other nuclear Receptors with distinct AF1-coactivators.
-
Transcription activation by the Ecdysone Receptor (EcR/USP): identification of activation functions.
Molecular Endocrinology, 2003Co-Authors: Lucy Cherbas, Peter CherbasAbstract:The Ecdysone Receptor is a heterodimer of the two nuclear Receptors EcR and ultraspiracle (USP). We have identified the regions of Drosophila EcR and USP responsible for transcriptional activation of a semisynthetic Eip71CD promoter in Kc cells. The isoform-specific A/B domains of EcR-B1 and B2, but not those of EcR-A or USP, exhibit strong activation activity [activation function 1 (AF1)], both in isolation and in the context of the intact Receptor. AF1 activity in isoform B1 derives from dispersed elements; the B2-specific AF1 consists of a 17-residue amphipathic helix. AF2 function was studied using a two-hybrid assay in Kc cells, based on the observation that potent hormone-dependent activation by the EcR/USP ligand-binding domain heterodimer requires the participation of both partners. Mutagenesis reveals that AF2 function depends on EcR helix 12, but not on the cognate USP region. EcR helix 12 mutants (F645A and W650A) exhibit a dominant negative phenotype. Thus, in the setting tested, the Ecdysone Receptor can activate transcription using the AF1 regions of EcR-B1 or -B2 and the AF2 region of EcR. USP acts as an allosteric effector for EcR, but does not contribute any intrinsic function.
-
transcription activation by the Ecdysone Receptor ecr usp identification of activation functions
Molecular Endocrinology, 2003Co-Authors: Lucy Cherbas, Peter CherbasAbstract:The Ecdysone Receptor is a heterodimer of the two nuclear Receptors EcR and ultraspiracle (USP). We have identified the regions of Drosophila EcR and USP responsible for transcriptional activation of a semisynthetic Eip71CD promoter in Kc cells. The isoform-specific A/B domains of EcR-B1 and B2, but not those of EcR-A or USP, exhibit strong activation activity [activation function 1 (AF1)], both in isolation and in the context of the intact Receptor. AF1 activity in isoform B1 derives from dispersed elements; the B2-specific AF1 consists of a 17-residue amphipathic helix. AF2 function was studied using a two-hybrid assay in Kc cells, based on the observation that potent hormone-dependent activation by the EcR/USP ligand-binding domain heterodimer requires the participation of both partners. Mutagenesis reveals that AF2 function depends on EcR helix 12, but not on the cognate USP region. EcR helix 12 mutants (F645A and W650A) exhibit a dominant negative phenotype. Thus, in the setting tested, the Ecdysone Receptor can activate transcription using the AF1 regions of EcR-B1 or -B2 and the AF2 region of EcR. USP acts as an allosteric effector for EcR, but does not contribute any intrinsic function.
-
Bombyx EcR (BmEcR) and Bombyx USP (BmCF1) combine to form a functional Ecdysone Receptor
Insect Biochemistry and Molecular Biology, 1996Co-Authors: Luc Swevers, Lucy Cherbas, Peter Cherbas, Kostas IatrouAbstract:Abstract The Drosophila Ecdysone Receptor (DmEcR) is a member of the nuclear Receptor superfamily; it functions as an obligate heterodimer with another nuclear Receptor, DmUSP. EcR homologs have now been cloned from several other insects. We report here that one such homolog, BmEcR from the commercial silkmoth, Bombyx mori , is a functional Ecdysone Receptor. Upon dimerization with BmCF1, the silkmoth homolog of DmUSP, BmEcR binds the radiolabeled steroid ligand 125 I-iodoponasterone A with K d = 1.1 nM, indistinguishable from that exhibited by DmEcR/DmUSP. BmEcR/BmCF1 forms a specific complex with an Ecdysone response element (EcRE) derived from the heat shock protein 27 ( hsp27 ) gene promoter of Drosophila ; and, as with DmEcR/DmUSP, formation of this complex is stimulated by the presence of 20-hydroxyEcdysone. Finally, BmEcR can substitute for DmEcR in an EcR-deficient Drosophila tissue culture line, stimulating trans -activation of an Ecdysone-inducible reporter gene construct. Thus, BmEcR and BmCF1 are the functional counterparts of DmEcR and DmUSP, respectively and, despite considerable sequence divergence between the Drosophila and Bombyx proteins, the counterparts are—at least qualitatively-functionally equivalent.
-
Functional Ecdysone Receptor Is the Product of EcR and Ultraspiracle Genes
Nature, 1993Co-Authors: Tso-pang Yao, Lucy Cherbas, Peter Cherbas, Barry M. Forman, Ze-yu Jiang, Jasmine Chen, Michael Mckeown, Ronald M. EvansAbstract:Although the biological activity of the insect moulting hormone Ecdysone, is manifested through a hormonally regulated transcriptional cascade associated with chromosomal puffing, a direct association of the Receptor with the puff has yet to be established. The cloned Ecdysone Receptor (EcR) is by itself incapable of high-affinity DNA binding or transcriptional activation. Rather, these activities are dependent on heterodimer formation with Ultraspiracle (USP) the insect homologue of vertebrate retinoid X Receptor. Here we report that native EcR and USP are co-localized on Ecdysone-responsive loci of polytene chromosomes. Moreover, we show that natural Ecdysones selectively promote physical association between EcR and USP, and conversely, that high-affinity hormone binding requires both EcR and USP. Replacement of USP with retinoid X Receptor produces heterodimers with distinct pharmacological and functional properties. These results redefine the Ecdysone Receptor as a dynamic complex whose activity may be altered by combinatorial interactions among subunits and ligand.
