REVIEW ARTICLE Gene regulation by tetracyclines Constraints of resistance regulation in bacteria shape TetR for application in eukaryotes Christian Berens and Wolfgang Hillen Lehrstuhl fu ¨ r Mikrobiologie, Institut fu ¨ r Mikrobiologie, Biochemie und Genetik, Friedrich-Alexander Universita ¨ t Erlangen-Nu ¨ rnberg; Germany The Tet repressor protein (TetR) regulates transcription of a family of tetracycline (tc) resistance determinants in Gram-negative bacteria. The resistance protein TetA, a membrane-spanning H + -[tcÆM] + antiporter, must be sen- sitively regulated because its expression is harmful in the absence of tc, yet it has to be expressed before the drugs’ concentration reaches cytoplasmic levels inhibitory for protein synthesis. Consequently, TetR shows highly speci- fic tetO binding to reduce basal expression and high affinity to tc to ensure sensitive induction. Tc can cross biological membranes by diffusion enabling this inducer to penetrate the majority of cells. These regulatory and pharmacological properties are the basis for application of TetR to selec- tively control the expression of single genes in lower and higher eukaryotes. TetR can be used for that purpose in some organisms without further modifications. In mam- mals and in a large variety of other organisms, however, eukaryotic transcriptional activator or repressor domains are fused to TetR to turn it into an efficient regulator. Mechanistic understanding and the ability to engineer and screen for mutants with specific properties allow tailoring of the DNA recognition specificity, the response to inducer tc and the dimerization specificity of TetR-based eukary- otic regulators. This review provides an overview of the TetR properties as they evolved in bacteria, the functional modifications necessary to transform it into a convenient, specific and efficient regulator for use in eukaryotes and how the interplay between structure ) function studies in bacteria and specific requirements of particular applica- tions in eukaryotes have made it a versatile and highly adaptable regulatory system. Keywords: antibiotic resistance; disease models; fusion pro- tein; inducible gene expression; ligand-binding specificity; mammalian cell lines; protein engineering; structure–activity relationship; Tet repressor; transgenic organism. Properties of bacterial Tet systems Efflux-mediated tetracycline resistance is always regulated in Gram-negative bacteria In Gram-negative bacteria, resistance to tetracyclines (tc) is mediated by the TetA protein, a proton-[tcÆMg] + anti- porter embedded in the cytoplasmic membrane [1,2]. Eleven tc resistance determinants (Tet classes A–E, G, H, J, Z, 30, and 33 [3–5]) share the organization of structural and regulatory genes (reviewed in [6]). In enteric bacteria, the efflux-encoding tetA genes are strictly regulated at the level of transcription by the tc-responsive Tet repressor (TetR). In the absence of inducer, TetR dimers bind to the operators tetO 1 and tetO 2 , shutting down transcription of its own gene, tetR, and of the resistance gene, tetA.Oncetchas entered the cell, it binds TetR with high affinity as a [tcÆMg] + complex [7]. This induces a conformational change in TetR [8] resulting in dissociation from tetO [9]. The following expression burst of TetA and TetR leads to a rapid reduction of the cytoplasmic tc concentration [10] which, in turn, shuts expression of both genes off again. Expression of TetA is fine-tuned in the presence of tc so that export overcomes the slow uptake (compare below). Regulation of Tc resistance is optimized for tightness and sensitivity Regulation of tet determinants is subject to strong, opposing selective pressures. Expression of the resistance protein TetA is detrimental to the cell [11,12]. Overexpression of this integral membrane protein is lethal for Escherichia coli [13], probably due to the collapse of the membrane potential [14]. Consequently, expression of TetA must be tightly repressed in the absence of the drug. However, when tc diffuses into the cell the resistance protein must be expressed before the cytoplasmic concentration of tc reaches the micromolar level necessary to inhibit translation. This requires: (a) high Correspondence to W.Hillen,Lehrstuhlfu ¨ r Mikrobiologie, Institut fu ¨ r Mikrobiologie, Biochemie und Genetik, Friedrich-Alexander Universita ¨ t, Staudtstr. 5, D-91058 Erlangen, Germany. Fax: +49 9131 8528082, Tel.: +49 9131 8528081, E-mail: whillen@biologie.uni-erlangen.de Abbreviations: tc, tetracycline; dox, doxycycline; atc, anhydrotetra- cycline; tTA, tetracycline-dependent transactivator; rtTA, reverse tetracycline-dependent transactivator; tTS, tetracycline-dependent trans-silencer; CMV, cytomegalovirus; GFP, green fluorescent protein. (Received 8 April 2003, revised 14 May 2003, accepted 15 May 2003) Eur. J. Biochem. 