- Email: [email protected]
Inducible gene expression systems for higher eukaryotic cells
Inducible gene expression systems for higher eukaryotic cells Manfred Gossen, Angelika L Bonin, Sabine Freundlieb and Hermann Bujard Zentrum for Molekulare Biologie der Universit~t Heidelberg, Heidelberg, Germany Of the wide variety of regulatory systems that have been developed to control gene expression in higher eukaryotes, those utilizing elements from prokaryotic systems presently achieve the highest specificity. In particular, systems based on the repressor/operator elements of an Escherichia coil tetracycline resistance operon appear broadly applicable. During the past year, the usefulness of these systems has been demonstrated not only at the cellular level, but also at the organismal level (i.e. in transgenic animals and plants). Current Opinion in Biotechnology 1994, 5:516-520
Introduction Systems that allow the stringent regulation of gene expression offer great advantages both for studying gene function and for tackling a diverse spectrum of problems in applied biology. In particular, tightly controlled expression (i.e. using a system where transcription can be shut off and then relieved in defined increments until high levels of expression are attained) is a prerequisite for a variety of studies and applications. Thus, to elucidate the function of a protein in vivo, it may not suffice to merely analyze the phenotype resulting from its 'off' and 'on' expression state. Many gene products participate in specific equilibria characteristic for a physiological or developmental state; therefore, it may be necessary to vary the intracellular concentration of a protein around a given value to recognize its true role in vivo. Similarly, to produce a protein with cytotoxic properties, quantitative control of its synthesis is not only required in the final fermentation process, but also in the initial construction and optimization of the production strain or cell line. An essential feature of such regulatory systems is the potential to control the activity of a gene in a reversible and temporally defined manner. This will open up new approaches for the analysis of differentiation and developmental processes. Moreover, such methodologies will have an impact on many aspects ofbiotechnology. They may, for instance, permit the development of new pharmacological models in transgenic animals, and doubtless provide the basis for various prospective human genetherapy regimens. In prokaryotes, such as Escherichia coli, regulatory systems that fulfill all of the above requirements have been known and in wide use for a number of years. In contrast, promising approaches for higher eukaryotic systems
have been described only recently, despite numerous attempts. In this review, we focus exclusively on systems in which an inducing stimulus (in general, a small effector molecule) directly governs the onset of transcription of a specific gene. Such effectors usually bind to transcriptional activators or repressors and modulate their DNAbinding properties. Thus, we do not cover other very useful regulatory approaches, including the hormonal control strategy of Picard et al. [1], the use of bivalent effect molecules to control signal transduction reported by Spencer et al. [2], and the use of so-called 'binary systems' for the regulation of defined genes in transgenic organisms [3-5]. For a more detailed discussion of these approaches, the reader is referred to reviews elsewhere in this issue by Picard (pp 511-515), Ramirez-Solis and Bradley (pp 528-533) and Sauer (pp 521-527).
Regulating gene activity in higher eukaryotic systems by endogenous factors A number of transcription control systems have been described, in which the expression of specific genes is directed by R N A polymerase II promoters susceptible to outside stimuli. These endogenous systems usually suffer from one, or both, of the following drawbacks: first, the inducing molecules (e.g. heavy-metal ions, steroid hormones or heat-shock proteins) evoke pleiotropic effects that render the analysis of the resulting phenotype difficult; second, most of the regulable promoters have activities that are too high in the non-induced state, the resulting 'leakiness' precluding them from many applications. Even so, when the primary goal is the induction of high levels of protein synthesis, and negative effects that interfere with the physiology of the cell can be tolerated, such systems may be well suited.
Abbreviations IPTG--isopropyl-l~-D-thiogalactopyranoside;R/O--repressor/operator;tTA--tetracycline-controlled transactivator.
516
© Current Biology Ltd ISSN 0958-1669
Inducible gene expression systemsfor higher eukaryotic cells Gossen et al. Because these approaches have been excellently reviewed by Yarronton [6], we would like to add here only a few general remarks. Most vertebrate polymerase II promoters are highly modular in structure. They interact with a variety of transcription factors, which themselves are involved in a multitude of interactions with other regulatory elements and promoters. Thus, it is difficult to conceive of a natural or artificial endogenous system that could be both controlled by outside stimuli in a monospecific way and at the same time, applicable in various cell types, tissues or even transgenic organisms. Even the drosophila ecdysone receptor transfected into mammalian cells [7] is reliant on the recruitment of other members of the hormone receptor superfamily to function efficiently [8]. One way out of this dilemma appears to be the utilization of regulatory elements from organisms distant in evolution, such as E. coli. Because a good chance exists that some foreign regulatory elements and their inducers may not interfere with the physiology of higher eukaryotic cells, it may be feasible to establish truly monospecific regulatory systems.
