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At least 37 records · Page 2Linked to original sources
PubMed2006

Development of a tightly regulated and highly inducible ecdysone receptor gene switch for plants through the use of retinoid X receptor chimeras.

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.

Arabidopsis↗
PubMed2002

The Plasmodium falciparum var gene switching rate, switching mechanism and patterns of parasite recrudescence described by mathematical modelling.

Recrudescing Plasmodium falciparum parasitaemia is attributed to the switching of PfEMP1, a variant antigen family encoded by the var gene repertoire, and the host's immune response. We have developed a mathematical model which incorporates var gene switching, and variant specific, non-variant specific and non-specific immunity. By conducting a sensitivity analysis of the model we have defined the parameter limits which produce chronic and recrudescing infections. We explore 3 switching mechanisms: ordered, random and uncoupled switching. We show that if var genes switch on and off independently at variable rates through the repertoire a chronic clinical infection is predicted. The fastest switching-on rate that produces a chronic infection is 0.03% per generation. The model predicts that non-variant specific immunity plays an important role in reducing disease severity. This work illustrates the complex relationship between the malaria parasite and its host and shows that var gene switching at rates substantially slower than 2% are essential for parasite survival.

Animals↗
PubMed2004

Stringent rosiglitazone-dependent gene switch in muscle cells without effect on myogenic differentiation.

We have developed a gene switch based on the human transcription factor peroxisome proliferator-activated receptor gamma (PPARgamma) and its activation by rosiglitazone. However, ectopic expression of PPARgamma has been demonstrated to convert myogenic cells into adipocyte-like cells and, more generally, may interfere with the physiology of the target tissue. Consequently we modified the DNA-binding specificity of PPARgamma, resulting in a transcription factor that we named PPAR*. We demonstrated by histological and molecular assessment of cell phenotype that the overexpression of PPAR* did not alter the myogenic differentiation program of G8 myoblasts. We showed that PPAR* does not transactivate promoters containing PPARgamma-responsive elements but transactivates promoters containing PPAR*-responsive elements that are at least 80% identical to a 20-bp consensus. We improved the rosiglitazone-dependent gene switch by tuning PPAR* expression with a scaffold/matrix attachment region and by expressing both PPAR* and the reporter gene under the control of PPAR*-responsive elements. Treatment of cultured murine muscle cells (myotubes) with rosiglitazone induced reporter gene expression from assay background up to the level attained by a CMV I/E promoter-enhancer. These results indicate the potential of the PPAR* gene switch for use in gene therapy applications.

Amino Acid Substitution↗
PubMed2006

Functional characterization of ecdysone receptor gene switches in mammalian cells.

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).

Animals↗
PubMed1997

Binary transgenic mouse model for studying the trans control of globin gene switching: evidence that GATA-1 is an in vivo repressor of human epsilon gene expression.

To test whether human GATA-1 (hGATA-1) is involved in the transcriptional control of globin gene switching, we produced transgenic mice overexpressing hGATA-1, crossed them with mice carrying a human beta-globin locus yeast artificial chromosome (beta YAC), and analyzed globin gene expression in their progeny. Mice carrying both the hGATA-1 and the beta YAC transgenes had normal levels of gamma- and beta-globin mRNA and no distortion in the rate or in the timing of gamma-to-beta switch, indicating that hGATA-1 is not involved in the developmental control of gamma- and beta-globin genes. In contrast, mice carrying the hGATA-1 and the beta YAC transgenes had 5- to 6-fold lower expression of the human epsilon globin gene compared with beta YAC mice lacking the hGATA-1 transgene. These results provide direct in vivo evidence that hGATA-1 is a specific repressor of human epsilon gene expression. Our findings also suggest that binary transgenic mouse systems based on overexpression of transcriptional factors can be used to investigate the trans control of human globin gene switching. Systems as the one we describe here should be useful in the study of any developmentally controlled human gene for which transgenic mice are available.

Animals↗
PubMed1999

Tissue-specific and developmental stage-specific DNA binding by a mammalian SWI/SNF complex associated with human fetal-to-adult globin gene switching.

SWI/SNF complexes in yeast and higher eukaryotes are thought to facilitate gene activation and transcription factor binding by disrupting repressive chromatin structures. Little is known, however, about how these complexes target specific genes for activation. We now have purified a specialized SWI/SNF-related complex (PYR complex) from murine erythroleukemia (MEL) cell nuclear extract that binds pyrimidine-rich elements at the human and murine beta-globin loci. PYR complex DNA-binding activity is restricted to definitive hematopoietic cells and is both DNA sequence- and length-dependent. Mass spectrometric identification of purified peptides and antibody supershift assays indicate that PYR complex contains at least four known mammalian SWI/SNF subunits: BAF57, INI1, BAF60a, and BAF170. PYR complex broadly footprints a 250-bp pyrimidine-rich element between the human fetal and adult beta-globin genes. A short intergenic deletion that removes this element from a human globin locus cosmid construct results in delayed human fetal-to-adult globin gene switching in transgenic mice. Taken together, the data suggest that PYR complex may act through this intergenic element to facilitate human fetal-to-adult globin gene switching, presumably by opening the locus in the region of the adult genes to permit the binding of beta-globin transcriptional activators.