270, 3109–3121 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03694.x affinity of TetR for both tetO andtctokeepthebasal expression level of tetA low and to ensure that its transcription is initiated at concentrations which are still subinhibitory for translation; (b) low affinity of the TetR– [tcÆMg] + complex for DNA; and (c) high-level, but short- term expression of TetA to initially reduce the internal concentration of tc. A low level of TetR is important for sensitive induction, since E.coli strains expressing high levels of TetR need high concentrations of tc for full induction [15]. These conflicting requirements are met by the genetic organization of the resistance determinants (reviewed in [6]) and by the ligand binding properties of TetR. High sensitivity towards tetracyclines [see Fig. 1 for the structures of tc, doxycycline (dox) and anhydro-tc (atc)] is achieved by the remarkable binding constant of TetR for [tcÆMg] + (K a  10 9 M )1 ), [doxÆMg] + (K a  10 10 M )1 )or [atcÆMg] + (K a  10 11 M )1 ) [7,9], about 10 3 )10 5 -fold higher than the affinity of the drugs to their intracellular target, the ribosome [16]. Binding of two molecules of [tcÆMg] + to a TetR dimer diminishes repressor affinity for tetO by about nine orders of magnitude to the unusually low background DNA binding level of less than 10 5 M )1 [9]. This high ratio of specific over nonspecific DNA binding enables TetR to bind tetO efficiently, even in larger genomes containing competing nonspecific DNA to a much higher degree than bacteria. Taken together, the evolutionary pressures on tc-dependent gene regulation have led to tight repression in the absence of tc, without compromising sensitivity of induction, so that regulated tc resistance determinants impose no burden on the fitness of E.coliin the absence of the antibiotic, but still mediate high levels of resistance to tc in its presence [12]. The structural change of TetR associated with induction by tetracycline is known X-ray crystal structures of free TetR [17], TetR complexed with different tetracyclines [18–21] and with tetO [8] have been determined at resolutions of 1.9–2.5 A ˚ , revealing the allosteric conformational change leading to induction. These results have been reviewed in detail [22] and have been compared to Lac repressor [23]. Thus, they are only summarized here (Fig. 2). The DNA reading head of TetR (magenta) is connected to the protein core (blue) by the helix a4 (green). Binding of [tcÆMg] + (yellow) to TetR unwinds the C-terminal residues of helix a6 (light blue), which bump into a4 and displace it. As the C terminus of a4isheldin place by contacts to tc, the displacement leads to a pendulum-like swing of the a4 N terminus increasing the distance between the recognition helices by 3 A ˚ , so that they do not fit into successive major grooves of DNA anymore [24]. These conformational changes are consistent with many noninducible TetR mutants [24,25], spectroscopic analysis of TetR in vitro [26], in vivo [27] and in vitro [28] disulfide trapping experiments. Furthermore, a movement of a9 closes the tc binding pocket after the drug has entered [17], and the loop between a8anda9 is also important for induction [29–31]. Tetracycline penetrates cells by diffusion Tetracyclines (Fig. 1) diffuse in their uncharged forms through lipid bilayers without the aid of protein channels [32–36]. Measuring the increase in fluorescence intensity of tc observed upon binding to TetR [7] allows us to determine the cytoplasmic concentration of tc and, thus, to calculate permeation coefficients for tc uptake into liposomes [(2.4 ± 0.6) · 10 )9 cmÆs )1 ] and whole E.coli cells [(5.6 ± 1.9) · 10 )9 cmÆs )1 ] [36]. These translate into half- equilibration times of 35 ± 15 min for tc to cross the membranes and are in good agreement with the half- equilibration time of 15 min measured for [ 3 H]tc-uptake in Bacillus subtilis [37], and the slow uptake of tc observed in Staphylococcus aureus [38]. Tetracycline diffusion through phospholipid membranes is, thus, slow and appears to be the rate-limiting step of uptake into cells [36]. The previously observed rapid uptake of tc [33,39] might rather reflect unspecific adsorption of tc to membrane surfaces [32,36]. A detailed model explaining the transport and accumulation of tc across the Gram-negative cell envelope has been presented by Nikaido and coworkers ([40,41] and references cited therein). In the medium, as well as in the periplasm and cytoplasm, tc is present in one uncharged and several charged or zwitterionic species, due to its three titratable groups (Fig. 1). The distribution between these species depends on the pH of the respective compartment [40]. The Fig. 1. Structures of tetracyclines used in eukaryotic gene regulation. (A) Structure of tetracycline with the pK a values of the three titratable groups. (B) Structure of doxycycline. (C) Structure of anhydrotetra- cycline. 3110 C. Berens and W. Hillen (Eur. J. Biochem. 270) Ó FEBS 2003 uncharged form of tc can penetrate the outer membrane directly. But the major fraction of tc equilibrates as a [tcÆM] + -complex rapidly through the outer membrane via porins, with the Donnan potential across the outer mem- brane leading to a two- to threefold accumulation of this charged complex in the periplasm. Tc then diffuses passively in its uncharged form through the cytoplasmic membrane. Due to the pH gradient across the cytoplasmic membrane, a larger fraction of the uncharged tc dissociates in the cytoplasm than in the periplasm. Since equilibrium is reached when the concentration of uncharged tc is identical in both compartments, this results in a higher intracellular concentration of [tcÆM] + , the biologically active compound. Again, accumulation of tc is the product of this passive equilibration across the inner membrane [40,41]. Tc-based gene regulation functions in different setups in many eukaryotic systems The evolved properties of TetR described above combined with the favorable pharmacokinetics of tetracyclines and their long record of safe use in clinical practice make the Tet system a good candidate to fulfill the criteria that are required for an ideal transcriptional regulator in eukaryotic cells as given by Saez and others [42,43]. Consequently, the past 15 years have seen the broad application of tc-dependent regulatory systems, mainly in mammalian cell culture, but to an increasing