Prokaryotic control elements as mediators of regulated gene expression in eukaryotic cells
The repression principle Elements of two inducible prokaryotic repressor/operator (R/O) systems--the E. coli lactose (lac) operon and the (Tn 10-derived) tetracycline (tet) operon - - have been utilized to regulate gene activity in eukaryotic cells. The most suitable inducer of the Lac R / O system appears to be isopropyl-~-D-thiogalactopyran0side (IPTG), which prevents the formation of the Lac R / O complex. Tetracycline and many of its derivatives function in an analogous way to the Tet R / O complex. Both the Lac R / O - I P T G system and the Tet R/O-tetracycline system have been employed to control the activity of polymerase II promoters according to the prokaryotic paradigm. In this paradigm, operator sequences are placed near the transcriptional start site such that the R / O complexes interfere with the formation of a transcription-competent complex (Fig. la). A number of early studies showed that both these R / O systems have significant potential [9-11]. Even so, in all Lac R/O-based systems (for a review see [6]), the induction exerted by IPTG is not satisfactory. In spite of the fact that the system can modulate expression by factors of up to 100, the induction process is slow, incomplete and frequently requires IPTG concentrations that approach cytotoxic levels. Following the same strategy, the Tet R / O regulatory system has been established in several systems, including yeast ([12,13]; A Bonin, H Bujard, unpublished data), dictyostehum [14], plant cells [15] and even tobacco plants [16,17]. The most impressive results were reported from the tobacco plant system, where expres-
sion could be modulated several hundred fold [16,17]. In addition, efficient control by Tet R / O of the expression of a toxic gene in transgenic tobacco plants has been demonstrated recently [18°]. Thus far, attempts to establish a corresponding regulatory system in mammalian cells have failed for reasons not fully understood (A Bonin, H Bujard, unpubhshed data). To control the onset of transcription via repression, it is crucial to achieve a high occupancy of the operator by the repressor. Thus, besides a number of parameters that must be favorable for R / O function, for example, the positioning of operator sequences within a promoter (for a discussion, see [19"']), high intracellular repressor concentrations must also prevail. Up to 106 repressor molecules can be maintained in tobacco plant cells [16], resulting in tight repression. In contrast, sufficiently high intracellular repressor concentrations have never been achieved in mammalian cells (A Bonin, H Bujard, unpublished data), and some results indicate that strong overexpression is not tolerated (A Bonin, H Bujard, unpubhshed data). Thus, one of the intrinsic problems of these repression systems is the difficulty of generating and maintaining high intracellular repressor concentration in various cells and tissues. As Gossen et al. [19"] and Weinmann et al. [20"'] discuss in more detail, stringent control of transcription in higher eukaryotic systems is achieved by promoter activation rather than by repression.
The activation principle A different approach, which uses prokaryotic R / O elements to regulate gene expression in mammalian cells, is based on the construction of fusion proteins between transcriptional transactivation domains (e.g. the transactivator of herpes simplex virus VP16) and the Lac repressor [6,21] or the Tet repressor [22], respectively. The LacR based transactivating system, although it functions in principle, again suffers from the intrinsic pharmacological and thermodynamic limitations~of all Lac R / O IPTG based systems (discussed in [19°°]). In contrast, the tetracycline-controlled transactivator (tTA) profits from favorable thermodynamic properties of the Tet R/O-tetracycline interaction [23]. At present, we consider the tTA system the most promising. Its mechanism of action is outlined in Fig. lb. The effector (i.e. tetracychne) inactivates the transactivator and thereby shuts off transcription from a minimal promoter, the function of which depends entirely on the binding of tTA to the tet operators placed -70bp upstream of the transcriptional start site. Removal of tetracychne leads to activation of transcription. A luciferase reporter gene controlled by a tTA-responsive promoter, which had been integrated into HeLa cells stably producing tTA, revealed that expression could be modulated by factors of up to 105 [22]. This high degree of regulation demonstrates high levels of expression in the activated state and very tight control of gene
517
518
Expressionsystems
(a)
P-'t
~
Repressor
- A n
--
+E
4 Enhancer
(~
Enhancer
//L_ TATA - + 4--
/ / - - TATA _ ~
I x
Operator
Operator
p --~
(b)
Transactivator ]-~ An - -
i
4
i
+1 TATA Operator
+1 *--F~
+1 - Operator
©1994
TATA
xl-<-
Current Opinion in Biotechnology
activity in the inactivated state. It should, however, be pointed out that the transcription unit containing the tTA-responsive promoter, which has all the properties of an 'enhancer trap', has to be integrated into a locus of the chromosome where no external activation can take place. Since the above studies were reported, the tTA regulatory system has been successfully applied in numerous laboratories at the cellular [24°*,25,26",27] as well as the organismal level. In transgenic tobacco plants, regulation that is both extremely tight and capable of inducing expression at a wide range of levels has been achieved [20*°]. Direct transfer of DNAs encoding tTA and the luciferase reporter unit into the heart muscle o f rats results i n more than 100-fold differences in luciferase activity in that tissue, depending on whether or not the rats are exposed to tetracycline [28*°]. Finally, double transgenic mice, which produce tTA and contain either the luciferase gene or a corresponding [~-galactosidase gene, exhibit tetracycline-controlled expression of reporter genes that can be modulated several thousand fold ([29"°]; A Kistner, H Bujard, unpublished
Fig. 1. Schematic representation of a repression system and an activation system. (a) In the repression system, the repressor (R) is synthesized under the control of an appropriate expression signal (P). When the effector (E; e.g. tetracycline) is absent, the repressor binds one, or several, strategically placed operator sequences (dyad symmetries are indicated by small arrows) within a promoter/enhancer region [the TATA box and transcriptional start site (+1) of the promoter are indicated] and interferes with transcription initiation. In the presence of effector, repression is relieved (RE), leading to transcription of the controlled gene (X). Tissue specificity of the system can be achieved by selection of a suitable P for expression of the repressor as well as an operator containing a promoterenhancer for gene X. (b) In the activation system, the transactivator, consisting of a repressor moiety and an activation domain (A), is synthesized under the control of an appropriate expression signal. In the absence of effector, it binds to the operator site(s) linked to a minimal promoter, thereby activating transcription. In the presence of effector, the transactivator dissociates from its binding site(s) and, depending on the quality of the minimal promoter and its site of integration in the genome, the transcription unit is silent. Tissue specificity of the system is achieved by selection of a suitable P for expression of the transactivator. Active transcription of the gene is indicated by the larger shaded crooked arrow at the transcriptional start site (+1).
data). Thus, the tTA regulatory system appears widely applicable, largely because o f the simplicity with which cell- and tissue-specific expression can be achieved. For completeness, it should be mentioned that transgenic mice carrying the gene o f either the Lac repressor or the Tet repressor have also been generated [30,31]. In light of the low levels of expression induced in systems applying negative rdgulation (see previous section), we must await further studies to ascertain whether this approach can be successful and generally applicable.