Animals↗
PubMed1997

Cyclosporin A inhibits early mRNA expression of G0/G1 switch gene 2 (G0S2) in cultured human blood mononuclear cells.

Cyclosporin A (CsA) may achieve its immunosuppressive effects by inhibiting the calcium- and calmodulin-dependent phosphatase calcineurin which is required for activation of target genes by members of the NFAT (nuclear factor of activated T cells) transcription factor family. Among these target genes is the gene encoding interleukin-2 (IL2), a cytokine facilitating progression through the G1 phase of the cell cycle. However, IL2 does not reverse CsA inhibition, suggesting that at least one other NFAT-sensitive gene may be involved. The human G0/G1 switch gene, G0S2, has potential NFAT-binding sites in the 5' flank and encodes a small basic potential phosphoprotein of unknown function. Using a sensitive, reverse transcription-polymerase chain reaction (RT-PCR) assay, G0S2 mRNA levels were assayed in cultured blood mononuclear cells. Freshly isolated cells contain high levels of G0S2 mRNA which rapidly decline. This "spontaneous stimulation" is also noted with some other G0S genes and has been attributed to some aspect of the isolation procedure. In cells that have been preincubated to lower mRNA levels, there is a transient increase in G0S2 mRNA, peaking between 1-2 h, in response to Concanavalin-A (ConA), or to the combination of phorbol ester (TPA), and the calcium ionophore, ionomycin. Both these responses are inhibited by CsA. Our results suggest that G0S2 expression is required to commit cells to enter the G1 phase of the cell cycle, and that, while not excluding other possible targets, early inhibition of G0S2 expression by CsA may be important in achieving immunosuppression. G0S2 may be of value as a reporter gene for analyzing the mechanism of action of CsA and its influence on the positive and negative selection of lymphocytes in response to self and not-self antigens.

Amino Acid Sequence↗
PubMed1992

Individual stage selector element mutations lead to reciprocal changes in beta- vs. epsilon-globin gene transcription: genetic confirmation of promoter competition during globin gene switching.

Biochemical and genetic analysis of the embryonic to adult beta-like globin gene switch in chickens has led to the hypothesis that competition between the promoters of the cis-linked epsilon- and beta-globin genes for interaction with a shared enhancer mediates the developmental changes in expression of beta-globin protein isotypes. To test specific predictions of this promoter competition model, a sensitive RNA/polymerase chain reaction assay has been used to investigate the effects of individual beta-globin promoter mutations on expression of the two linked genes in transiently transfected erythroid cells. Mutations that attenuated adult beta-globin transcription resulted concomitantly in a proportional increase in expression of the embryonic epsilon-globin gene. Consistent with the model, mutations disrupting the binding sites for either of two adult stage-specific transcription factors (NF-E4 and beta CTF) indicate that these sites are essential both for induction of beta-globin gene expression and for indirect suppression (through promoter competition) of epsilon-globin transcription in definitive (adult) erythroid cells. These results provide direct evidence that stage-specific transcription factors affect the equilibrium existing between multiple interacting globin cis-regulatory elements. We conclude that promoter competition is an important mechanism through which developmental regulation of chicken beta-globin gene switching is achieved and that such competitive interactions may prove to be generally applicable to the regulation of a variety of other temporally or spatially restricted gene expression patterns.

Animals↗
PubMed1993

The DNA-binding domain of two bZIP transcription factors, the Epstein-Barr virus switch gene product EB1 and Jun, is a bipartite nuclear targeting sequence.

The Epstein-Barr virus BZLF1 gene product EB1 (also called ZEBRA and Zta), is a transcription factor belonging to the bZIP (basic domain leucine zipper) family of nuclear proteins. Translocation to the nucleus of EB1 (J. Becker, U. Leser, M. Marschall, A. Langford, W. Jilg, H. Gelderblom, P. Reichart, and H. Wolf, Proc. Natl. Acad. Sci. USA 88:8332-8336, 1991) and of two other bZIP proteins, c-Jun and c-Fos (P. Roux, J.-M. Blanchard, A. Fernandez, N. Lamb, P. Jeanteur, and M. Piechaczyk, Cell 63:341-351, 1990), has been shown to be subject to regulation. We show here that for both EB1 and Jun the nuclear targeting signals (NTS) in the proteins' primary sequences are two clusters of positively charged amino acids. These clusters, called BRA and BRB, are necessary and sufficient to direct beta-galactosidase to the nuclear compartment and act as a bipartite NTS. They are conserved among all the bZIP proteins, and although they are not identical, they probably share the same function. Site-directed mutagenesis studies made on these basic clusters suggest that they also act as a bipartite NTS in the EB1 protein. Our results also demonstrate that in EB1 and Jun, these bipartite NTS are superimposed with bipartite DNA-binding domains, since BRA and BRB are required in vitro for direct and specific contact between these proteins and their DNA-binding sites.