degree in transgenic organisms like plants, yeasts, protozoan parasites, slime molds, flies, and rodents. These topics have been extensively reviewed [42–52]. The following section presents an overview of the basic Tet systems used to regulate gene expression in eukaryotes. Gene regulation by TetR in eukaryotes The most basic and first published application of tc-mediated gene regulation in eukaryotes is transcriptional repression by unmodified TetR [53]. Here, TetR most likely acts by interfering sterically with binding of RNA polymerase or auxiliary transcription factors [42,54]. To achieve this, one or more tetO elements are placed in proximity of either the TATA box or the transcriptional start site of the respective target gene and TetR is expressed concomitantly by a strong, constitutive promoter. Promot- ers of all three eukaryotic RNA polymerases have been targeted in the manner described. Unfortunately, as will become evident in the following paragraph, the published, successful approaches do not yet allow formation of a simple strategy for establishing a TetR-repressed system, although they clearly point out that the positioning of the tetO boxes is crucial for efficient regulation. In Leishmania donovani, an RNA polymerase I promo- ter was brought under tc-control by placing a single tetO site 2–24 bp upstream of the transcriptional start site [55], whereas in Trypanosoma brucei at least one tetO element had to be inserted at a position +2 or )2relativetothe transcription start site of an RNA polymerase I-like promoter [56]. For RNA polymerase III-mediated tran- scription of suppressor tRNA genes, induction factors between two- to fivefold were observed in Saccharomyces cerevisiae, Dictyostelium discoideum and carrot protoplasts when tetO was introduced within 10 bp upstream of the transcriptional start site [57–59]. A regulated version of the human U6 snRNA promoter, also transcribed by RNA polymerase III, was developed by replacing sequences between the proximal sequence element and the transcrip- tional start site with tetO [60]. Flanking the TATA-box with two operators completely abolished transcriptional activity. In contrast, introduction of a single tetO element affected transcription only slightly, but led to up to 25-fold repression in the presence of TetR. A regulated U6 snRNA promoter with a defined expression window [61,62] would be a very powerful tool as this promoter is used to express the small interfering RNA [63] needed for silencing gene Fig. 2. Structure of the TetR–[tcÆMg] + complex. Tet repressor is shown as a ribbon diagram with one monomer in gray and the other monomer color-coded as follows: The DNA-binding region is in magenta, the helix connecting it with the protein core is in green. The protein core is dark blue, with the helix a6 in light blue. Tetracycline is displayed as space-filling CPK model in yellow. For clarity, the helices a1–a10 of one monomer are num- beredandtheNandCterminiofbothsub- units are indicated. The coordinates were taken from the PDB entry 2TRT [18]. Ó FEBS 2003 Gene regulation by tetracyclines (Eur. J. Biochem. 270) 3111 expression by RNA interference [64]. Repression of RNA polymerase II promoters exerted by TetR is strongest in plants [65,66], mammalian cells [67] and fungi like Schizo- saccharomyces pombe [68,69] when multiple tet operators are positioned within a region from 5 bp upstream to 35 bp downstream of the TATA-element. In contrast, placement of one to four tet operators immediately downstream of the transcription initiation site has been shown to be most effective in the parasitic protozoa Entamoeba histolytica [70,71], Toxoplasma gondii [72] and Giardia lamblia [73]. Gene regulation by TetR-based transregulators While unmodified TetR acts as a transcriptional repressor in plants and lower eukaryotes, it can be, but not always is efficient in mammalian cells [67,74]. A consistently func- tional version for yeasts, flies and mammalian cell lines is TetR fused to an eukaryotic regulatory domain, such as an acidic activation domain (Fig. 3A; tTA or Tet-Off) [75–80] or a repression domain (Fig. 3B; tTS) [81–83]. The trans- activator tTA directs expression from a tc-dependent promoter that contains seven repeats of a tetO 2 element from the transposon Tn10. The palindromic centers of two adjacent operators are separated by 41 bp. This element is fused to a minimal promoter, typically derived from the human cytomegalovirus (CMV) immediate early promoter [75]. When both components are stably integrated into proper chromosomal loci of mammalian cell lines, tran- scription from the hybrid promoter is silent in the presence of more than 10 ngÆmL )1 dox. Removal of dox leads to binding of tTA to tetO and subsequent activation of transcription. Regulatory factors of up to five orders of magnitude can be reached with sensitive reporter genes like firefly luciferase [75]. Luciferase activity is expressed within 4 h of removal of tc and about 20% of the steady-state level is reached after 12 h. While the use of a strong, constitutive promoter (CMV IE, EF-1a, Ubiquitin C) is common in cell culture applications, the use of tissue-specific promoters in transgenic animals provides spatial control to the Tet system, restricting expression of the Tet transregulator and, subsequently, the transgene to the desired tissue [84,85]. In Drosophila, usage of the Gal4-UAS system to control Tet transregulator expression allows the generation of spatially delimited expression patterns by simple crossing with one of the many Gal4 driver lines available in the Drosophila research community [86]. One concern has