Subtype selective tetracycline agonists and their application for a two-stage regulatory system.
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Doxycycline↗
Biomedical subjects
Christian Berens
Publications and source records attributed to Christian Berens.
A gene regulation system with four distinct expression levels.
BACKGROUND: The amount of a particular protein, and not just its presence or absence, frequently determines the outcome of a developmental process or disease progression. These dosage effects can be studied by conditionally expressing such proteins at different levels. With typical gene regulation systems like the Tet-On system, intermediate expression levels can be obtained by varying the effector concentration. However, this strategy is limited to situations in which these concentrations can be precisely controlled and, thus, not suited for animal models or gene therapy approaches. Here, we present a Tet transregulator setup that allows establishment of four levels of promoter activity largely independent of effector concentration. METHODS: A newly introduced transsilencer is combined with a reverse transactivator. As the regulators respond differentially to tetracycline derivatives, four expression levels are obtained by adding different effectors. To facilitate integration of the components, we generated versatile all-in-one vectors. Apart from a cassette expressing the transregulators and a selection marker, these vectors encode a bidirectional, regulated promoter driving expression of GFP and the gene of interest. The features of this stepwise regulation system were analyzed by transient and stable transfections of human cell lines. RESULTS: We demonstrate in a variety of experimental settings that coexpression of these transregulators leads to robust stepwise regulation. Depending on the respective effectors, four expression levels are achieved with different responsive promoters, cell lines and target genes. CONCLUSIONS: This system shows that a promoter can be adjusted to different activities and provides an excellent strategy to investigate protein dosage effects.
Cell Line↗
Random insertion of a TetR-inducing peptide tag into Escherichia coli proteins allows analysis of protein levels by induction of reporter gene expression.
The insertion element InsTipalpha was constructed to generate protein expression data. It randomly fuses the TetR-inducing peptide Tip to the affected reading frame. Fusion protein expression is quantified by Tet-regulated reporter gene expression. The expression patterns of tagged Escherichia coli genes fully agree with published data from transcriptional fusions or microarrays, validating the Tip tag approach.
DNA Transposable Elements↗
Reproducible doxycycline-inducible transgene expression at specific loci generated by Cre-recombinase mediated cassette exchange.
Comparative analysis of mutants using transfection is complicated by clones exhibiting variable levels of gene expression due to copy number differences and genomic position effects. Recombinase-mediated cassette exchange (RMCE) can overcome these problems by introducing the target gene into pre-determined chromosomal loci, but recombination between the available recombinase targeting sites can reduce the efficiency of targeted integration. We developed a new LoxP site (designated L3), which when used with the original LoxP site (designated L2), allows highly efficient and directional replacement of chromosomal DNA with incoming DNA. A total of six independent LoxP integration sites introduced either by homologous recombination or retroviral delivery were analyzed; 70-80% of the clones analyzed in hamster and human cells were correct recombinants. We combined the RMCE strategy with a new, tightly regulated tetracycline induction system to produce a robust, highly reliable system for inducible transgene expression. We observed stable inducible expression for over 1 month, with uniform expression in the cell population and between clones derived from the same integration site. This system described should find significant applications for studies requiring high level and regulated transgene expression and for determining the effects of various stresses or oncogenic conditions in vivo and in vitro.
Animals↗
Stringent doxycycline-dependent control of gene activities using an episomal one-vector system.
Conditional expression systems are of pivotal importance for the dissection of complex biological phenomena. Here, we describe a novel EBV-derived episomally replicating plasmid (pRTS-1) that carries all the elements for conditional expression of a gene of interest via Tet regulation. The vector is characterized by (i) low background activity, (ii) high inducibility in the presence of doxycycline (Dox) and (iii) graded response to increasing concentrations of the inducer. The chicken beta actin promoter and an element of the murine immunoglobin heavy chain intron enhancer drive constitutive expression of a bicistronic expression cassette that encodes the highly Dox-sensitive reverse tetracycline controlled transactivator rtTA2(S)-M2 and a Tet repressor-KRAB fusion protein (tTS(KRAB)) (silencer) placed downstream of an internal ribosomal entry site. The gene of interest is expressed from the bidirectional promoter P(tet)bi-1 that allows simultaneous expression of two genes, of which one may be used as surrogate marker for the expression of the gene of interest. Tight down regulation is achieved through binding of the silencer tTS(KRAB) to P(tet)bi-1 in the absence of Dox. Addition of Dox releases repression and via binding of rtTA2(S)-M2 activates P(tet)bi-1.