Concluding remarks The value of any system for controlling gene activity is ultimately judged by its applicability. According to this criterium, at present, transcriptional control via tTA appears to be the method o f choice for tackling a large spectrum o f problems. This, however, by no means precludes the urgent need for improving other presently available systems and for developing new ones. For ex-
Inducible gene expression systemsfor higher eukaryotic cells Gossen et ample, a disadvantage of the tTA system is that activation of transcription follows the removal of tetracycline, a process intrinsically slower than uptake of an effector for induction of gene expression. Using the current tTA system, activation of gene expression by addition of tetracycline can be achieved via a tetracycline-regulated antisense RNA. In an alternative approach using the lacR based transactivation system, Bairn et al. [32] succeeded in generating a reverse phenotype for the transactivator. The IacR/VP16 derivative LAP267, which is induced by IPTG, is another, in principle, elegant regulatory system. Because this transactivator can only be fully exploited at elevated temperatures and because IPTG induction is hampered by the problems discussed above, it has not found wide application, however. Nevertheless, isolation of transactivators showing a reverse phenotype may be possible in the tetracycline system. Such a system would actually not supersede the present tTA system; rather, it would complement it. A great advantage of the tetracycline-based regulatory system is the availability of a large number of tetracycline derivatives with various properties. This will be particularly useful for in vivo studies because tissue penetration, crossing of the blood-brain barrier or crossing of the placenta may be optimized by selecting appropriate compounds that have well characterized pharmacological properties. In conclusion, we foresee that the tetracyclinedependent regulation will be only the first example of a number of rather generally applicable regulatory systems for controlling gene expression in higher eukaryotes. As independent regulation of the activity of several genes is a major aim, we anticipate that the application of these inducible gene expression systems to eukaryotic gene studies will open up exciting new perspectives in biology and medicine.
al.
Specific Recombination in Transgenic Mice. Proc Nat/Acad Sci USA 1992, 89:6232-6236.
6.
Yarronton GT: Inducible Vectors for Expression in Mammalian Cells. Curt Opin Biotechnol 1992, 3:506-511.
7.
Christopherson
KS, Mark
MR,
Bajaj V,
Godowski
PJ:
Ecdysteroid-Dependent Regulation of Genes in Mammalian Cells by a Drosophila Ecdysone Receptor and Chimeric Transactivators. Proc Nat/ Acad Sci USA 1992, 89:6314-6318. 8.
Yao TP, Segraves WA, Or• AE, McKeown M, Evans RM:
Drosophila Ultraspiracle Modulates Ecdysone Receptor Function via Heterodimer Formation. Cell 1992, 71:63-72. 9.
Hu MCT, Davidson N: The Inducible Lac Operator-Repressor System is Functional in Mammalian Cells. Cell 1987, 48:555-566.
10.
Brown M, Figge J, Hansen U, Wright C, Jeang KT, Khoury G, Livingston DM, Roberts TM: Lac Repressor Can Regulate Expression from a Hybrid SV40 Early Promoter Containing a Lac Operator in Animal Cells. Ceil 1987, 49:603-612.
11.
Figge J, Wright C, Collins CJ, Roberts TM, Livingston DM: Stringent Regulation of Stably Integrated Chloramphenicol Acetyl Transferase Genes by E. coil lac Repressor in Monkey Cells. Ceil 1988, 52:713-722.
12.
Faryar K, Gatz C: Construction of a Tetracycline-Inducible Promotor in Schizosaccaromyes pombe. Curt Genet 1992,
21:345-349. 13.
Dingermann T, Frank-Stoll U, Werner H, Wissmann A, Hillen W, Jacquet M, Marschalek R: RNA Polymerase III Catalysed
Transcription Can be Regulated in Saccharomycescerevislae by the Bacterial Tetracycline Repress•r-Operator System. EMBO J 1992, 11:1487-1492. 14.
Dingermafln T, Werner H, Schlitz A, Z0ndod I, Nerke K, Knecht D, Marschlek R: Establishment of a System for Conditional
Gene Expression using an Inducible tRNA Suppressor Gene. A4ol Cell Biol 1992, 12:4038-4045.
15.
Gatz C, Quail PH: TnlO-Encoded Tet Repressor Can Regulate an Operator-Containing Plant Promoter. Proc Nat/ Acad Sci USA 1988, 85:1394-1397.
16.
Gatz C, Kaiser A, Wendenburg R: Regulation of a Modified
CaMV 35S Promoter by the TnlO-Encoded let Repressor in Transgenic Tobacco. Mol Gen Genet 1991, 227:229-237. 17.
Gatz C, Frohberg C, Wendenburg R: Stringent Repression and
Homogeneous De-Repression by Tetracycline of a Modified CaMV 35S Promoter in Intact Transgenic Tobacco Plants. Plant J 1992, 2:397-404. 18.
R~der FT, Schmglling T, Gatz C: Efficiency of the Tetracycline-
References and recommended reading
•
Dependent Gene Expression System--Complete Suppression and Efficient Induction of the rolB Phenotype in Transgenic Plants. Mo/ Gen Genet 1994, 243:32-38.
Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest o• of outstanding interest
First application of a tetracycline-controlled gene expression system in a transgenic organism.