been the expression levels of Tet transregulators as influenced by a potentially low mRNA stability or efficiency of translation. This was recently addressed by generating a synthetic coding sequence for tetR. Potential splice donor and acceptor sites identified by sequence analysis, several potential endonuclease cleavage sites, and potential stable hairpin structures in the mRNA were eliminated and human codon usage was used [87–90]. The consequence of this optimization protocol is a higher protein level in Drosophila, HeLa and HEK293 cells. Another concern voiced was that the CMV-derived minimal promoter was not transcriptionally silent under all experimental conditions [91–93]. This promoter leakiness can be caused by promoter-dependent or integration site- dependent effects and has been discussed in detail [94]. Promoter-dependent leakiness has been addressed by the use of alternative minimal promoters [75,95,96]. In transient transfection experiments, these show lower basal activities Fig. 3. Regulation of gene expression by Tet transregulators. The promoter proximal tetO boxes are represented by black boxes. The transregulators are shown as follows: the DNA reading heads are in light gray, the inducer-binding and dimerization domain is in dark gray, activation domains are black boxes, and the silencing domain is stippled. The conformational change leading to the loss of DNA-binding activity is pictured as a light gray box. High-level activated transcription is displayed by a bold arrow, low-level basal transcription by a dotted arrow. (A) tTA. (B) tTS. (C) rtTA. 3112 C. Berens and W. Hillen (Eur. J. Biochem. 270) Ó FEBS 2003 than P tet -1, but also do not reach its maximal activation level. Thus, the regulatory window for target gene expres- sion is shifted and expanded due to the stronger reduction of the basal activity. Integration site-dependent leakiness has been attributed to enhancers located close to the integration site of the target gene construct. Besides screening additional clones until one harboring the desired properties is found, the problem has been approached by insulating P tet -1 from external activating signals through insertion of a chicken lysozyme matrix attachment region just upstream of P tet -1 [87] or by flanking the target gene expression unit with either chicken b-globin insulators [90] or SCS and SCS’ boundary elements from Drosophila [86]. A different strategy was adopted by engineering a tc-controlled trans-silencer protein [81]. Fusion of the KRAB domain of Kox1 [97] to TetR yielded a hybrid protein called tTS, that not only substantially repressed basal transcription from P tet -1 even if the tet operators were located 3 kbp distant from the minimal promoter, but also efficiently down-regulated gene expression from a CMV enhancer-driven P tet -1 [83]. This strategy therefore appears to be more versatile in coping with unwanted target gene expression than the promoter adaptation proposed above. In addition and in contrast to tTA, the tc-dependent silencing of complex promoters offers the unique possibility of reversibly down-regulating the expression of cellular genes on top of their normal regulation. The KRAB domain is inactive in S. cerevisiae and Drosophila where it was replaced with repression domains from the proteins SSN6 [82], knirps, giant or dCtBP [83]. The expression of transfected genes can be rapidly repressed in mammalian cells by epigenetic mechanisms [98]. Although this Ôtransgene silencingÕ is not specific for the Tet system, it is often observed for genes under tc control due to its frequent usage as conditional expression system. Approaches to achieve stable gene expression have been to: (a) screen many transfected clones; (b) the use of lentiviral vectors [99]; (c) replace the viral promoters that direct expression of the transregulators with promoters of human origin [100]; (d) use chromatin insulator sequences to protect transgene expression [98]; or (e) couple transgene expression to a selectable marker via an IRES element [101] or by fusion of the transregulator with green fluorescent protein (GFP) [102]. Note that in this fusion protein GFP is connected to the DNA-binding domain of TetR which can interfere with nonspecific DNA-binding activity of TetR at low levels of dox (see Fig. 2A in [102] and [78]). The few published examples make it impossible to recommend one of the strategies for use in establishing homogenous expression of transgenes, but silencing of transregulator expression is not completely suppressed by the use of lentiviral vectors [103] or insulator sequences [101]. Modifications of the Tet transregulators The TetR–VP16 fusion works very well in many cases, but may not be optimal for all applications. Structure–function studies based on powerful selection and screening systems in E.coli [104,105] and in S. cerevisiae [88] have lead to a profound understanding of how DNA binding, inducer binding and dimerization function in TetR. This informa- tion can be used to find solutions to some of the problems and limitations that arise for Tet system applications in eukaryotes. Alterations of the activator domain of tTA Especially for gene therapy, concern about a viral protein is often voiced, as humoral as well as cellular immune response against the VP16 protein has been found in herpes simplex infected humans [106–108]. Thus, immune res- ponses against transactivators containing the VP16 domain cannot be rigorously excluded, although they have not been observed so far in a mouse model using reverse tTA (rtTA; Tet-On) [109]. Two solutions circumventing this concern have been developed: (a) the VP16 domain has been replaced by three repeats of a minimal activation domain derived from a 12-amino acid activating patch of the VP16 protein (tTA2 [76]); and (b) a variety of human activator domains from the acidic, glutamine-rich, serine/threonine- rich and proline-rich functional groups were tested for their ability to replace the VP16 domain. When fused to TetR, only acidic activation domains were highly active [78–80]. Minor activation was observed with the serine/threonine- rich domains from the transcription factors ITF-1, ITF-2, and MTF-1. Transactivators with activation potentials spanning more than three orders of magnitude have been generated by combination of various minimal activation domains (see above; [76]). They are attractive for combined Ôknock-in/knock-outÕ strategies to convey tissue-specific expression of the transactivator, while at the same time inactivating expression of the genomic copy of the target gene. Expression of the regulatory protein is then an invariant function of the genomic locus and, if too high, can lead to ÔsquelchingÕ [110]. This can be addressed by employing a transactivator with reduced activation poten- tial as these are tolerated in the cell at higher concentrations [76]. Conversion of TetR to reverse TetR Eukaryotic gene regulation by tTA shows a high dynamic range and works consistently well, but has several practical drawbacks. Tc has to be continually present to keep expression of the gene of interest downregulated. Although tc is not toxic at the levels utilized in gene regulation, prolonged exposure to the antibiotic is not always desirable in transgenic animals nor is it possible in gene therapy. Furthermore, induction of target genes is mostly slow as it requires removal of the drug from the culture or organism. To be able to control the time point of induction more precisely, and since organisms are more easily saturated with an effector than depleted of it [111,112], reverse TetR variants which bind tetO onlyinthepresenceoftcwere searched for and found (Fig. 3C). Screening in E.coli[113] and in S. cerevisiae [88] revealed that a small number of mutations in TetR can lead to that phenotype [113]. Once this was discovered, intensive screening led to rtTA alleles in which the initial disadvantages of occasional background expression and low sensitivity for dox were eliminated [88]. The rtTA-S2 allele was obtained by screening for reduced background expression and rtTA-M2 was the result of screening for higher sensitivity towards dox starting from Ó FEBS 2003 Gene regulation by tetracyclines (Eur. J. Biochem. 270) 3113 the alterations in rtTA-S2 that are responsible for the reverse phenotype [88]. None of the exchanges found in these new alleles were present in the original rtTA. The mutations leading to the reverse phenotype are located at the interface between the DNA reading head and the protein core or in the last turn of helix a6 that undergoes a conformational change upon inducer binding. Structural analysis of the DNA-bound form of TetR has led to the proposal that the mutations present in rtTA [113] restrict the repressor to a noninducible conformation and lock the DNA-binding domains in the position necessary for oper- ator binding [8]. Taken together, the phenotype of rtTA can be improved and designed by using appropriate screens. Tet transregulators vary in their sensitivity towards tetracyclinic inducers The tTA and rtTA variants presently employed in eukary- otic gene regulation display differential sensitivity towards tc and its derivatives. While tTA can be induced by tc, dox and atc [114], reverse transactivators respond only to dox and atc [113] and tTS G is about twofold less sensitive to dox than rtTA [115]. The response range of tTA to dox (0.1– 10 ngÆmL )1 ) is clearly lower and, more importantly, non- overlapping with that of rtTA to dox (100–3000 ngÆmL )1 ) [114], but slightly overlapping with that of the more sensitive rtTA2 s -M2 allele (2–200 ngÆmL )1 ) [88]. The molecular mechanisms responsible for these different sensitivities are presently unknown. The isolation of a tc-like antagonist for TetR [116] and the demonstration of its activity in transgenic plants [117] make it seem likely that alternative inducers for TetR can be identified by screening. The DNA binding specificity of Tet-transregulators can be changed Structure–function analyses of TetR–tetO interactions had shown that only few changes (shown in Fig. 4) in the DNA binding helix–turn–helix motif of TetR suffice to switch the recognition specificity from the 19-base pair wild-type tetO to variants containing symmetric exchanges of bases at position 4 (tetO-4C [118]) and position 6 (tetO-6C [119]). The TetR mutants were converted into the transactivators tTA2 4C or tTA2 6C and minimal promoters P tet4 and P tet6 were constructed with the respective tetO variants [114]. DNA binding of the modified transactivators is efficient; in transient transfections in HeLa cells, they specifically achieve induction factors between 2000 and 8000 and are, thus, as active as wild-type tTA2. Moreover, they are also highly specific, as they induce the converse operator less than twofold [114]. Modulation of the DNA-binding specificity is not confined to tTA. Alleles specific for the 4C- [120] and 6C-tet operators [114] have been constructed with rtTA and also regulate tc-dependent expression units efficiently. This now leaves us with different tTA- and rtTA- operator combinations capable of controlling gene expres- sion tightly over a wide range of inducer concentrations. Mastering subunit recognition of TetR Comparison of the