Alexander S. Raikhel - One of the best experts on this subject based on the ideXlab platform.
-
the competence factor βftz f1 potentiates Ecdysone Receptor activity via recruiting a p160 src coactivator
Molecular and Cellular Biology, 2006Co-Authors: Jinsong Zhu, Li Chen, Guoqiang Sun, Alexander S. RaikhelAbstract:Hormones provide generalized signals that are interpreted in a specific spatial and temporal manner by a developing or reproducing multicellular organism. The ability to respond to hormones is determined by the competence of a cell or a tissue. The βFtz-F1 orphan nuclear Receptor acts as a competence factor for the steroid hormone 20-hydroxyEcdysone (20E) in Drosophila melanogaster metamorphosis and mosquito reproduction. The molecular nature of the βFtz-F1 action remains unclear. We report that the protein-protein interaction between βFtz-F1 and a p160/SRC coactivator of the Ecdysone Receptor, FISC, is crucial for the stage-specific expression of the 20E effector genes during mosquito reproduction. This interaction dramatically increases recruitment of FISC to the functional Ecdysone Receptor in a 20E-dependent manner. The presence of βFtz-F1 facilitates loading of FISC and the Ecdysone Receptor on the target promoters, leading to enhanced local histone H4 acetylation and robust activation of the target genes. Thus, our results reveal the molecular basis of competence for the stage-specific 20E response.
-
The Competence Factor βFtz-F1 Potentiates Ecdysone Receptor Activity via Recruiting a p160/SRC Coactivator
Molecular and Cellular Biology, 2006Co-Authors: Jinsong Zhu, Li Chen, Guoqiang Sun, Alexander S. RaikhelAbstract:Hormones provide generalized signals that are interpreted in a specific spatial and temporal manner by a developing or reproducing multicellular organism. The ability to respond to hormones is determined by the competence of a cell or a tissue. The βFtz-F1 orphan nuclear Receptor acts as a competence factor for the steroid hormone 20-hydroxyEcdysone (20E) in Drosophila melanogaster metamorphosis and mosquito reproduction. The molecular nature of the βFtz-F1 action remains unclear. We report that the protein-protein interaction between βFtz-F1 and a p160/SRC coactivator of the Ecdysone Receptor, FISC, is crucial for the stage-specific expression of the 20E effector genes during mosquito reproduction. This interaction dramatically increases recruitment of FISC to the functional Ecdysone Receptor in a 20E-dependent manner. The presence of βFtz-F1 facilitates loading of FISC and the Ecdysone Receptor on the target promoters, leading to enhanced local histone H4 acetylation and robust activation of the target genes. Thus, our results reveal the molecular basis of competence for the stage-specific 20E response.
-
Two distinct subpopulations of Ecdysone Receptor complex in the female mosquito during vitellogenesis
Molecular and Cellular Endocrinology, 1999Co-Authors: Ken Miura, Sheng-fu Wang, Alexander S. RaikhelAbstract:The native functional Ecdysone Receptor complex, a heterodimer of the Ecdysone Receptor (EcR) and ultraspiracle (USP) proteins, was identified in the fat body of adult female mosquitoes, Aedes aegypti, through electrophoretic mobility shift assays (EMSA) using previously characterized Drosophila Ecdysone response elements (EcREs). The use of different salt concentrations during preparation of nuclear extracts enabled us to characterize two distinct subpopulations of the Receptor complex, one of which was high salt-sensitive and responsive to exogenous 20-hydroxyEcdysone (20E), and the other of which was high salt-resistant and refractory to exogenous 20E. Salt-sensitivity correlated with ligand responsiveness. Developmental EMSA analyses demonstrated that previtellogenic fat body nuclei and nuclei from the termination phase of vitellogenesis with low 20E titer contained solely high-salt-sensitive, ligand responsive complexes, which could be recovered in nuclear extracts (NEs) only by low salt tissue homogenization, suggesting these complexes were unliganded. In contrast, the fat body nuclei from stages of active vitellogenesis with high 20E titer contained almost exclusively high salt-resistant, ligand refractory complexes, implying these complexes were liganded; the nuclei from the intermediate stages, early and late phases of vitellogenesis, contained a mixture of the two subpopulations. The developmental profile of fully activated, ligand refractory Receptor complexes closely correlated with that of yolk protein expression, suggesting an intimate involvement of the Ecdysone Receptor complex in both the induction and maintenance of high level expression of yolk protein genes.