Picard D, Salser SJ, Yamamoto KR: A Movable and Regulalable
Trends Biochem Sci 1993, 18:471-475. Reviews the utility of prokaryotic regulatory elements in regulating expression in higher eukaryotic cells. One of the issues in this review is the comparison of the basic requirements of activating systems and repressing systems.
1.
Inactivation Function Within the Steroid Binding Domain of the Glucocortlcoid Receptor. Cell 1988, 54:1073-1080. 2.
Spencer DM, Wandless TJ, Schreiber SL, Crabtree GR: Controlling Signal Transduction with Synthetic Ligands. Science 1993,
262:1019-1024. 3.
19. •-
Proc Nat/Acad Sci USA, 1989, 86:5473-5477.
4.
Ornitz DM, Moreadith RW, Leder P: Binary System for Regulating Transgene Expression in Mice: Targeting int-2 Gene Expression with Yeast GAL4/UAS Control Elements. Proc Nat/ Acad Sci USA 1991, 88:698-702.
5.
Lakso M, Sauer B, Mosinger B, Lee EJ, Manning RW, Yu SH, Mulder KL, Westphal H: Targeted Oncogene Activation by Site-
Higher Eukaryotic Cells by Prokaryotic Regulatory Elements.
20.
Weinmann P, Gossen M, Hillen W, Bujard H, Gatz C: A
••
Chimeric Transactivator Allows Tetracycline-Responsive Gene Expression in Whole Plants. Plant J 1994, 5:559-569.
Byrne GW, Ruddle FH: Multiplex Gene Regulation: A Two-
Tiered Approach to Transgene Regulation in Transgenic Mice.
Gossen M, Bonin AL, Bujard H: Control of Gene Activity in
This work describes the function of the tTA system in transgenic plants, comparing its activity as a repressor and an activator. After proper adaptation, the function of the tTA system is demonstrated in transgenic plants. The extreme tightness and the wide range of regulation achieved with the tTA system is also discussed with respect to the tet repression system in tobacco. 21.
I.abow MA, Baim SB, Shenk T, Levine AJ: Conversion of the Lac
Repressor into an AIIosterically Regulated Transcriptional Activator for Mammalian Cells. Mol Cell Bio11990, 10:3343-3356.
519
520
Expression systems 22.
Gossen M, Bujard H: Tight Control of Gene Expression in Mammalian Cells by Tetracycline-Responsive Promoters. Proc
23.
Hillen W, Wissmann A: TetR-teIO Interaction. In Protein-Nucleic Acid Interaction. Topics in Molecular and Structural Biology, vol 10. Edited by Saenger W, Heinemann U. London: Macmillan Press; 1989:143-162.
Natl Acad Sci USA 1992, 89:5547-5551.
Resnitzky D, Gossen M, Bujard H, Reed SI: Acceleration of the G1/S Phase Transition by Expressionof Cyclins D1 and E with an Inducible System. Mol Cell Biol 1994, 14:1669-1679. This work makes use of the potential of the tet system to temporally control the activity of a regulator element and permits insights into the role of this element in a temporal program.
29. n
Furth PA, Onge LSt, B~ger H, Gruss P, Gossen M, Kistner A, Bujard H, Hennighausen L: Temporal Control of Gene Expression in Transgenic Mice by a Tetracycline Responsive Promoter. Proc Nat/ Acad Sci USA 1994, in press. Demonstrates that expression in mice transgenic for tTA can be controlled by tetracyclines. Reporter units containing the luciferase or the [3-galactosidase gene are expressed under control of a tTA-dependent promoter.
24. •*
25.
Wimmel A, Lucibello FC, Sewing A, Adolph S, MCiller R: Inducible Acceleration of G(1) Progressionthrough TetracyclineRegulated Expression of Human Cycline E. Oncogene 1994, 9:995-997.
26. n
Haase SB, Heinzel SS, Caius MP: Transcription Inhibits the
Replication of Autonomously Replicating Plasmids in Human Cells. Mol Cell Biol 1994, 14:2516-2524. This work demonstrates a direct correlation between level of induction by tetracycline and a phenotype. 27.
Fr~ih K, Gossen M, Wang K, Bujard H, Peterson PA, Yang Y: Displacement of Housekeeping Proteasome Subunits by MHC-Encoded LMPs: A Newly Discovered Mechanism for Modulating the Multicatalytic Protelnase Complex. EMBO ] 1994, 13:3236-3244.
Fishman GI, Kaplan ML, Buttrick PM: Tetracycline-Regulated Cardiac Gene Expression in Vivo. J Clio Invest 1994, 93:1864-1868. Description of the adaption of the tTA system to heart muscle specific expression. After direct injection of DNAs encoding tTA into the heart muscle of rats, expression control by tetracycline is demonstrated in vivo. 28.
•.
30.
Epstein-Baak R, Lin Y, 8radshaw V, Cohn M: Inducible Transformation of Cells from Transgenic Mice ExpressingSV40 Under lac Operon Control. Cell Growth Diff 1992, 3:127-134.
31.
Byrne GW, Kagan D: Tetracycline Regulated Gene Expression in Transgenic Mice: Control of Growth Hormone Expression. In Abstracts of Papers Presented at the 1992 Meeting on "Mouse Molecular Genetics'. Cold Spring Harbor: Cold Spring Harbor Laboratory Press; 1992:254.
32.
Bairn SB, Labow MA, Levine AJ, Shenk T: A Chimeric Mammalian Transactivator Based on the lac Repressor that is Regulated by Temperature and Isopropyl I~-D-Thlogalactopyranoside. Proc Natl Acad Sci USA 1991, 88:5072-5076.