TetR primary structures reveals 38–90% identical amino acids overall, but only 18% in the four-helix bundle involved in dimerization. Detailed structural infor- mation [19] of the dimerization interface [121] suggested that TetR proteins from individual classes would not readily form heterodimers. The modular architecture of TetR allows the combination of a class B DNA-binding domain with the inducer-binding and/or dimerization domains of Tet repressors from other classes [121]. Fusion to the reading head from TetR(B) increases activity of Tet repressors from several other classes [121] and ensures tight binding to the tetO boxes from Tn10 [122]. Class B TetR does not form heterodimers with Tet repressors from classes D [121], E [93,114,120], or G [115]. The fusion points can be chosen with some flexibility; functional chimeras have been obtained either by connecting the entire protein core from TetR(D) or TetR(E) to a TetR(B) DNA-reading head [114,120,121] or by replacing the four-helix bundle formed by the helices a8anda10 from both subunits (see Fig. 2), with the respective region from TetR(G) [115]. The resulting transrepressors or transactivators regulate gene expression efficiently and do not form heterodimers as demonstrated in DNA-retardation assays [114], immunoprecipitation and FACS analysis [115] opening up the possibility to introduce two or more TetR-based regulatory proteins into the same cell without having to cope with the disadvantages of heterodimer formation [114,115]. Combinatorial Tet regulation solves special problems and allows sophisticated applications The previous section has shown that DNA-binding speci- ficity, subunit recognition and response to the inducer can be altered in TetR. Fig. 5 gives an overview of the present state of the Tet modules that are available for use and the following section presents a few principles of how the modular nature of the transregulators can be exploited to address specific experimental requirements and open up new applications for conditional regulation. Expression can be switched between two alleles of one gene The expression of two genes or of two alleles of one gene can be controlled in a mutually exclusive manner by combining different dimerization domains, different operator-binding Fig. 4. Operator specificity combinations for the Tet system. The pri- mary structure of the TetR(B) recognition helix a3 and the flanking loops is given in standard one-letter abbreviations. The entire sequence of tetO 2 is shown with the palindromic center marked by an asterisk and the base numbering shown above one operator half-side. The exchanges in TetR and tetO are highlighted in inverse print for each matching pair (wt, 4C, 6C). 3114 C. Berens and W. Hillen (Eur. J. Biochem. 270) Ó FEBS 2003 specificities and by exploiting the differential sensitivity of Tet transregulators towards tetracyclines [114]. Interference between the two expression units is excluded by using a tTA allele with an alternative class E or G dimerization domain and by furnishing rtTA with a modified DNA-binding domain that contacts the tetO-4C operator in P tet4 speci- fically. Expression of the wild-type allele, for example, is placed under tTA control and represents the normal state of the cell. A knockout situation can be generated by adding either tc or, alternatively, atc or dox at concentrations between 10 and 100 ngÆmL )1 which dissociates tTA from the promoter but does not lead to DNA binding by rtTA [114]. Maintaining the intermediate concentration of dox needed to shut down expression of both alleles will be feasible in cell culture applications. In transgenic animals, however, the necessary fine-tuning of a dox or atc concen- tration may prove impossible suggesting instead the use of tc to shut down tTA-dependent gene expression without interfering with regulation by rtTA. To switch to the expression of the mutant allele requires atc or dox concen- trations of 1 lgÆmL )1 or more. Such a dual control system can provide valuable insights into developmental and pathogenic processes. One can imagine shutting down expression of a tumor suppressor while inducing expression of an oncogene to study cancerogenesis. Switching off expression of the oncogene after tumor formation can establish whether the respective protein is a valid target for therapeutic inter- vention. One could also switch from a wild-type to a mutant allele at a defined developmental state of the organism and then return to wild-type expression at a later stage. This type of regulatory circuit can also deliver an additional degree of freedom to gene therapeutic strat- egies ) one regulatory circuit may be used to control a therapeutic gene, while the other may be exploited to serve as a suicide switch to terminate the treatment once the therapeutic goal has been reached or, if necessary, in case of emergency. One gene can be regulated stringently by conversely acting transrepressor and transactivator Detectable levels of transgene expression in animals or cells in which the transactivator is not active can limit the usefulness of any conditional expression system for mode- ling complex biological processes or evaluating the effects of a gene product. For the Tet system, this Ôtransgene leakageÕ has been attributed either to basal activity of the respective tetO-based minimal promoter used (see above; [115]); or, in systems with rtTA, to residual binding of the reverse transactivator to tetO in the absence of dox [123,124]. A stringently controlled regulatory system can now be accomplished by combining a trans-silencer with a reverse transactivator, since heterodimer