Amino Acid Sequence↗
PubMed1988

Butyrate infusions in the ovine fetus delay the biologic clock for globin gene switching.

The switch from fetal to adult hemoglobin expression is regulated in many mammalian species by a developmental clock-like mechanism and determined by the gestational age of the fetus. Prolonging fetal globin gene expression is of considerable interest for therapeutic potential in diseases caused by abnormal beta-globin genes. Butyric acid, which is found in increased plasma concentrations in infants of diabetic mothers who have delayed globin gene switching, was infused into catheterized fetal lambs in utero during the time of the normal globin gene switch period. The globin gene switch was significantly delayed in three of four butyrate-treated fetuses compared with controls and was entirely prevented in one fetus in whom the infusion was begun before the globin switch was under way. These data provide a model for investigating and arresting the biologic clock of hemoglobin switching.

Animals↗
PubMed1989

Butyric acid modulates developmental globin gene switching in man and sheep.

The developmental switch from production of fetal (gamma) to adult (beta) globin occurs on a normally set biologic clock which proceeds even if the adult (beta) globin genes are defective. Preventing or reversing the globin gene switch would be beneficial for subjects with abnormal beta globin genes. We have now identified a class of agents which, when present in elevated plasma concentrations during gestation, appears to inhibit the gamma beta globin gene switch in developing humans. Further investigation has shown that butyric acid and related compounds can increase gamma globin and decrease beta globin expression in erythroid cells cultured from subjects with diseases of abnormal beta globin. Butyrate compounds were therefore infused in an in vivo fetal animal model, and the globin switch was inhibited in most and reversed in some fetal lambs. These data suggest that inhibiting expression of abnormal beta globin genes may be possible in future generations. Histone modification may be a mechanism of action involved. The developmental switch from production of gamma globin to beta globin results in significant morbidity when the beta globin genes are defective. The globin switch has therefore been extensively studied, appearing to be set on a biologic clock and proceeding despite the site of blood production and solely on the basis of gestational age. We previously found that this developmental gene switch is delayed in human fetuses developing in the presence of maternal diabetes. A number of metabolites present in abnormal concentrations in these infants were therefore tested for effects on globin expression.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylation↗
PubMed1983

c-myc Gene rearrangements involving gamma immunoglobulin heavy chain gene switch regions in murine plasmacytomas.

In murine plasmacytomas, the c-myc gene has frequently been found to undergo rearrangement by virtue of a T(12;15) chromosome translocation. The immunoglobulin heavy chain gene switch region (S alpha) constitutes the target for most of these recombinations particularly in IgA producing plasmacytomas. We sought to identify non-S alpha myc target sites in several IgG producing tumors. The c-myc target in MPC-11 (a BALB/c IgG2b producing plasmacytoma) has been cloned, localized to the Igh-C locus and identified as the gamma 2a heavy chain gene switch region (S gamma 2a). Furthermore, by Southern blot hybridization, we have determined that the S gamma 2b region is the c-myc target in two NZB IgG2b producing plasmacytomas. The potential relation between Ig class expressed and c-myc translocation target is discussed.

Animals↗
PubMed1997

Chimeric receptors as gene switches.

There is a recognized need for eukaryotic molecular gene switches that are tightly regulated by the administration of small molecule drugs. The modular nature of intracellular receptor proteins has allowed the recent development of chimeric receptors that fulfill this need. These switches will help dissect the roles that specific proteins play in signaling pathways, cell differentiation and development. Some of these switches will also be used in gene therapy to regulate therapeutic gene expression.

Animals↗
PubMed2005

The G0/G1 switch gene 2 is a novel PPAR target gene.

PPARs (peroxisome-proliferator-activated receptors) alpha, beta/delta and gamma are a group of transcription factors that are involved in numerous processes, including lipid metabolism and adipogenesis. By comparing liver mRNAs of wild-type and PPARalpha-null mice using microarrays, a novel putative target gene of PPARalpha, G0S2 (G0/G1 switch gene 2), was identified. Hepatic expression of G0S2 was up-regulated by fasting and by the PPARalpha agonist Wy14643 in a PPARalpha-dependent manner. Surprisingly, the G0S2 mRNA level was highest in brown and white adipose tissue and was greatly up-regulated during mouse 3T3-L1 and human SGBS (Simpson-Golabi-Behmel syndrome) adipogenesis. Transactivation, gel shift and chromatin immunoprecipitation assays indicated that G0S2 is a direct PPARgamma and probable PPARalpha target gene with a functional PPRE (PPAR-responsive element) in its promoter. Up-regulation of G0S2 mRNA seemed to be specific for adipogenesis, and was not observed during osteogenesis or myogenesis. In 3T3-L1 fibroblasts, expression of G0S2 was associated with growth arrest, which is required for 3T3-L1 adipogenesis. Together, these data indicate that G0S2 is a novel target gene of PPARs that may be involved in adipocyte differentiation.