M Gossen, University of California at Berkeley, 401 Barker Hall, Berkeley, California 94720-3202, USA. AL Bonin, BASF Bioresearch Corporation, 100 Research Drive, Worcester, Massachusetts 01605-4314, USA. S Freundlieb and H Bujard, Zentrum ftir Molekulare Biologie der Universit~it Heidelberg, lm Neuenheimer Feld 282, 69120 Heidelberg, Germany.
Introduction Systems that allow the stringent regulation of gene expression offer great advantages both for studying gene function and for tackling a diverse spectrum of problems in applied biology. In particular, tightly controlled expression (i.e. using a system where transcription can be shut off and then relieved in defined increments until high levels of expression are attained) is a prerequisite for a variety of studies and applications. Thus, to elucidate the function of a protein in vivo, it may not suffice to merely analyze the phenotype resulting from its 'off' and 'on' expression state. Many gene products participate in specific equilibria characteristic for a physiological or developmental state; therefore, it may be necessary to vary the intracellular concentration of a protein around a given value to recognize its true role in vivo. Similarly, to produce a protein with cytotoxic properties, quantitative control of its synthesis is not only required in the final fermentation process, but also in the initial construction and optimization of the production strain or cell line. An essential feature of such regulatory systems is the potential to control the activity of a gene in a reversible and temporally defined manner. This will open up new approaches for the analysis of differentiation and developmental processes. Moreover, such methodologies will have an impact on many aspects ofbiotechnology. They may, for instance, permit the development of new pharmacological models in transgenic animals, and doubtless provide the basis for various prospective human genetherapy regimens. In prokaryotes, such as Escherichia coli, regulatory systems that fulfill all of the above requirements have been known and in wide use for a number of years. In contrast, promising approaches for higher eukaryotic systems
have been described only recently, despite numerous attempts. In this review, we focus exclusively on systems in which an inducing stimulus (in general, a small effector molecule) directly governs the onset of transcription of a specific gene. Such effectors usually bind to transcriptional activators or repressors and modulate their DNAbinding properties. Thus, we do not cover other very useful regulatory approaches, including the hormonal control strategy of Picard et al. [1], the use of bivalent effect molecules to control signal transduction reported by Spencer et al. [2], and the use of so-called 'binary systems' for the regulation of defined genes in transgenic organisms [3-5]. For a more detailed discussion of these approaches, the reader is referred to reviews elsewhere in this issue by Picard (pp 511-515), Ramirez-Solis and Bradley (pp 528-533) and Sauer (pp 521-527).
Regulating gene activity in higher eukaryotic systems by endogenous factors A number of transcription control systems have been described, in which the expression of specific genes is directed by R N A polymerase II promoters susceptible to outside stimuli. These endogenous systems usually suffer from one, or both, of the following drawbacks: first, the inducing molecules (e.g. heavy-metal ions, steroid hormones or heat-shock proteins) evoke pleiotropic effects that render the analysis of the resulting phenotype difficult; second, most of the regulable promoters have activities that are too high in the non-induced state, the resulting 'leakiness' precluding them from many applications. Even so, when the primary goal is the induction of high levels of protein synthesis, and negative effects that interfere with the physiology of the cell can be tolerated, such systems may be well suited.
Abbreviations IPTG--isopropyl-l~-D-thiogalactopyranoside;R/O--repressor/operator;tTA--tetracycline-controlled transactivator.
516
© Current Biology Ltd ISSN 0958-1669
Inducible gene expression systemsfor higher eukaryotic cells Gossen et al. Because these approaches have been excellently reviewed by Yarronton [6], we would like to add here only a few general remarks. Most vertebrate polymerase II promoters are highly modular in structure. They interact with a variety of transcription factors, which themselves are involved in a multitude of interactions with other regulatory elements and promoters. Thus, it is difficult to conceive of a natural or artificial endogenous system that could be both controlled by outside stimuli in a monospecific way and at the same time, applicable in various cell types, tissues or even transgenic organisms. Even the drosophila ecdysone receptor transfected into mammalian cells [7] is reliant on the recruitment of other members of the hormone receptor superfamily to function efficiently [8]. One way out of this dilemma appears to be the utilization of regulatory elements from organisms distant in evolution, such as E. coli. Because a good chance exists that some foreign regulatory elements and their inducers may not interfere with the physiology of higher eukaryotic cells, it may be feasible to establish truly monospecific regulatory systems.