formation and concomit- ant phenotype blurring will be prevented if the trans-silencer is equipped with a dimerization domain from the TetR classes E or G. Thus, both transregulators bind in a mutually exclusive manner. Gene expression is actively repressed in the absence of dox by the binding of tTS E /tTS G to the minimal promoter. Upon addition of dox, tTS E /tTS G dissociates from tetO, allowing the reverse transactivator to bind and activate transcription. This setup efficiently reduces background expression in yeast [82], in mammalian cell lines [93,115,120] and in transgenic animals [125–127], while affecting the maximal expression level only slightly [128] or not at all [93]. Transgenes can be expressed in a graded or in a binary manner Transcriptional control has generally been assumed to operate as a binary switch with on/off characteristics [129,130], but several examples displaying graded changes in gene expression have recently been published [131,132]. The manner of gene expression might well be a key factor in programs of cell differentiation or stimulus response. Different regulatory setups of the Tet system allow a transgene to be expressed in one or the other manner [133– 135], enabling not only an analysis of a gene’s function, but also of its mode of expression. When tTA and rtTA are expressed constitutively in mammalian cells and also in S. cerevisiae, they drive transgene expression in a dose- dependent, graded manner [133,135]. However, when rtTA was expressed in S. cerevisiae under conditions of positive feedback using an autoregulatory circuit, the cell population was clearly divided into regulator-expressing and nonex- pressing cell pools [135]. In mammalian cells, the combined usage of tTS G and rtTA also led to bimodal expression of the GFP reporter (see Fig. 3 in [134]). Although not formally proven, we assume that a bimodal expression pattern will not be observed for all repressor/activator combinations, but only for those in which the sensitivity of tTS for the inducer is lower than that of the rtTA allele used, as is the case for tTS G (compare the dose–response curve of tTS E and rtTA of Fig. 4 in [93] with the one for tTS G and rtTA of Fig. 2 of [134]). This will ensure that rtTA is preloaded with inducer and ready to activate transcription the moment the dox concentrations needed for binding to Fig. 5. The Tet toolbox. TetR modules and regulatory domains are displayed with the possible combinations. The different binding func- tions of TetR were coded in different shades of gray and placed at their approximate position in the protein, but not drawn to scale. The TetR variants characterized were classified in the corresponding module. The regulatory domains that can be fused to TetR are coded in dif- ferent shades of gray according to their viral, human, insect or fungal origin. Note that not every possible combination of modules need result in a transregulator with acceptable regulatory properties. Ó FEBS 2003 Gene regulation by tetracyclines (Eur. J. Biochem. 270) 3115 tTS G are reached and tetO is subsequently released. In principle, only two regulatory states are observed: either the tetO sites are fully occupied with tTS G and gene expression is shut off, or they are saturated with the rtTA variant, resulting in full transcriptional activation. The consequence is a binary expression pattern of the target gene. While this setup already works with rtTA, the effect should be even more pronounced with rtTA2 s -M2, as its inducer response range overlaps completely with that of tTS G . Highlighting the regulatory potential and looking into the future The properties and the adaptability of Tet regulation as presented in the previous sections allow its use in many different applications. We would like to demonstrate this enormous variability by referring to a few key studies that, in our opinion, highlight the potential of Tet regulation. Regulation by tetracyclines is sensitive and efficient enough to control target gene expression in pathogenic organisms even when they have been injected into a mammalian host. The role of individual genes in infection and pathogenesis can, thus, be probed and their validity as targets for therapeutic intervention determined in an in vivo disease model [136]. This has not only been demonstrated for trypanosomes [137,138], but also for common human pathogens like Staphylococcus aureus [139] and Candida glabrata [140]. In the fungus, squalene synthase [136] and sterol 14a-demethylase [141] were, thus, shown not to be ideal targets for antifungal development. The successful expression of the diphtheria toxin A subunit by tTA/P tet -1 in transgenic mice has demonstrated the stringency of regulation that can be reached with the Tet system [142]. Although mouse lines that carried the target transgene were obtained at an approximately 10-fold lower frequency than normal, those that were established regula- ted the transgene efficiently. Induction of toxin expression led to cell death and development of cardiomyopathies. Stringent control of transgene expression using rtTA has also been achieved in HeLa cells for the Shiga toxin B subunit [143], for the proapoptotic gene PUMA in SAOS-2 and H1299 cell lines [144] and, using rtTA2 s -S2 in transgenic mice, for Cre-recombinase [145]. The strength of a true conditional system ) the possibi- litytoswitchgeneexpressiononandoffatleisureand repeatedly ) represents a powerful method with which to explore the relationship between mutant protein expression and disease progression. This has become evident upon studying transgenic mouse models for cancer and