Adipocytes↗
PubMed2002

A single point mutation in ecdysone receptor leads to increased ligand specificity: implications for gene switch applications.

The ecdysone receptor (EcR), a member of the nuclear receptor superfamily, plays an important role in regulating development and reproduction in insects. The EcR binds to ecdysteroids and regulates transcription of genes that contain ecdysone response elements. The EcR has been used to develop inducible gene switches for efficient regulation of foreign genes in applications such as gene therapy, protein production, and functional genomics. An EcR [Choristoneura fumiferana EcR (CfEcR)] homology model was constructed, and 17 amino acid residues were identified as critical for 20-hydroxyecdysone binding. Mutation of these amino acids followed by analysis of these mutants in transactivation (in insect and mammalian cells and in vivo in mice) and ligand-binding assays identified one particular mutant (A110P) that failed to respond to steroids, but its response to the diacylhydrazine nonsteroidal ligands RG-102240 (GS(TM)E) and RG-102317 was unaffected. This steroid-insensitive EcR mutant has potential gene switch applications in insects and plants that have endogenous ecdysteroids. In addition, this mutant would be also useful for developing orthogonal EcR-ligand pairs for simultaneous regulation of multiple genes in the same cell.

Amino Acid Sequence↗
PubMed1996

Bi-directional gene switching with the tetracycline repressor and a novel tetracycline antagonist.

We have screened a panel of tetracycline (tc)-like compounds for their potential use with tc-repressor (tetR) based gene switches. The interaction between tc and tetR appears quite specific, as only tc itself and its close homologues anhydro-tc and doxycycline strongly inhibited DNA binding. However, a single tc-like compound, GR33076X, increased DNA binding of the tetR-VP16 fusion protein, both in eukaryotic cells and in bacteria. We provide evidence that this antagonist of tetracycline is potentially useful for accelerated gene switching, especially in whole animals.

Base Sequence↗
PubMed1991

Developmental regulation of fetal to adult globin gene switching in human fetal erythroid x mouse erythroleukemia cell hybrids.

Human fetal erythroid x murine erythroleukemia cell hybrids undergo human fetal (gamma) to adult (beta) globin gene switching in vitro under the control of a mechanism located on human chromosome 11. We investigated whether this mechanism acts in cis or in trans by preparing hybrid cells containing marked fragments of the gamma and beta genes known to switch in transgenic mice. In these cells the chromosomally introduced human globin locus undergoes the fetal to adult globin gene switch. In contrast, the marked globin gene fragments were expressed at all stages of hybrid development. These results suggest that either the mechanism of switching acts in cis or that sequences present in the chromosomal globin locus but missing from the transfected globin gene fragments mediate its action.

Animals↗
PubMed2005

Inducible control of transgene expression with ecdysone receptor: gene switches with high sensitivity, robust expression, and reduced size.

The ecdysone receptor (EcR)-based gene regulation system is a tool for controlling gene expression. To improve the sensitivity of this system, we evaluated many two-hybrid format synthetic gene constructs in which the GAL4 DNA binding domain was fused to the ligand binding domain of the Choristoneura fumiferana EcR mutant V390I/Y410E (GEvy), and various activation domains--VP16, p53, p65, or E2F-i--were fused to the EF domains of chimeric human RXR. These gene switches were assayed in NIH3T3 cells, HEK293 cells, and in mouse quadriceps in the presence of the nonsteroidal inducer RG-115819 or GS-E. All of the two-hybrid format constructs had no or very low background in the "off" condition and high luciferase reporter gene expression levels in "on" conditions. Extremely high sensitivity was achieved, with EC50 values in the subnanomolar range and with maximal induction at 10 nM RG-115819. Co-expression of both receptor genes with encephalomyocarditis virus (EMCV) or eIF4G internal ribosome entry site (IRES) sequences gave robust induction levels. To reduce the size of the switch construct, we tested single receptor formats, in which any of 14 different activation domains were fused to GEvy. We identified several switches with acceptable levels of basal and maximal induction levels. The gene switches described here provide receptor configuration options suitable for gene function studies, therapeutic protein production in cell culture, transgenic mouse models, and gene/cell therapy.

Animals↗