Prokaryotic control elements as mediators of regulated gene expression in eukaryotic cells
The repression principle Elements of two inducible prokaryotic repressor/operator (R/O) systems--the E. coli lactose (lac) operon and the (Tn 10-derived) tetracycline (tet) operon - - have been utilized to regulate gene activity in eukaryotic cells. The most suitable inducer of the Lac R / O system appears to be isopropyl-~-D-thiogalactopyran0side (IPTG), which prevents the formation of the Lac R / O complex. Tetracycline and many of its derivatives function in an analogous way to the Tet R / O complex. Both the Lac R / O - I P T G system and the Tet R/O-tetracycline system have been employed to control the activity of polymerase II promoters according to the prokaryotic paradigm. In this paradigm, operator sequences are placed near the transcriptional start site such that the R / O complexes interfere with the formation of a transcription-competent complex (Fig. la). A number of early studies showed that both these R / O systems have significant potential [9-11]. Even so, in all Lac R/O-based systems (for a review see [6]), the induction exerted by IPTG is not satisfactory. In spite of the fact that the system can modulate expression by factors of up to 100, the induction process is slow, incomplete and frequently requires IPTG concentrations that approach cytotoxic levels. Following the same strategy, the Tet R / O regulatory system has been established in several systems, including yeast ([12,13]; A Bonin, H Bujard, unpublished data), dictyostehum [14], plant cells [15] and even tobacco plants [16,17]. The most impressive results were reported from the tobacco plant system, where expres-
sion could be modulated several hundred fold [16,17]. In addition, efficient control by Tet R / O of the expression of a toxic gene in transgenic tobacco plants has been demonstrated recently [18°]. Thus far, attempts to establish a corresponding regulatory system in mammalian cells have failed for reasons not fully understood (A Bonin, H Bujard, unpubhshed data). To control the onset of transcription via repression, it is crucial to achieve a high occupancy of the operator by the repressor. Thus, besides a number of parameters that must be favorable for R / O function, for example, the positioning of operator sequences within a promoter (for a discussion, see [19"']), high intracellular repressor concentrations must also prevail. Up to 106 repressor molecules can be maintained in tobacco plant cells [16], resulting in tight repression. In contrast, sufficiently high intracellular repressor concentrations have never been achieved in mammalian cells (A Bonin, H Bujard, unpublished data), and some results indicate that strong overexpression is not tolerated (A Bonin, H Bujard, unpubhshed data). Thus, one of the intrinsic problems of these repression systems is the difficulty of generating and maintaining high intracellular repressor concentration in various cells and tissues. As Gossen et al. [19"] and Weinmann et al. [20"'] discuss in more detail, stringent control of transcription in higher eukaryotic systems is achieved by promoter activation rather than by repression.
The activation principle A different approach, which uses prokaryotic R / O elements to regulate gene expression in mammalian cells, is based on the construction of fusion proteins between transcriptional transactivation domains (e.g. the transactivator of herpes simplex virus VP16) and the Lac repressor [6,21] or the Tet repressor [22], respectively. The LacR based transactivating system, although it functions in principle, again suffers from the intrinsic pharmacological and thermodynamic limitations~of all Lac R / O IPTG based systems (discussed in [19°°]). In contrast, the tetracycline-controlled transactivator (tTA) profits from favorable thermodynamic properties of the Tet R/O-tetracycline interaction [23]. At present, we consider the tTA system the most promising. Its mechanism of action is outlined in Fig. lb. The effector (i.e. tetracychne) inactivates the transactivator and thereby shuts off transcription from a minimal promoter, the function of which depends entirely on the binding of tTA to the tet operators placed -70bp upstream of the transcriptional start site. Removal of tetracychne leads to activation of transcription. A luciferase reporter gene controlled by a tTA-responsive promoter, which had been integrated into HeLa cells stably producing tTA, revealed that expression could be modulated by factors of up to 105 [22]. This high degree of regulation demonstrates high levels of expression in the activated state and very tight control of gene
517
518
Expressionsystems
(a)
P-'t
~
Repressor
- A n
--
+E
4 Enhancer
(~
Enhancer
//L_ TATA - + 4--
/ / - - TATA _ ~
I x
Operator
Operator
p --~
(b)
Transactivator ]-~ An - -
i
4
i
+1 TATA Operator
+1 *--F~
+1 - Operator
©1994
TATA
xl-<-
Current Opinion in Biotechnology
activity in the inactivated state. It should, however, be pointed out that the transcription unit containing the tTA-responsive promoter, which has all the properties of an 'enhancer trap', has to be integrated into a locus of the chromosome where no external activation can take place. Since the above studies were reported, the tTA regulatory system has been successfully applied in numerous laboratories at the cellular [24°*,25,26",27] as well as the organismal level. In transgenic tobacco plants, regulation that is both extremely tight and capable of inducing expression at a wide range of levels has been achieved [20*°]. Direct transfer of DNAs encoding tTA and the luciferase reporter unit into the heart muscle o f rats results i n more than 100-fold differences in luciferase activity in that tissue, depending on whether or not the rats are exposed to tetracycline [28*°]. Finally, double transgenic mice, which produce tTA and contain either the luciferase gene or a corresponding [~-galactosidase gene, exhibit tetracycline-controlled expression of reporter genes that can be modulated several thousand fold ([29"°]; A Kistner, H Bujard, unpublished
Fig. 1. Schematic representation of a repression system and an activation system. (a) In the repression system, the repressor (R) is synthesized under the control of an appropriate expression signal (P). When the effector (E; e.g. tetracycline) is absent, the repressor binds one, or several, strategically placed operator sequences (dyad symmetries are indicated by small arrows) within a promoter/enhancer region [the TATA box and transcriptional start site (+1) of the promoter are indicated] and interferes with transcription initiation. In the presence of effector, repression is relieved (RE), leading to transcription of the controlled gene (X). Tissue specificity of the system can be achieved by selection of a suitable P for expression of the repressor as well as an operator containing a promoterenhancer for gene X. (b) In the activation system, the transactivator, consisting of a repressor moiety and an activation domain (A), is synthesized under the control of an appropriate expression signal. In the absence of effector, it binds to the operator site(s) linked to a minimal promoter, thereby activating transcription. In the presence of effector, the transactivator dissociates from its binding site(s) and, depending on the quality of the minimal promoter and its site of integration in the genome, the transcription unit is silent. Tissue specificity of the system is achieved by selection of a suitable P for expression of the transactivator. Active transcription of the gene is indicated by the larger shaded crooked arrow at the transcriptional start site (+1).
data). Thus, the tTA regulatory system appears widely applicable, largely because o f the simplicity with which cell- and tissue-specific expression can be achieved. For completeness, it should be mentioned that transgenic mice carrying the gene o f either the Lac repressor or the Tet repressor have also been generated [30,31]. In light of the low levels of expression induced in systems applying negative rdgulation (see previous section), we must await further studies to ascertain whether this approach can be successful and generally applicable.