Animals↗
Development of a transactivator in hepatoma cells that allows expression of phase I, phase II, and chemical defense genes.
Precise control of the level of protein expression in cells can yield quantitative and temporal information on the role of a given gene in normal cellular physiology and on exposure to chemicals and drugs. This is particularly relevant to liver cells, in which the expression of many proteins, such as phase I and phase II drug-metabolizing enzymes, vary widely between species, among individual humans, and on exposure to xenobiotics. The most widely used gene regulatory system has been the tet-on/off approach. Although a second-generation tet-on transactivator was recently described, it has not been widely investigated for its potential as a tool for regulating genes in cells and particularly in cells previously recalcitrant to the first-generation tet-on approach, such as hepatocyte-derived cells. Here we demonstrate the development of two human (HepG2 and HuH7) and one mouse (Hepa1c1c7) hepatoma-derived cell lines incorporating a second-generation doxycycline-inducible gene expression system and the application of the human lines to control the expression of different transgenes. The two human cell lines were tested for transient or stable inducibility of five transgenes relevant to liver biology, namely phase I (cytochrome P-450 2E1; CYP2E1) and phase II (glutathione S-transferase P1; GSTP1) drug metabolism, and three transcription factors that respond to chemical stress [nuclear factor erythroid 2 p45-related factors (NRF)1 and 2 and NFKB1 subunit of NF-kappaB]. High levels of functional expression were obtained in a time- and dose-dependent manner. Importantly, doxycycline did not cause obvious changes in the cellular proteome. In conclusion, we have generated hepatocyte-derived cell lines in which expression of genes is fully controllable.
Amino Acid Sequence↗
An optimized conditional suicide switch using doxycycline-dependent expression of human tBid.
We have examined in a HeLa model system whether tight doxycycline-dependent expression of human tBid can be used as a fast and efficient suicide switch. A stably transfected cell line expressing human tBid in a strictly dox-dependent manner showed fast and efficient killing with up to 98 % specific cell death 24 h after induction. The survival rate was only 0.6 % after eleven days of dox treatment. Very low amounts of tBid were sufficient to induce apoptosis indicating that stringent control of the dox-dependent suicide gene is essential for cell survival in the absence of inducer. Using human tBid, an endogenous effector protein with low immunogenic potential, and doxycycline, an inducer with low cytotoxicity and a long record of safe use in humans, this conditional suicide switch provides a tool for various applications, e.g., adoptive immune transfer.
Apoptosis↗
A peptide triggers allostery in tet repressor by binding to a unique site.
Regulatory proteins often communicate with each other to manage various cellular processes. Such interactions mostly rely on the recognition of small peptide motifs. The activity of other regulatory proteins depends on small molecular weight effectors and allostery. We demonstrate the in vivo regulation of the tetracycline-dependent Tet repressor by an oligopeptide fused to the N or C terminus of thioredoxin A. The binding site of the peptide overlaps but is not identical with the tetracycline binding site. Several TetR mutants that are non-inducible by tetracycline also respond to the peptide. This demonstrates for the first time the conversion of a small molecular weight effector-dependent regulator to a protein-protein contact-dependent potential member of designed signaling chains.
Allosteric Site↗
Inducible DNA-loop formation blocks transcriptional activation by an SV40 enhancer.
It is well established that gene expression in eukaryotes is controlled by sequence-dependent binding of trans-acting proteins to regulatory elements like promoters, enhancers or silencers. A less well understood level of gene regulation is governed by the various structural and functional states of chromatin, which have been ascribed to changes in covalent modification of core histone proteins. And, much on how topological domains in the genome take part in establishing and maintaining distinct gene expression patterns is still unknown. Here we present a set of regulatory proteins that allow to reversibly alter the DNA structure in vivo and in vitro by adding low molecular weight effectors that control their oligomerization and DNA binding. Using this approach, we completely regulate the activity of an SV40 enhancer in HeLa cells by reversible loop formation to topologically separate it from the promoter. This result establishes a new mechanism for DNA-structure-dependent gene regulation in vivo and provides evidence supporting the structural model of insulator function.