neuro- logical disorders. Here, the use of tTA and rtTA to control expression of an oncogene revealed for solid tumors [146,147] and for leukemias [148,149] that the oncogene is not only necessary for tumor formation but also for tumor maintenance, suggesting pharmacological inactivation of oncogenes as a possible therapeutic strategy for cancer. This assumption has been substantiated by the unexpected observation that, after having gone through one cycle of MYC-gene expression and silencing, reactivation of the oncogene does not lead to tumor regrowth, but rather to apoptosis [150]. Similar effects have also been found for neurological disorders. In a conditional model of Hunting- ton’s disease, mice expressing a mutated huntingtin fragment in the brain demonstrated that its continuous supply was needed to maintain the characteristic neuro- pathology and behavioral phenotype, raising the possibility that the disease may be reversible by targeting the causative agent [151]. Regulation by the Tet system has also had a significant impact on behavioral studies. Expression of constitutively active mutant forms of the calcium/calmodulin dependent kinase II or calcineurin in the brain of adult mice resulted in altered synaptic plasticity and impairments in spatial memory storage and retrieval, but these deficits were fully reversed when transgene expression was suppressed [84,152]. Because expression of the transgene was limited to the hippocampus, this structure was additionally proven to be the site responsible for the behavioral effects. In a different example, knockout mice lacking the serotonin 1A receptor show increased anxiety-like behavior which could be rescued by conditional expression, but only if the receptor was synthesized during the early postnatal period in the hippocampus and cortex [153]. Nevertheless, improvement and additions to the Tet system, among them the regulatory components, are still possible and necessary. Promoter development has not received the same degree of attention as the transregulators. The number of tetO elements and their spacing [154], as well as the linker sequence separating the operators [155] have not been optimized yet. It remains to be seen if an ÔidealÕ minimal promoter with no intrinsic leakiness supporting very high-level activation can be identified or designed. Fortunately, screens for regulators with improved prop- erties can now be performed in eukaryotic systems [88] and, as an example, the isolation of novel Tet regulators which recognize nonantibiotically active tetracyclines or even nontetracyclinic inducers, would be of great benefit. They would not only facilitate gene therapy applications which, at the moment, can be impaired by the use of tetracyclines in anti-infective therapy or their misuse as growth promoting additives to animal food. If these novel inducers are not only ecologically safe, but also easy and nonexpensive to manufacture, the inducer–regulator pair- ing could also be useful in insect population control using dominant, repressible, lethal genetic systems [156,157] and might even introduce regulation by the Tet system to crop plants. They would add to the repertoire of transregulators and finally, since multiple dimerization and DNA-binding specificities are already present, allow fully independent expression control of more than one gene by the Tet system. A major experimental challenge will be to express a target gene within its physiological window, which might depend on environmental stimuli and even change during develop- ment, since over- or underexpression often results in altered phenotypes [131] or pathologies. While tc-controlled expres- sion can mimic the natural level [146], this must not always be the case. A solution might be precise promoter targeting by tetO elements, to minimally interfere with gene expres- sion. This will be difficult and will require extensive knowledge about the influence of chromatin structure on gene expression and its sensitivity to perturbation, partic- ularly when regulatory regions are modified [158]. But, if successful, this approach will provide an additional degree of freedom to manipulate gene expression, as the existing 3116 C. Berens and W. Hillen (Eur. J. Biochem. 270) Ó FEBS 2003 transregulators can be used to activate or silence gene expression, in addition to and independent of the promo- ter’s natural expression pattern. Conclusion The Tet system is the most widely used regulatory system for conditional gene expression at the moment. The increasing number of: (a) cell lines stably transfected with tTA and rtTA; (b) cell lines harboring tTA or rtTA that have been derived from transgenic mice; and (c) transgenic mice expressing either the transregulators via cell-type specific promoters or a target gene under P tet -1 control will greatly facilitate genetic studies by allowing combination of the existing components instead of having to generate all cell and mouse lines, a costly and time-consuming process. Ongoing improvement of the existing components as well as the continuous addition of new components to extend its applicability have turned the Tet system into a highly versatile and flexible regulatory system that can be adapted to many different applications. 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ARTICLE Gene regulation by tetracyclines Constraints of resistance regulation in bacteria shape TetR for application in eukaryotes Christian Berens and Wolfgang. regulate gene expression in eukaryotes. Gene regulation by TetR in eukaryotes The most basic and first published application of tc-mediated gene regulation in eukaryotes