Concluding remarks The value of any system for controlling gene activity is ultimately judged by its applicability. According to this criterium, at present, transcriptional control via tTA appears to be the method o f choice for tackling a large spectrum o f problems. This, however, by no means precludes the urgent need for improving other presently available systems and for developing new ones. For ex-
Inducible gene expression systemsfor higher eukaryotic cells Gossen et ample, a disadvantage of the tTA system is that activation of transcription follows the removal of tetracycline, a process intrinsically slower than uptake of an effector for induction of gene expression. Using the current tTA system, activation of gene expression by addition of tetracycline can be achieved via a tetracycline-regulated antisense RNA. In an alternative approach using the lacR based transactivation system, Bairn et al. [32] succeeded in generating a reverse phenotype for the transactivator. The IacR/VP16 derivative LAP267, which is induced by IPTG, is another, in principle, elegant regulatory system. Because this transactivator can only be fully exploited at elevated temperatures and because IPTG induction is hampered by the problems discussed above, it has not found wide application, however. Nevertheless, isolation of transactivators showing a reverse phenotype may be possible in the tetracycline system. Such a system would actually not supersede the present tTA system; rather, it would complement it. A great advantage of the tetracycline-based regulatory system is the availability of a large number of tetracycline derivatives with various properties. This will be particularly useful for in vivo studies because tissue penetration, crossing of the blood-brain barrier or crossing of the placenta may be optimized by selecting appropriate compounds that have well characterized pharmacological properties. In conclusion, we foresee that the tetracyclinedependent regulation will be only the first example of a number of rather generally applicable regulatory systems for controlling gene expression in higher eukaryotes. As independent regulation of the activity of several genes is a major aim, we anticipate that the application of these inducible gene expression systems to eukaryotic gene studies will open up exciting new perspectives in biology and medicine.
al.
Specific Recombination in Transgenic Mice. Proc Nat/Acad Sci USA 1992, 89:6232-6236.
6.
Yarronton GT: Inducible Vectors for Expression in Mammalian Cells. Curt Opin Biotechnol 1992, 3:506-511.
7.
Christopherson
KS, Mark
MR,
Bajaj V,
Godowski
PJ:
Ecdysteroid-Dependent Regulation of Genes in Mammalian Cells by a Drosophila Ecdysone Receptor and Chimeric Transactivators. Proc Nat/ Acad Sci USA 1992, 89:6314-6318. 8.
Yao TP, Segraves WA, Or• AE, McKeown M, Evans RM:
Drosophila Ultraspiracle Modulates Ecdysone Receptor Function via Heterodimer Formation. Cell 1992, 71:63-72. 9.
Hu MCT, Davidson N: The Inducible Lac Operator-Repressor System is Functional in Mammalian Cells. Cell 1987, 48:555-566.
10.
Brown M, Figge J, Hansen U, Wright C, Jeang KT, Khoury G, Livingston DM, Roberts TM: Lac Repressor Can Regulate Expression from a Hybrid SV40 Early Promoter Containing a Lac Operator in Animal Cells. Ceil 1987, 49:603-612.
11.
Figge J, Wright C, Collins CJ, Roberts TM, Livingston DM: Stringent Regulation of Stably Integrated Chloramphenicol Acetyl Transferase Genes by E. coil lac Repressor in Monkey Cells. Ceil 1988, 52:713-722.
12.
Faryar K, Gatz C: Construction of a Tetracycline-Inducible Promotor in Schizosaccaromyes pombe. Curt Genet 1992,
21:345-349. 13.
Dingermann T, Frank-Stoll U, Werner H, Wissmann A, Hillen W, Jacquet M, Marschalek R: RNA Polymerase III Catalysed
Transcription Can be Regulated in Saccharomycescerevislae by the Bacterial Tetracycline Repress•r-Operator System. EMBO J 1992, 11:1487-1492. 14.
Dingermafln T, Werner H, Schlitz A, Z0ndod I, Nerke K, Knecht D, Marschlek R: Establishment of a System for Conditional
Gene Expression using an Inducible tRNA Suppressor Gene. A4ol Cell Biol 1992, 12:4038-4045.
15.
Gatz C, Quail PH: TnlO-Encoded Tet Repressor Can Regulate an Operator-Containing Plant Promoter. Proc Nat/ Acad Sci USA 1988, 85:1394-1397.
16.
Gatz C, Kaiser A, Wendenburg R: Regulation of a Modified
CaMV 35S Promoter by the TnlO-Encoded let Repressor in Transgenic Tobacco. Mol Gen Genet 1991, 227:229-237. 17.
Gatz C, Frohberg C, Wendenburg R: Stringent Repression and
Homogeneous De-Repression by Tetracycline of a Modified CaMV 35S Promoter in Intact Transgenic Tobacco Plants. Plant J 1992, 2:397-404. 18.
R~der FT, Schmglling T, Gatz C: Efficiency of the Tetracycline-
References and recommended reading
•
Dependent Gene Expression System--Complete Suppression and Efficient Induction of the rolB Phenotype in Transgenic Plants. Mo/ Gen Genet 1994, 243:32-38.
Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest o• of outstanding interest
First application of a tetracycline-controlled gene expression system in a transgenic organism.
Picard D, Salser SJ, Yamamoto KR: A Movable and Regulalable
Trends Biochem Sci 1993, 18:471-475. Reviews the utility of prokaryotic regulatory elements in regulating expression in higher eukaryotic cells. One of the issues in this review is the comparison of the basic requirements of activating systems and repressing systems.
1.
Inactivation Function Within the Steroid Binding Domain of the Glucocortlcoid Receptor. Cell 1988, 54:1073-1080. 2.