Base Sequence↗
A short autonomous repression motif is located within the N-terminal domain of CTCF.
The vertebrate transcription factor CTCF is not only involved in transcriptional activation, insulation and genomic imprinting, but also in transcriptional repression. Sequence motifs mediating these activities have not been identified so far. We have mapped a short repression motif to residues 150-170 within the N-terminal domain of CTCF. This motif is active in HeLa, HEK293 and COS-7 cell lines where it is both sufficient and necessary for silencing either an SV40-, or a CMV-enhancer. It also represses the basal activity of an SV40 core promoter. Since this autonomous repression motif displays no sequence similarity to any other regulatory protein, it represents a yet unknown co-repressor recruiting motif.
Animals↗
Transactivator mutants with altered effector specificity allow selective regulation of two genes by tetracycline variants.
A set of Tet repressor (TetR) based eukaryotic transactivators that respond to 4-de(dimethylamino)-6-deoxy-6-demethyl-tetracycline (cmt3) but no longer to tetracycline (tc) is presented. The novel transactivators exhibit high activation in absence of an effector and a 200-fold reduction of reporter gene activity in the presence of cmt3. The most cmt3-sensitive mutant was coexpressed with a tc-responsive Tet transregulator harbouring an altered DNA recognition specificity. Use of cmt3 and tc yields independent control of expression of two genes in the same cell without crosstalk.
Cell Division↗
A theophylline responsive riboswitch based on helix slipping controls gene expression in vivo.
Riboswitches are newly discovered regulatory elements which control a wide set of basic metabolic pathways. They consist solely of RNA, sense their ligand in a preformed binding pocket and perform a conformational switch in response to ligand binding resulting in altered gene expression. We have utilized the enormous potential of RNA for molecular sensing and conformational changes to develop novel molecular switches with predetermined structural transitions in response to the binding of a small molecule. To validate these in vivo, we exploit the distance-dependent inhibitory potential of secondary structure elements placed close to the bacterial ribosome binding site. We created a translational control element by combining the theophylline aptamer with a helical communication module for which a ligand-dependent one-nucleotide slipping mechanism had been proposed. This structural element was inserted at a position just interfering with translation in the non ligand-bound form. Addition of the ligand then shifts the inhibitory element to a distance which permits efficient translation. We present here a novel regulatory mechanism in the first rationally designed, in vivo active RNA switch. Its use of a slippage mechanism to control gene expression makes it different from natural riboswitches which are based on sequestration or antitermination.
Base Sequence↗
Comparison of tetracycline and tigecycline binding to ribosomes mapped by dimethylsulphate and drug-directed Fe2+ cleavage of 16S rRNA.
OBJECTIVES: The new antibiotic tigecycline (9-t-butylglycylamido-minocycline; GAR-936) overcomes most of the known tetracycline resistance mechanisms. Here we analyse its mode of antibiotic action by probing 70S ribosomes of Escherichia coli with dimethylsulphate (DMS) and Fe(2+)-mediated cleavage to identify binding sites of tetracycline and tigecycline. METHODS: Fe(2+)-mediated cleavage makes use of the ability of Fe2+ to replace the Mg2+ ion complexed with tetracyclines. After addition of H2O2, Fe2+ generates short-lived, highly reactive hydroxyl radicals that can cleave RNA close to the tetracycline binding sites. RESULTS: We identified three prominent Fe(2+)-mediated cleavage sites in helices 29 and 34, and in the internal loop of helix 31 of 16S rRNA in the presence of tetracycline or tigecycline. Qualitatively, these sites are modified identically by both antibiotics, but quantitative differences observed in the cleavage intensities indicate that the drugs bind in slightly different orientations. These results are supported by DMS modification, mutational analysis of 16S rRNA and structural modelling of tigecycline at a tetracycline-binding site in the 30S ribosomal subunit. CONCLUSIONS: Both derivatives bind to identical or overlapping sites and probably share the same mode of antibiotic action. The fact that tigecycline overcomes most of the known tetracycline resistance mechanisms is interpreted as a result of steric hindrance due to the large substituent at position 9.
Base Sequence↗
Gene regulation by tetracyclines.