Spencer DM, Wandless TJ, Schreiber SL, Crabtree GR: Controlling Signal Transduction with Synthetic Ligands. Science 1993,
262:1019-1024. 3.
19. •-
Proc Nat/Acad Sci USA, 1989, 86:5473-5477.
4.
Ornitz DM, Moreadith RW, Leder P: Binary System for Regulating Transgene Expression in Mice: Targeting int-2 Gene Expression with Yeast GAL4/UAS Control Elements. Proc Nat/ Acad Sci USA 1991, 88:698-702.
5.
Lakso M, Sauer B, Mosinger B, Lee EJ, Manning RW, Yu SH, Mulder KL, Westphal H: Targeted Oncogene Activation by Site-
Higher Eukaryotic Cells by Prokaryotic Regulatory Elements.
20.
Weinmann P, Gossen M, Hillen W, Bujard H, Gatz C: A
••
Chimeric Transactivator Allows Tetracycline-Responsive Gene Expression in Whole Plants. Plant J 1994, 5:559-569.
Byrne GW, Ruddle FH: Multiplex Gene Regulation: A Two-
Tiered Approach to Transgene Regulation in Transgenic Mice.
Gossen M, Bonin AL, Bujard H: Control of Gene Activity in
This work describes the function of the tTA system in transgenic plants, comparing its activity as a repressor and an activator. After proper adaptation, the function of the tTA system is demonstrated in transgenic plants. The extreme tightness and the wide range of regulation achieved with the tTA system is also discussed with respect to the tet repression system in tobacco. 21.
I.abow MA, Baim SB, Shenk T, Levine AJ: Conversion of the Lac
Repressor into an AIIosterically Regulated Transcriptional Activator for Mammalian Cells. Mol Cell Bio11990, 10:3343-3356.
519
520
Expression systems 22.
Gossen M, Bujard H: Tight Control of Gene Expression in Mammalian Cells by Tetracycline-Responsive Promoters. Proc
23.
Hillen W, Wissmann A: TetR-teIO Interaction. In Protein-Nucleic Acid Interaction. Topics in Molecular and Structural Biology, vol 10. Edited by Saenger W, Heinemann U. London: Macmillan Press; 1989:143-162.
Natl Acad Sci USA 1992, 89:5547-5551.
Resnitzky D, Gossen M, Bujard H, Reed SI: Acceleration of the G1/S Phase Transition by Expressionof Cyclins D1 and E with an Inducible System. Mol Cell Biol 1994, 14:1669-1679. This work makes use of the potential of the tet system to temporally control the activity of a regulator element and permits insights into the role of this element in a temporal program.
29. n
Furth PA, Onge LSt, B~ger H, Gruss P, Gossen M, Kistner A, Bujard H, Hennighausen L: Temporal Control of Gene Expression in Transgenic Mice by a Tetracycline Responsive Promoter. Proc Nat/ Acad Sci USA 1994, in press. Demonstrates that expression in mice transgenic for tTA can be controlled by tetracyclines. Reporter units containing the luciferase or the [3-galactosidase gene are expressed under control of a tTA-dependent promoter.
24. •*
25.
Wimmel A, Lucibello FC, Sewing A, Adolph S, MCiller R: Inducible Acceleration of G(1) Progressionthrough TetracyclineRegulated Expression of Human Cycline E. Oncogene 1994, 9:995-997.
26. n
Haase SB, Heinzel SS, Caius MP: Transcription Inhibits the
Replication of Autonomously Replicating Plasmids in Human Cells. Mol Cell Biol 1994, 14:2516-2524. This work demonstrates a direct correlation between level of induction by tetracycline and a phenotype. 27.
Fr~ih K, Gossen M, Wang K, Bujard H, Peterson PA, Yang Y: Displacement of Housekeeping Proteasome Subunits by MHC-Encoded LMPs: A Newly Discovered Mechanism for Modulating the Multicatalytic Protelnase Complex. EMBO ] 1994, 13:3236-3244.
Fishman GI, Kaplan ML, Buttrick PM: Tetracycline-Regulated Cardiac Gene Expression in Vivo. J Clio Invest 1994, 93:1864-1868. Description of the adaption of the tTA system to heart muscle specific expression. After direct injection of DNAs encoding tTA into the heart muscle of rats, expression control by tetracycline is demonstrated in vivo. 28.
•.
30.
Epstein-Baak R, Lin Y, 8radshaw V, Cohn M: Inducible Transformation of Cells from Transgenic Mice ExpressingSV40 Under lac Operon Control. Cell Growth Diff 1992, 3:127-134.
31.
Byrne GW, Kagan D: Tetracycline Regulated Gene Expression in Transgenic Mice: Control of Growth Hormone Expression. In Abstracts of Papers Presented at the 1992 Meeting on "Mouse Molecular Genetics'. Cold Spring Harbor: Cold Spring Harbor Laboratory Press; 1992:254.
32.
Bairn SB, Labow MA, Levine AJ, Shenk T: A Chimeric Mammalian Transactivator Based on the lac Repressor that is Regulated by Temperature and Isopropyl I~-D-Thlogalactopyranoside. Proc Natl Acad Sci USA 1991, 88:5072-5076.
M Gossen, University of California at Berkeley, 401 Barker Hall, Berkeley, California 94720-3202, USA. AL Bonin, BASF Bioresearch Corporation, 100 Research Drive, Worcester, Massachusetts 01605-4314, USA. S Freundlieb and H Bujard, Zentrum ftir Molekulare Biologie der Universit~it Heidelberg, lm Neuenheimer Feld 282, 69120 Heidelberg, Germany.