Gene regulation by tetracyclines has become a widely-used tool to study gene functions in pro- and eukaryotes. This regulatory system originates from Gram-negative bacteria, in which it fine-tunes expression of a tetracycline-specific export protein mediating resistance against this antibiotic. This review attempts to describe briefly the selective pressures governing the evolution of tetracycline regulation, which have led to the unique regulatory properties underlying its success in manifold applications. After discussing the basic mechanisms we will present the large variety of designed alterations of activities which have contributed to the still growing tool-box of components available for adjusting the regulatory properties to study gene functions in different organisms or tissues. Finally, we provide an overview of the various experimental setups available for pro- and eukaryotes, and touch upon some highlights discovered by the use of tetracycline-dependent gene regulation.
Allosteric Regulation↗
Tetracycline derivatives: alternative effectors for Tet transregulators.
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Bacterial Proteins↗
Single-chain Tet transregulators.
We demonstrate here that the Tet repressor (TetR), a dimeric allosterical regulatory protein, can be converted to a fully functional monomer when connected by a 29 amino acid linker. TetR-based transregulators are widely used to regulate gene expression in eukaryotes. They can be fused to form single-chain (sc) Tet transregulators with two TetR moieties and one eukaryotic regulatory domain. Sc variants of transactivator and transsilencer exhibit the same regulatory properties as their respective dimeric counterparts in human cell lines. In particular, the reverse 'tet-on' phenotype of rtTA variants is also present in the sc variants. Coexpression of a reverse transactivator and sc transsilencer leads to reduced background expression and shows full activation upon induction. The data demonstrate that sc Tet transregulators exhibit the phenotype of their respective dimers and lack functional interference when coexpressed in the same cell.
Cell Line↗
Conditional gene expression by controlling translation with tetracycline-binding aptamers.
We present a conditional gene expression system in Saccharomyces cerevisiae which exploits direct RNA-metabolite interactions as a mechanism of genetic control. We inserted preselected tetracycline (tc) binding aptamers into the 5'-UTR of a GFP encoding mRNA. While aptamer insertion generally reduces GFP expression, one group of aptamers displayed an additional, up to 6-fold, decrease in fluorescence upon tc addition. Regulation is observed for aptamers inserted cap-proximal or near the start codon, but is more pronounced from the latter position. Increasing the thermodynamic stability of the aptamer augments regulation but reduces expression of GFP. Decreasing the stability leads to the opposite effect. We defined nucleotides which influence the regulatory properties of the aptamer. Exchanging a nucleotide probably involved in tc binding only influences regulation, while mutations at another position alter expression in the absence of tc, without affecting regulation. Thus, we have developed and characterized a regulatory system which is easy to establish and controlled by a non-toxic, small ligand with good cell permeability.
5' Untranslated Regions↗
Conditional cell suicide using dox-dependent caspase-2 expression.
BACKGROUND: Adoptive immune transfer is used as an efficient treatment modality to achieve a graft-versus-leukemia effect in persisting or relapsing residual leukemic disease. Safety considerations dictate the need for equipping the transferred cells with a conditional suicide mechanism to eliminate donor T cells when graft-versus-host disease occurs. We have examined in a model system using HeLa cells whether doxycycline (dox)-dependent expression of pro-apoptotic proteins could be used as a potential new strategy for conditional cell elimination. METHODS: Four constructs encoding pro-apoptotic proteins were tested in transient transfections to identify suitable cell death inducers. Murine caspase-2 placed under Tet-control was chosen for stable transfection into cell lines carrying different dox-dependent transregulators. The efficiency of cell death induction and the expression patterns of caspase-2 were analyzed in the respective clones. RESULTS: Different levels of induced cell death were obtained depending on the properties of the transregulators used to control target gene expression. High expression levels of caspase-2 in the presence of dox were required to achieve efficient induction of cell death, while tight repression in the absence of inducer was not necessary for cell survival. Dox treatment for 48 h resulted in 94% cell death indicating a very efficient conditional suicide mechanism. CONCLUSIONS: We propose that the principle of using pro-apoptotic cellular proteins placed under appropriate dox-dependent regulation may represent an alternative conditional suicide mechanism to the frequently used herpes simplex virus thymidine kinase (HSV-TK)/ganciclovir-system, which harbors immunological and toxicological risks.
Animals↗